identifier: Hamid, Jawed <https://research.library.mun.ca/view/creator_az/Hamid=3AJawed=3A=3A.html>   (1983) Characterization of extracellular proteases of psychrotrophic pseudomonads.  Masters thesis, Memorial University of Newfoundland.

abstract: For this study 8 cultures of P. fluorescens were selected. A protease was isolated from bacterial culture number T20 and was partially purified by a combination of ammonium sulfate precipitation, gel filtration on Sephadex G-200 and affinity column chromatography. The molecular weight of this protease was estimated to be 43,000 by gel filtration on Sephadex G-200. The proteases investigated in this study were active over a wide range of temperatures (5-45°C) with maximum activity at the temperature of 35°C. The pH optimum for T20 protease was 7.2; other proteases were also maximally active at neutral pH. All of the proteases investigated retained considerable activity after a heat exposure of ten minutes at 100°C. The T20 protease was stable over a wide range of pH (5.5-9.0) when stored at 10°C for four days. All of the proteases were found to be metalloproteases requiring divalent cations for activity and were inactivated slightly by sulfhydryl reagents. The synthesis of all proteases by the bacteria was stimulated by addition of milk to the growth medium. The protease activity of all isolates began to increase in early logarithmic phase and continued to increase till late stationary phase, and then began to decline. The proteases from various isolates were found to possess similar antigenic determinants. Each bacterial culture produced only one extracellular protease.


identifier: Senanayake, S. P. J. Namal <https://research.library.mun.ca/view/creator_az/Senanayake=3AS=2E_P=2E_J=2E_Namal=3A=3A.html>   (1995) Composition and physicochemical properties of oat starches.  Masters thesis, Memorial University of Newfoundland.

type: Thesis NonPeerReviewed

format: application/pdf application/pdf

abstract: Starches from two new oat varieties namely AC Stewart (Avena  sativa L.) and NO 753-2 (Avena nuda L.) were isolated and some of their characteristics determined. The total amount of starch lipids (TSL) extracted by acid hydrolysis was 1.60% (w/w) in both starches. The free lipid content (extracted by chloroform-methanol (CM) 2:1 v/v at 25°C) was 0.36 and 0.30% (w/w) in NO 753-2 and AC Stewart starches, respectively. The bound lipid content (extracted by hot n-propanol-water 3:1 v/v from the residue left after CM extraction) was 1.27 and 1.37% (w/w) in NO 753-2 and AC Stewart starches, respectively. The total amylose contents were nearly the same (~23%, w/w) in both starches, of which 14.1 (NO 753-2) and 15.3% (AC Stewart) were complexed by native lipids. The starches differed widely in their degree of swelling, gelatinization transition temperatures and enthalpy, susceptibility towards acid and enzyme hydrolysis, gel strength, paste viscosity, thermal stability and retrogradation characteristics. The results suggest that the  extent of interaction of starch chains within the amorphous and crystalline regions of the granule was stronger in NO 753-2 than in AC Stewart.


identifier: Atreya, Prabhakara Lakshmi <https://research.library.mun.ca/view/creator_az/Atreya=3APrabhakara_Lakshmi=3A=3A.html>   (1987) Conformational aspects of proline hydroxylation in collagen biosynthesis : studies with synthetic peptides.  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: The hydroxylation of selected proline residues by prolylhydroxylase (E.C. 1.14.11.2) is a crucial posttranslational event in the biosynthesis of collagen, an important protein of the connective tissues. Hydroxyproline (Hyp) offers additional stability to the unique triple-helical conformation of collagen, which in turn is necessary for the functional viability of the protein, at physiological temperatures. -- Earlier studies on the substrate specificity  of prolylhydroxylase have been intriguing. It was proposed earlier that prolylhydroxylase recognized the folded β-turn conformation, formed at the Pro-Gly segments in the nascent procollagen chains (Brahmachari and Ananthanarayanan, 1979). The present thesis involves the further elucidation of conformational aspects of proline hydroxylation in vitro, using chicken prolylhydroxylase and Pro-containing synthetic peptides. -- Pure prolylhydroxylase was obtained from 13-day old chicken embryos using established procedures. Pro-containing linear oligopeptides were characterized in different solvents, using circular dichroism (CD) and infrared (IR) spectroscopy. These studies have indicated the existence of two conformation, namely an extended conformation similar to that of poly (Pro) (PP-II) and a folded β-turn, in these peptides. The interaction between the enzyme and the oligopeptides of known conformation was studied by the following reactions: (1) hydroxylation of the peptides themselves and (2) the capability of these peptides to compete with the standard substrate, for the active site of prolylhydroxylase.  It was found that peptides with either β-turn or extended conformation alone can act only as inhibitors. On the other hand, peptides with both these conformations can also serve as substrates for the enzyme, in addition to being competitive inhibitors. -- Based on these observations, a model is proposed for the conformational criteria of enzymatic proline hydroxylation. According to this model, the enzyme requires the presence of PP-II like extended conformation followed by folded β-turns in the substrate molecules. The PP-II structure is necessary at the binding site of the enzyme, while the β-turn structure is necessary at the catalytic site. Peptides with either one of these structures can act only as inhibitors since they can fulfill only part of the conformational requirement. These studies are of importance since they help to define the observed substrate specificity of prolylhydroxylase, in precise conformational terms. -- The structure-function relationship of the prolylhydroxylase itself and it interaction with substrates and cosubstrates, in conformational terms, are also studied by CD and fluorescence spectroscopy. The implications of these studies in understanding the substrate specificity of prolylhydroxylase are discussed.


identifier: Shamsuzzaman, Kazi M. <https://research.library.mun.ca/view/creator_az/Shamsuzzaman=3AKazi_M=2E=3A=3A.html>   (1983) Isolation, properties and the use of a chymosin-like enzyme from harp seal (Pagophilus groenlandicus).  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: Four zymogens of acidic proteases A, B, C, and C’ were isolated from the gastric mucosa of harp seal (Pagophilus groenlandicus) by ion exchange chromatography on a DEAE Sephadex A-50 column. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phynylalanine-triethylene-tetramine and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (E.C.3.4.23.4) and porcine pepsin (E.C.3.4.23.1). -- Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and high performance liquid  chromatography but was heterogenous as judged by polyacrylamide gel electrophoresis in the absence of SDS. Zymogens A and C had molecular weights of 26,300 ± 1540 and 37,100 ± 1075 daltons respectively as estimated by gel filtration but 33,800 + 1800 and 44,000 + 2100 daltons respectively as estimated by SDS polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. The energies of activation of protease A and calf chymosin were 15.6 and 13.1 Kcal/more calculated from the milk clotting activity of the enzymes. -- Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk clotting to proteolytic activity than those of seal protease C and porcine pepsin, clotted milk up to pH 7.0, and had a pH optimum of 2.2-3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on APDT and lost activity in 6M urea. These results indicate that protease A is chymosin-like rather than pepsin-like. -- Experimental Cheddar cheese made with protease A, calf rennet, crude seal gastric protease (SGP) and Fromase (Musor miehei protease) had comparable yields and proximate compositions. Sensory analysis of experimental cheeses after 30 weeks of aging showed that the product made with crude SGP had higher sensory scores than those made with the other coagulants. Chemical analyses showed more extensive degradation of protein in cheeses made with calf rennet and Fromase than in the cheeses made with the seal proteases. The electrophoretic patterns of the proteins from cheeses made with seal proteases were qualitatively similar to those of calf rennet cheese. It was concluded that protease A and seal gastric proteases are promising as rennet subtitutes.


identifier: Qian, Dalong <https://research.library.mun.ca/view/creator_az/Qian=3ADalong=3A=3A.html>   (1993) Effects of bacterial endotoxin on liver mass and hepatocyte volume.  Masters thesis, Memorial University of Newfoundland.

abstract: Hepatomegaly is common in infections. An increase in liver mass, which cannot be accounted for by the net increase of hepatic protein and lipid contents, has been reported to be a common tumour necrosis factor a (TNFα)- mediated host response after such stress stimuli as endotoxemia, sepsis, trauma and chronic inflammation in either fasted or fed rats. The increase of hepatocyte volume has also been associated with, in experiments in vitro, alterations in hepatic metabolic functions such as protein and carbohydrate  metabolism. There are, however, no previous studies carried out on alterations in liver mass and hepatocyte volume in vivo as a host response of endotoxic animals which undergo profound metabolic changes in liver. -- In this study, a single intraperitoneal injection of E. coli lipopolysaccharide (LPS, 0127:B8) at a sublethal dose (3 mg/kg, LD₅) was found to introduce a generally time- and dose-dependent increase in hepatic mass and water content of  fasted rats compared to corresponding saline-injected controls. Twenty-four hr after LPS treatment, the absolute and relative weight of livers removed by complete hepatectomy remain unaltered in fasted endotoxic rats but they decrease by 20% in the fasted saline-controls, compared   to that in the normal rats fed ad libitum. The difference in  the mean liver weight between the endotoxin- and saline-treated animals is 2.0-2.5 g, i.e. 20-25% of the total liver  mass of 300-350 g rats. The hepatic wet/dry weight ratio is significantly higher in endotoxic livers than that in the other two groups. The hepatic water content increases by almost the same extent (27%) as the increase (26%) in the total liver mass 24 hr after treatment, indicating that the effect of E. coli LPS is mainly on the hepatic water content, rather than on the dry mass components. -- Analyses  of hepatic dry mass components and hepatic cellular spaces show that only one third (0.79 g) of the increase (2.18 g) in the total liver mass can be accounted for by the net increase of the dry mass components (0.38 g) plus the blood/plasma and/or interstitial fluid (0.41 ml) in the extrahepatocellular space; and hepatic K⁺ content, the predominant intracellular cation, increases by 25%, 24 hr after LPS injection. These suggest that an expansion of the hepatocellular space is mainly responsible for the increase in the liver mass of endotoxic rats. This hypothesis is also supported by morphometric data from an image analysis system  demonstrating that the mean transsectional area and the mean  volume is 8% and 12% larger, respectively, in the mononucleate hepatocytes from the endotoxic livers. -- The phenomenon of increases in the hepatic mass, water content and wet/dry weight ratio after LPS treatment can be reproduced by a single intravenous administration of rhTNF-α (25 μg/250 g B.W.) and prevented by the pretreatment of endotoxic rats with an intraperitoneal injection of goat anti-mTNFα serum (20 mg/300 g B.W.). The mean transsectional  area and mean volume is 11% and 17% larger, respectively, in  mononucleate hepatocytes from the livers of endotoxic rats treated with goat non-immune IgG compared to the anti-mTNFα serum-treated rats. These data suggest that effects of E. coli LPS on the increase in hepatic mass and hepatocyte volume are mediated by cytokine TNF-α. -- Twenty-four hr after E. coli LPS injection, the concentrations of hepatic adenine nucleotides in the endotoxic rats are not significantly different from either the saline-controls or the normal fed rats. There are no significant changes in the  activity of SGPT or in the plasma ratio of lactate/pyruvate and β-hydroxybutyrate/acetoacetate during the 24 hr after LPS injection. Those observations argue against "compensatory hyperplasia" resultinq from acute liver damage  due to direct endotoxin toxicity or hepatocyte swelling due to tissue hypoxia and impaired energy status in the liver. Metabolic changes mediated by insulin, TNF-α and/or other "hepatotrophic factors" may be responsible for the increase of liver mass after E. coli LPS administration.


identifier: Sarkar, Mohan Lal <https://research.library.mun.ca/view/creator_az/Sarkar=3AMohan_Lal=3A=3A.html>   (1987) Effects of inflammation and dexamethasone on various aspects of glycoprotein biosynthesis.  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: The concentration of acute-phase proteins in plasma is significantly increased in response to inflammatory agents. Almost all acute-phase proteins are glycoproteins in which the carbohydrate moiety is attached to the peptide through an asparagine nitrogen and are synthesized via dolichol-linked intermediates. Dexamethasone, a potent synthetic glucocorticoid, has been shown to influence the biosynthesis  of these acute-phase glycoproteins in experiments with animals and in vitro. -- This thesis concerns various aspects of the glycosylation of acute-phase proteins during inflammation and dexamethasone treatment in the rat. -- Turpentine-induced inflammation caused increased sialyl and galactosyltransferase activities in the liver, while in serum, only the sialyltransferase activity was increased. -- The formation of several dolichol-linked intermediates such as dolichol phosphate mannose, dolichol pyrophosphate N-acetyl chitobiose and dolichol pyrophosphate oligosaccharides was increased in cultured hepatocytes, or their homogenates, isolated from inflamed rats. Dexamethasone treatment of hepatocytes from control and inflamed animals also caused an increased formation of these  intermediates. The increase in the formation of dolichol-linked intermediates in inflammation was attributed to increased endogenous dolichol phosphate levels. In contrast,  the increased levels of the intermediates in dexamethasone treatment were not due to the endogenous dolichol phosphate but were most likely due to the induction of glycosyltransferases involved in glycoprotein biosynthesis. These conclusions were based on the results obtained from the following experiments: 1) estimation of the endogenous dolichol phosphate, 2) formation of dolichol phosphate mannose in presence of increasing amounts of exogenous dolichol phosphate, and 3) formation of dolichol and dolichol phosphate from mevalonate. -- Experiments in hepatocytes with actinomycin D and cycloheximide suggested that the increase in dolichol-linked intermediates was dependent on the increased synthesis of glycosylatable polypeptides of the acute-phase proteins. -- Nucleotide sugar pyrophosphatase activities were increased only in dexamethasone treated hepatocytes, whereas, the nucleotide sugar levels remained unaltered during both inflammation and  dexamethasone treatment. The present study showing increased  synthesis of dolichol-linked intermediates during the biosynthesis of acute-phase proteins in response to inflammation and dexamethasone treatment has provided new information on the role of the dolichol pathway in glycoprotein synthesis.


identifier: Mishra, Ram Kishore <https://research.library.mun.ca/view/creator_az/Mishra=3ARam_Kishore=3A=3A.html>   (1973) Effects of adrenocortical hormones on RNA metabolism in rat kidney and liver.  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: The turnover of RNA in various subcellular fractions of kidney and liver of normal, adrenalectomized and adrenalectomized plus adrenocortical hormone treated rats was investigated. The turnover rates were measured by the injection of a single dose of ¹⁴C-orotic acid and then following the loss of radioactivity from purified nuclei, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, free polysomes, total ribosomes and sRNA. Single exponential decay patterns were observed in all  the subcellular fractions of both tissues. The half-lives (t½) of the RNA for above fractions of normal kidneys were 7.4, 5.8, 5.1, 5.0, 5.4, 4.1, and 4.6 days, respectively, The t½ values for adrenalectomized rat kidneys were 9.2, 7.3, 6.1, 6,0, 5.8, 6.0, and 4.9 days, respectively. The half-lives (t½) of the RNA of normal rat liver nuclei, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, polysomes, total ribosomes and sRNA were 8.7, 6.5, 5.4, 6.4, 4.4, 4.9, and 4.8 days, respectively, The corresponding values for adrenalectomized rat livers were 12.0, 8.5, 6.9, 6.5, 5.8, 6.1, and 5.9 days,  respectively. -- The slower turnover rate of RNA in adrenalectomized rat kidney and liver suggests a slower rate  of RNA synthesis (steady state approximation) . Daily administration of aldosterone or deoxycorticosterone to adrenalectomized rats restored the turnover rates to normal in kidney and daily administration of corticosterone or hydrocortisone reversed the effects of adrenalectomy in liver indicating a tissue specificity of mineralocorticoids and glucocorticoids. -- Aggregate DNA-dependent RNA polymerase activity in nuclei of both tissues was decreased by adrenalectomy and restored by administration of aldosterone or deoxycorticosterone in kidney and by corticosterone or hydrocortisone in liver. To further investigate the mechanism of RNA polymerase stimulation by aldosterone in kidney, the polymerases were purified from various groups of rats and activity was determined using different sources of DNA. It was found that the DNA from aldosterone treated rats was transcribed more efficiently than from other sources. Fractionation of (¹⁴C-aldosterone injected) kidney chromatin revealed the presence of radioactivity in the non histone acidic protein and DNA fractions suggesting the possible binding of aldosterone, or  aldosterone-receptor complex or metabolite(s) of aldosterone  to these chromatin fractions which may result in enhanced template activity of DNA.


identifier: Omar, Rabeea Fahmy <https://research.library.mun.ca/view/creator_az/Omar=3ARabeea_Fahmy=3A=3A.html>   (1990) Mechanism of ochratoxin a-stimulated lipid peroxidation.  Masters thesis, Memorial University of Newfoundland.

abstract: Ochratoxin A (OTA) is a mycotoxin and a nephrotoxic carcinogen. The mechanism by which it stimulates lipid peroxidation was investigated in a reconstituted system consisting of phospholipid vesicles (liposomes), flavoprotein NADPH-cytochrome P-450 reductase, EDTA, Fe³⁺ ions, and NADPH. Lipid peroxidation, measured either as malondialdehyde formed or by oxygen uptake, was greatly stimulated in the presence of OTA, Omission of EDTA lowered the extent of lipid peroxidation but did not eliminate it. Fluorometric and spectrophotometric studies demonstrated the  formation of a 1:1 Fe³⁺-OTA complex. The rate of reduction of Fe³⁺ to Fe²⁺ was greatly enhanced in the presence of OTA and there was a further increase in the rate when EDTA was also included. Cytochrome P-450 (an enzyme normally present in microsomes) was found to effectively replace EDTA in the reconstituted system and its role in microsomal lipid peroxidation was also implicated suggesting that this hemoprotein could play an important role in OTA-stimulated lipid peroxidation in vivo. -- ESR studies showed that the Fe³⁺-OTA complex produced hydroxyl radicals in the presence of NADPH and NADPH-cytochrome P-450 reductase. The lack of any diminution of lipid peroxidation by catalase and several  hydroxyl radical scavengers suggests that hydroxyl radical production by the Fe³⁺-OTA complex may not be a significant factor in the lipid peroxidation in vitro, but these results  do not preclude hydroxyl radicals produced by the Fe³⁺-OTA complex from playing an important role in the toxicity of OTA. -- Structure-activity relationship studies indicated that the presence of a free carboxyl group and chlorine atom  as well as the L-Phe moiety on OTA contributed significantly  to the stimulatory effect on lipid peroxidation. Earlier studies had shown an absolute requirement for a free phenolic hydroxyl group on OTA. -- My results indicate that OTA stimulates lipid peroxidation by complexing with Fe³⁺ and facilitating its reduction. Subsequent to oxygen binding, an iron-oxygen complex of undetermined nature initiates lipid peroxidation. The extent of OTA-dependent lipid peroxidation in vivo and its role in the toxicity of OTA remain to be determined.


identifier: Shamsuddin, Zuzzer Ali <https://research.library.mun.ca/view/creator_az/Shamsuddin=3AZuzzer_Ali=3A=3A.html>   (1985) Metabolism and possible toxicity of 2, 6-dimethylnaphthalene, a component of crude oil.  Masters thesis, Memorial University of Newfoundland.

abstract: In process


identifier: Tinker, Donald Andrew <https://research.library.mun.ca/view/creator_az/Tinker=3ADonald_Andrew=3A=3A.html>   (1984) Metabolism in the domestic fowl (Gallus domesticus) : hepatic and renal concentrations of metabolites, and measurements of metabolite concentrations in abdominal blood vessels supplying and draining leg, liver and kidney.  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: Physiological levels of blood gases can be maintained in an anesthetized, abdominally opened fowl by means of a simple unidirectional ventilation technique.  Hepatic ATP levels and ATP/ADP ratios in control birds measured using this ventilation technique have been found to be high (1.9 micromole/g and 1.4 respectively) and are comparable to those reported in rats.  Hepatic lactate levels and the lactate:pyruvate ratio were lower than is reported for non-ventilated chickens.  Elevations of the tissue concentration of AMP, fructose-1,6-bisphosphate and inorganic phosphate and fall in ATP, in ischemic liver are consistent with an activation of glycogen phosphorylase, phosphofructokinase and pyruvate kinase - all indicative of the increased glycolytic flux in ischemic chicken liver. -- A six-day fast in chickens results in a 55% drop in hepatic glucose, an increase in the measures of cellular energy level in the liver, and a shift to a more oxidized hepatic cytosolic NAD⁺/NADH ratio.  In contrast, glucose levels, cellular energy level indices and the redox state of the kidney cytosolic NADH system remain unchanged after a six-day fast.  It is concluded that the relative availability of cytosolic reducing equivalents results in the process of gluconeogenesis from amino acids and pyruvate being more favored in the kidney of a fasted chicken than in its liver. -- Measurements of afferent-efferent differences across liver in the fed and fasted states suggest a significant uptake of glucose and lactate and most amino acids.  A negative afferent-efferent difference for urate across liver in the fasted, and not in the fed state, indicates that deamination of amino acids is more prevalent in the fasted liver. -- Measurements of arteriovenous differences across muscle indicate that this tissue releases amino acids into the circulation in the fasted state and takes up no glucose.  In the fed state no amino acids are released by muscle, although glucose is taken up. -- In the fed state there is no significant afferent-efferent difference across kidney for glucose.  Measurements of afferent-efferent differences across kidney suggest that in the fasted state there is a significant release of glucose and an uptake of amino acids, urate, and lactate.  If the renal portal component of total renal blood flow is assumed to be greater than 30% then when the sum is calculated of the gluconeogenic amino acids for which there is a significant uptake across kidney, the value (in glucose equivalents), is equal to the figure for the release of glucose.  It is concluded that the kidney is a major gluconeogenic organ in the fasted chicken.


identifier: Grewal, Kuldip <https://research.library.mun.ca/view/creator_az/Grewal=3AKuldip=3A=3A.html>   (1987) A study of doxorubicin toxicity.  Masters thesis, Memorial University of Newfoundland.

title: A study of doxorubicin toxicity

abstract: We examined myocardial injury following Doxorubicin use via analysis with the Automated Hitachi Analyzer of serum activity changes of enzymes specific for myocardial tissue. Sprague-Dawley rats were administered either saline, 3, 6, or 9 mg/kg doses of Doxorubicin by intraperitoneal injection. Blood samples were collected at times 0 and 48 hours posttreatment. There was an increase in serum creatine  kinase (except the 9 mg/kg injected rats), lactate dehydrogenase (LD), including elevations of LD 1 and LD 2 isoenzymes and aspartate aminotransferase (AST)activities at  48 hours post Doxorubicin injection. Increasing drug dosage resulted in increasing myocardial tissue concentration of the drug as measured by high performance liquid chromatography. This suggests increasing Doxorubicin dosage leads to increased myocardial tissue concentration of drug and is associated with tissue damage and release of cardiac-specific enzymes. -- Serum CK isoenzyme separation by electrophoresis revealed the presence of an additional band cathodic to CK-MM following administration of 6 and 9 mg/kg doses of the drug. Creatine kinase isoenzyme analysis of mitochondria also demonstrated this band in a position corresponding to that observed in the drug-treated animals. We suggest that at these doses mitochondrial damage resulted  in the release of mitochondrial CK. -- At a drug dose of 9 mg/kg, increased serum alanine aminotransferase (ALT) and LD  4 and LD 5 isoenzymes was observed suggesting hepatotoxicity. -- In rats, 24 hour urine specimens were analyzed for various biochemical parameters with the use of the Automated Hitachi Analyzer, the Astra-8 Automated Analyzer and the Chemstrip 9 Dipstick screen test, to detect  evidence of renal damage. The only abnormality noted was a decreased urine volume with increased dose of the drug. -- In the New Zealand white rabbit model, we demonstrated a serum CK-MB activity greater than five percent of total CK activity in all rabbits 48 hours subsequent to treatment with 10 mg/kg Doxorubicin. There was no such effect in the saline-treated controls. Myoglobin was detected in the serum  48 hours following a 10 mg/kg dose using the Rapitex Myoglobin kit. The origin of CK-MB was believed to be cardiac muscle; however the source of myoglobin could have been either skeletal muscle or myocardium.


identifier: Subrahmanyam, Vangala Venkata Satya <https://research.library.mun.ca/view/creator_az/Subrahmanyam=3AVangala_Venkata_Satya=3A=3A.html>   (1986) Hydrogen peroxide/peroxidase-mediated co-oxidation (a potential pathway for the activation of carcinogenic and toxic xenobiotics in the cell).  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: Products of the metabolism of arylamine carcinogens ¹⁴C-benzidine, N-¹⁴C-methylaminoazobenzene and ¹⁴C-aminofluorene and ¹⁴C-phenol bind irreversibly to nuclear DNA in intact polymorphonuclear leukocytes activated by the tumor promoter  phorbol myristate acetate. Little binding occurred under anaerobic conditions indicating that the binding was dependent upon oxygen. Sulfhydryl inhibitors p-chloromercuribenzoate and N-ethylmaleimide inhibited oxygen uptake as well as decreased the binding of arylamine carcinogens and phenol to DNA in leukocytes activated by tumor promoter. Phenolic antioxidants butylated hydroxyanisole or nordihydroguaiaretic acid also had a similar inhibitory effect. Azide increased both the oxygen uptake and binding, presumably as a result of intracellular catalase inhibition. However, higher concentrations of azide  and cyanide inhibited binding without affecting the oxygen uptake indicating that the binding is catalysed by myeloperoxidase. Granules isolated from the activated leukocytes catalysed a cyanide-sensitive binding of benzidine to calf-thymus DNA in the presence of hydrogen peroxide. -- Products of the metabolism of ¹⁴C-phenol also bind irreversibly to calf-thymus DNA in the presence of horseradish peroxidase and hydrogen peroxide. Binding also occurred to the homopolyribonucleotides polyriboadenylic acid, polyriboguanylic acid, polyribocytidylic acid and polyribouridylic acid, suggesting that the binding is relatively non-specific with respect to nucleotide bases. -- DNA, binding of ¹⁴C-phenol oxidized with horseradish peroxidase/hydrogen peroxide was prevented by glutathione and N-acetylcysteine and the mechanism was shown to involve reduction of the activated phenol intermediates and the formation of conjugates with glutathione and N-acetylcysteine. Ascorbate prevented binding by reduction of the activated phenol intermediates. -- Phenol oxidation by horseradish peroxidase/hydrogen peroxide initially results in p,p'-biphenol and o,o'-biphenol formation and subsequently results in polymer formation. o,o'-Biphenol is the major product formed but is rapidly oxidized to polymer,  particularly in the presence of phenol. Phenol oxidation with bone marrow homogenate and hydrogen peroxide also results in 0,0'-biphenol as the major product. p,p'-Biphenol is very rapidly oxidized to p,p'-biphenoquinone which can also be involved in polymer formation. -- Enzymic or acid-catalyzed hydrolysis of DNA releases the bound products. o,o'-Biphenol, but not p,p'-biphenol binds to DNA following peroxidase-catalysed oxidation. Enzymic hydrolysis of the DNA, to which the products of the oxidation of o,o'-biphenol  had bound, resulted in the release of products derived from the biphenol. -- Peroxidase also catalysed the formation of active oxygen species in the presence of NADH or GSH and traces of hydrogen peroxide and arylamine or phenolic substrates. Some oxygen activation occurred with some arylamines even in the absence of NADH or GSH. Oxygen consumption was proportional to the NADH oxidized or GSSG formed. Approximately 0.80 and 0.40 moles of oxygen were consumed per mol of NADH or GSH oxidized respectively. The requirement for trace amounts of hydrogen peroxide and arylamine or phenolic substrate suggests that the redox cycling resulted in hydrogen peroxide formation. It is proposed that initially formed phenoxv radicals or arylamine cation radicals oxidize NADH or GSH to radicals which react with oxygen to form superoxide radicals and hydrogen peroxide. -- Non-carcinogenic arylamines mesidine, aniline and 1-naphthylamine were poor at initiating redox cycling in  the presence of NADH or GSH, whereas carcinogenic 2-naphthylamine, 4-aminobiphenyl, methylaminoazobenzene, N,N'-dimethyl p-toluidine or 2-aminofluorene were highly effective in initiating redox cycling in this system with resultant hydrogen peroxide formation. -- Only phenol and o,o'-biphenol were effective among phenolic substrates in redox cycling with resultant hydrogen peroxide formation. p,p'-Biphenol, hydroquinone and catechol were ineffective in  activating oxygen. p,p'-Biphenol formed a glutathione conjugate when oxidized with horseradish peroxidase and hydrogen peroxide in the presence of glutathione. The major conjugate formed was identified as 3-S-(glutathion-yl)-p,p'-biphenol using FAB-Mass spectroscopy and NMR spectroscopy.


identifier: Mabrouk, Gehan M. Y. <https://research.library.mun.ca/view/creator_az/Mabrouk=3AGehan_M=2E_Y=2E=3A=3A.html>   (1996) Hormonal regulation of glycine cleavage enzyme system.  Masters thesis, Memorial University of Newfoundland.

abstract: The glycine cleavage enzyme complex is a mitochondrial enzyme that is known to be activated by hormones such as glucagon. The effects of several glucagon-related peptides such as glucagon-like peptide-1 (7-36) amide, oxyntomodulin and glicentin, as well as miniglucagon, on the glycine cleavage system were examined in isolated rat hepatocytes. Oxyntomodulin and glicentin were found to stimulate the glycine cleavage system flux through their interaction with the glucagon receptor. Glucagon-like peptide-1 and miniglucagon had no effect on the glycine cleavage system. -- Although the stimulation of the glycine cleavage system by  glucagon has been demonstrated previously, it is not known how the hormonal signal is transmitted to the mitochondria. This question is examined in this thesis. The cell-permeable  protein phosphatase inhibitor, okadaic acid, was found to stimulate the flux through the glycine cleavage system. The protein kinase A agonist, Sp-cAMPS was also found to stimulate the glycine cleavage system flux, an effect that was inhibited by the protein kinase A antagonist, Rp-8-Br-cAMPS. These results suggest that protein kinase A dependent  phosphorylation of cytosolic protein(s) can affect the glycine cleavage system. The role of intracellular calcium in the regulation of the glycine cleavage system was examined using the calcium-mobilizing hormones, vasopressin and angiotensin II, both of which stimulate the glycine cleavage system. Finally, the role of protein kinase C in the regulation of glycine cleavage system was examined using the phorbol ester, Phorbol 12-myristate 13-acetate (PMA). PMA had no effect on the glycine cleavage system or on the glucagon-stimulated glycine cleavage system flux, which suggests no role for protein kinase C in the activation of the glycine cleavage system.


identifier: Squires, Stephen Arthur <https://research.library.mun.ca/view/creator_az/Squires=3AStephen_Arthur=3A=3A.html>   (1994) Hormonal regulation of hepatic glutaminase.  Masters thesis, Memorial University of Newfoundland.

abstract: Liver glutaminase, a mitochondrial enzyme, is known to be activated by hormones such as glucagon. Diabetes mellitus is  a metabolic disease which is characterized by alterations in  the insulin:glucagon ratio and, therefore, represents an in vivo situation in which to study the effects of hyperglucagonemia on liver glutaminase. Glutaminase flux was found to be increased in isolated hepatocytes and in isolated mitochondria from streptozotocin-induced diabetic rats. -- An important question is how the hormonal signal is  transmitted into the mitochondria. One possibility examined is whether cAMP-mediated phosphorylation of cytosolic proteins can stimulate a mitochondrial enzyme. This was examined using the cell-permeable protein phosphatase inhibitors okadaic acid and calyculin A. In the presence of 1 mM NH₄C1, an obligatory positive effector for liver glutaminase, both inhibitors increased glutaminase flux in isolated hepatocytes. This effect was stable, existing in mitochondria isolated from okadaic acid-treated hepatocytes.  The protein kinase A agonist, Sp-cAMPS, was also found to stimulate glutaminase flux in isolated hepatocytes. This stimulation was inhibited by the protein kinase A antagonist, Rp-cAMPS. These results suggest that the cAMP-dependent phosphorylation of cytosolic protein(s) can affect  mitochondrial glutamine metabolism. This may also give insight into the mechanisms whereby hormones, such as glucagon, stimulate mitochondrial glutamine metabolism.


identifier: Porter, Edward L. <https://research.library.mun.ca/view/creator_az/Porter=3AEdward_L=2E=3A=3A.html>   (1988) Influence of reproductive status, sex, and petroleum hydrocarbon exposure on hepatic and extrahepatic biotransformation enzymes of the cunner, Tautogolabrus adspersus.  Masters thesis, Memorial University of Newfoundland.

abstract: Laboratory and field trials have validated liver mixed function oxygenase (MFO) induction as a sensitive biomonitor  for petroleum hydrocarbon exposure. However, basal enzyme levels are known to vary seasonally and the usage of extra-hepatic tissues for biological monitoring has received little attention. Laboratory experiments were conducted in the summer of 1985 to determine the induction potential of ethoxyresorufin O-deethylase (EROD) in liver, kidney, gill, and heart tissues of cunners (Tautogolabrus adspersus) exposed to no. 2 fuel oil (diesel oil) during the reproductive season. Although basal levels varied, induction was readily resolved in hepatic and extrahepatic tissues of both male and female cunners during prespawning, early spawning and late spawning. The induction potential of heart (7-18 fold) was much greater than liver (4-6 fold), kidney (4-7 fold) and gill (2-5 fold). Male cunners displayed a much higher induction potential compared to females in heart  EROD during prespawning (18 versus 13 fold) and early spawning (12 versus 7 fold) but no significant sex differences were observed during late spawning. Sex differences were also evident in liver and kidney tissues throughout the reproductive season, males exhibiting higher enzyme activities than females in each case. -- The potential for induction of a conjugating enzyme, glutathione  S-transferase (GST), was also studied. Unlike EROD, GST was refractory to induction in all tissues of male and female cunners throughout gonad maturation/spawning. Depression of GST was observed in the heart tissue of diesel exposed males during early spawning and male liver and kidney during late spawning. -- These experiments show the feasibility of using extrahepatic as well as hepatic MFO enzymes for monitoring studies even during the reproductive season.


identifier: El-Karawy, Nagwa M. <https://research.library.mun.ca/view/creator_az/El-Karawy=3ANagwa_M=2E=3A=3A.html>   (1996) In vitro assessment of acute and chronic toxicity of lovastatin to cultured HepG2 hepatoma cells.  Masters thesis, Memorial University of Newfoundland.

abstract: The investigations reported here highlight the cytotoxic effect of 3-Hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) Reductase inhibition by lovastatin on the cultured human hepatoma cell line HepG2. This investigation focused on the toxic effects of lovastatin at exposure levels at and above those currently used clinically. The laboratory approaches used included electron microscopy, cell cycle analysis by flow cytometry, investigation of peripheral cell damage by enzyme leakage and other protein studies, and analysis of intracellular and extracellular lipids for various exposure concentrations and times. The experiments were also extended  to consider the effects of adding oleic acid to the cell cultures as a nutritional supplement to enhance lipogenesis.  The findings are discussed in light of current understanding  on the significance of protein prenylation on the cell cycle  and the mechanisms of cell death by necrosis and apoptosis. The lower lovastatin concentrations used in this study (0.1 - 2.5 μM) did not kill cells, even after exposure for 8 days. However, if cell cultures containing otherwise nontoxic lovastatin concentrations were supplemented with oleic acid/BSA, cell viability was significantly reduced at all lovastatin concentrations (α=0.05). This work shows that  lovastatin supplemented by fatty acid can significantly affect HepG2 cell morphology and functions. The results of this work suggest also that HepG2 cells could be used as an efficient and practical model system to investigate the process of cell death in vivo. Also, it is a convenient model for investigations on mevalonate-dependent cellular mechanisms. This study provides evidence to support the potential role of lovastatin treatment in cancer management.


identifier: Shehnaz, Darakhshanda <https://research.library.mun.ca/view/creator_az/Shehnaz=3ADarakhshanda=3A=3A.html>   (1984) Activation of N,N-Dimethylaminoazobenzene catalyzed by peroxidase.  Masters thesis, Memorial University of Newfoundland.

abstract: The mechanism for the metabolic activatiou of N,N-dimethylaminoazobenzene is unknown. Although cytochrome P-450 dependent mixed function oxygenases are important in the  activation of arylamines, there are target tissues for arylamines which do not contain these oxygenases. These tissues do contain peroxidases. Therefore a one electron oxidation mechanism was investigated by studying a Horseradish Peroxidase/hydrogen peroxide catalyzed metabolism of aminoazobenzenes [i.e; N-methyl-4-aminoazobenzene (MAB), N,N-dimethyl-4-aminoazobenzene (DAB),  aminoazobenzene (AB)], and by following the binding of the activated products to exogenous DNA. In order to further explore the mechanism, the effect of biological and phenolic  antioxidants on metabolism and binding of the activated products of [¹⁴C] MAB was studied. The oxidation products were analyzed by spectrophotometry, high pressure liquid chromatography and thin layer chromatography. The major product obtained from both MAB and DAB at pH 7.4 was a dimer  of MAB, i.e; N-methyl,N(MAB)-4-aminoazobenzene. The product obtained at pH 5.0 was 4'-(MAB)-N-methyl-4-aminoazobenzene. It is suggested that DAB is first N-demethylaced, and then follows the same metabolic pathway as does MAB. Evidence suggests that AB is also oxidized to a dimer with N-N linkage at pH 7.4 and C-N linkage at pH 5.0. -- The binding of the activated products to calf thymus DNA was observed spectrophotometrically. [¹⁴C] MAB was used to determine the quantitative binding by liquid scintillation counting. DNA is attacked by a free radical of MAB, and also by other radicals formed in chain propagating reactions with the MAB radical. The maximum binding of [¹⁴C] MAB to homo polyriboguanylic acid among the four homo polyribonucleotides suggests the preferential binding of the  activated products occurs at the guanine residues in DNA. -- The phenolic and biological antioxidants inhibit the DNA binding either by inhibiting the total oxidation of MAB or by converting the reactive metabolites to detoxication products.


identifier: Parry, David Morris <https://research.library.mun.ca/view/creator_az/Parry=3ADavid_Morris=3A=3A.html>   (1980) Ammoniagenesis in the rat kidney during recovery from metabolic acidosis.  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: The regulation of renal glutamine metabolism in relation to acid-base status was investigated in rats. The model of NH₄Cl-induced acidosis was used and two new experimental situations introduced. Recovery from acidosis was investigated in rats allowed to drink H₂O in lieu of NH₄Cl and the process of recovery was accelerated by administration of NaHCO₃ (1.5 mmol/lOOg body wt). Animals allowed to recover from acidosis were again challenged with various acid loads and the responses of these rechallenged rats compared to the responses of naive-challenged rats. -- Urinary ammonia excretion, total renal ammonia production and glutamine extraction returned to normal by about 24h in animals which were allowed to recover from metabolic acidosis by drinking H₂O. In comparison, in animals administered NaHCO₃ at the start of recovery, these parameters were back to normal by 8h. Rats which were permitted three days to recover from metabolic acidosis were  able to excrete more acid as ammonium salt than animals challenged with NH₄Cl for the first time, when challenged with high acid loads. There was no difference in response to  lower acid loads. -- Decreases in the renal contents of glutamine, α-ketoglutarate and malate were found during metabolic acidosis and increases in giutamine, glutamate, α-ketoglutarate, malate, citrate, lactate, phosphoenolpyruvate and 3-phosphoglycerate occurred during recovery. No significant difference in the renal content of metabolites was observed between rats administered NaHCO₃ and rats administered the same amount of NaCl during recovery. -- The  renal activities of phosphoenolpyruvate carboxykinase (PEPCK), glutaminase (PDG) and glutamate dehydrogenase (GDH)  in these animals were also investigated. PEPCK activity increased identically in naive-challenged and rechallenged rats. During recovery PEPCK activity returned to normal by about 16h and no significant difference in activity could be  discerned between animals intubated with NaHCO₃ and animals  intubated with NaCl. Immunotitration of PEPCK revealed that the increased activity during acidosis and the decreased activity during recovery are due to changes in the content of this enzyme. The activities of PDG and GDH increased during acidosis and remained elevated long after renal glutamine metabolism returned to normal, in vivo. -- Ammonia  formation by isolated mitochondria and the fluxes through PDG and GDH increased during metabolic acidosis. However, normal rates of ammonia production in vivo were attained 12-24h after the induction of acidosis while no acceleration of  GDH flux in isolated mitochondria was evident at this time. In the most physiological medium used (1 mM glutamine, 3 mM glutamate, 4 mM phosphate) ammonia formation and GDH flux decreased coincidentally during recovery and preceeded the fall in PDG flux. The differences in renal ammoniagenesis between naive-challenged rats and rechallenged rats and between rats intubated with NaCl and NaHCO₃ during recovery  could not be accounted for by metabolic differences in isolated mitochondria. In the most unphysiological medium used (1 mM glutamine, 20 mM phosphate) the mitochondrial capacity to metabolize glutamine remained elevated for at least 15 days of recovery. -- These observations suggest that renal glutamine metabolism is not controlled simply by changes in the contents of PDG or PEPCK or by the mitochondrial events which are responsible for the changes in the fluxes through PDG or GDH in isolated mitochondria. The changes observed in αketoglutarate are consistent with its purported regulatory role.


identifier: Nag, Kaushik <https://research.library.mun.ca/view/creator_az/Nag=3AKaushik=3A=3A.html>   (1991) An epifluorescence microscopic surface balance to observe monolayers at the air-water interface.  Masters thesis, Memorial University of Newfoundland.

abstract: The details of the design and construction of an epifluorescence microscopic surface balance and some preliminary observations on lipid monomolecular layers performed using this balance are discussed. The balance consists of a Teflon trough with a computer - controlled movable barrier mounted on a vibration reducing platform. The balance is equipped with an epifluorescence microscopic attachment by which visual observation of fluorescent probes  in the monolayer is possible. Barrier movement can be utilized to give monolayer compression and expansion velocity from 20 mm²/sec up to 600 mm²/sec. A Wilhelmy dipping plate connected to a force transducer is used to measure surface tension in the monolayer during compression and expansion. The epifluorescence microscope is coupled to a charge couple device in tandem with a microchannel plate which permits observation of the low light level fluorescence from the monolayer. Images of the monolayer under compression were visualized, stored, digitized and processed using a video unit and operator interactive software. -- Using the balance preliminary studies of phase behaviour have been performed for monolayers of dipalmitoyl phosphatidylcholine (DPPC) and other lipids. The phase changes were observed by incorporating 1 mol% of a fluorescent probe, NBD-PC, into the lipid forming the monolayer. This probe had no appreciable effect on the isotherms and surface tension properties of DPPC in monolayers. DPPC was observed to undergo a number of phase transitions when compressed in monolayers. The liquid - expanded to liquid - condensed phase transition was visualized as the formation of dark patches (domains) from a  fluorescent background. The domains increased in average size from 100 μ² to 1500 μ² in a nonlinear manner as a function of area occupied per molecule of the DPPC, when monolayers were compressed at a rate of 20 mm²/sec. The size, shape and growth of these domains were quantitatively characterized and found to be dependent on compression rates. At a fast speed of compression (4 A²/molecule/sec) the domains were smaller in size, more irregularly shaped, and unimodally distributed. At a slow compression speed (0.13 A²/molecule/sec) the domains were larger in size and more regular in shape. The shapes of the domains were found to change dramatically when a small amount (2 mol %) of cholesterol was incorporated in the monolayer. No domain formation was observed in monolayers for lipids with one or two unsaturated chains.


identifier: Barrowman, Gwynedd <https://research.library.mun.ca/view/creator_az/Barrowman=3AGwynedd=3A=3A.html>   (1993) Gastrointestinal absorption of Ochratoxin A.  Masters thesis, Memorial University of Newfoundland.

abstract: The mycotoxin Ochratoxin A (OTA), is a widespread contaminant of cereals, beans and meat products. Its solubility in the aqueous phase depends on ionization of the  phenolic hydroxyl group whose pK, was found to be 7.05. Earlier researchers have demonstrated the importance of the portal venous route in OTA absorption. Absorption in the presence of lipids is of interest because lipids form a significant proportion of the diet in countries where OTA contamination occurs. When OTA was administered to conscious rats in corn oil rather than in sodium bicarbonate, the lymphatic route played a greater role in the transport of absorbed OTA. In the absence of bile, OTA transport in lymph  was not increased even in the presence of corn oil, suggesting that OTA handling by the lymphatic route depends on micellar solubilization in mixed bile salt/lipid micelles. When carried in lymph, OTA reaches the general circulation without being subjected to "first-pass" hepatic metabolism. -- Key words: ochratoxin A; nephrotoxin; lymphatic transport


identifier: Hall, Jennifer Lynn <https://research.library.mun.ca/view/creator_az/Hall=3AJennifer_Lynn=3A=3A.html>   (1987) Intestinal absorption: studies of absorption of certain natural lipids and lipophilic xenobiotics.  Masters thesis, Memorial University of Newfoundland.

abstract: The uptake of nutrient lipids by the absorptive cell of the small intestinal mucbsa is influenced by a number of factors. Some lipophilic xenobiotic compounds are also absorbed by the enterocyte in conjunction with the nutrient lipid. The interaction that occurs between these compounds and nutrient lipids is of toxicological and physiological interest. The chylomicron appearance time (the interval between introduction of ¹⁴C-oleic acid and the first appearance of ¹⁴C-labeled chylomicrons in thoracic duct lymph) is an effective tool in monitoring changes in the handling of the first lipid molecules being processed by the enterocyte. The chylomicron appearance time in control animals is highly reproducible and agrees well with previously published work. -- Polycyclic aromatic hydrocarbons such as 7,12-dimethylbenz[a|anthracene (DMBA) are known carcinogens and are readily absorbed from the gastrointestinal tract. Due to their lipophilic nature, a substantial proportion of the absorbed xenobiotic can be transported in lymph chylomicrons. Of the PAHs tested, only DMBA was found to exert any effect on the chylomicron appearance time resulting in an appearance time of 23.08 ± 2.10 minutes as compared to a control value of 10.81 ± 1.03 minutes (p < 0.001). These results indicate that the inclusion of DMBA in a nutrient lipid test meal significantly retards the normal process of uptake and/or processing of nutrient lipids within the enterocyte. -- Exposure to 10μmol DMBA several hours prior to test meal administration, still resulted a delay in chylomicron appearance time. Triglyceride flux analysis for three hours following test meal administration indicates a transient effect of DMBA since the total triglyceride outputs in both control and DMBA-treated rats were not significantly different. -- Subsequent studies used the chylomicron appearance time technique to determine the effects of other lipophilic compounds on lipid absorption Cholesterol had no effect on the chylomicron appearance time but the hydrophobic surfactant Pluronic L-81 delayed the the appearance of the radiolabel [16.60 ± 0.77 minutes, p < 0.01]. This result supports the report attributing hypolipidemic activity to this compound. -- The efficiency of the α-glycerophosphate pathway compared with the monoglyceride pathway for triglyceride resynthesis was examined using the chylomicron appearance time technique. Three groups of rats were used for this study. Group A received a lipid emulsion test meal containing 1-monoolein as its source of monoglyceride. Group B received a test meal  containing 2-monoolein and Group C received a test meal without any monoglyceride present. The chylomicron appearance times for these three groups did not differ significantly. This indicates that the α-glycerophosphate pathway is capable of handling the first few molecules of lipid as efficiently as the monoglyceride pathway despite the higher energy cost required to do so.


identifier: Squires, E. James <https://research.library.mun.ca/view/creator_az/Squires=3AE=2E_James=3A=3A.html>   (1984) Isolation and characterization of gastric proteases from the Greenland cod (Gadus ogac).  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: The Greenland cod Gadis ogac is a sub-arctic species that thrives year round in the North Atlantic.  It is postulated that the gastric proteases of this fish have several properties in common with the gastric proteases from mammalian species but also have several characteristics unique to fish species.  Therefore, the gastric proteases and their zymogens were isolated from the stomach mucosa of the Greenland cod Gadus ogac and their properties compared to mammalian gastric proteases and the gastric proteases of other fish.  Attempts were also made to purify porcine pepsin A and porcine gastricsin from a crude commercial pepsin preparation.  The properties of the purified gastricsin fraction obtained differed substantially from the literature data on porcine gastricsin so that direct comparisons of many of the properties of gastricsin and the cod proteases could not be made. -- The zymogens of three gastric proteases were separated and purified by Sephadex G100 chromatography at pH 7, chromatofocusing and, after activation of the zymogens, Sephadex G75 chromatography at pH 2.5.  The zymogens of protease 1, 2, and 3 had isoelectric points of > 7.5, 6.2 and 5.2 respectively.  The zymogens of the Greenland cod gastric proteases were activated much more rapidly at low temperature than porcine pepsinogen.  All three of the cod proteases had more alkaline pH optima with protein substrates than porcine pepsin, especially with methylated protein substrates.  The pH optima of cod protease 2 and 3 and porcine pepsin with peptide substrates were all near pH 2 while the pH optimum of cod protease 1 with APDT was near pH 3.  The specific activities of the individual cod proteases at 26 °C with protein substrates were generally lower than porcine pepsin.  However, a mixture of the cod proteases had activity with the protein substrates that was greater than the sum of the activities of the individual proteases.  Cod protease 2 and 3 were active on a number of peptide substrates that are good substrates for gastricsin while cod protease 1 was active only with APDT (N-acetyl-phenylalanine-diiodotyrosine) of all the peptide substrates investigated.  The milk clotting activities of the cod proteases were much greater than that of porcine pepsin and the cod proteases had CU/PU ratios (the ratio of the clotting activity to hydrolytic activity with hemoglobin) that were 20-50 times higher than porcine pepsin.  The individual cod proteases hydrolysed hemoglobin to a greater extent than porcine pepsin, indicating their wider substrate specificity.  Porcine pepsin had a high Vmax and low Km' compared to cod protease 1 which had low to moderate Vmax and Km' with all substrates.  Cod protease 2 had comparatively high Vmax and Km' with hemoglobin as substrate and moderate to low Vmax and Km’ with the other substrates.  Cod protease 3 had comparatively low to moderate Vmax and Km' with hemoglobin and casein, high Vmax and Km' with methylated hemoglobin and low Vmax and high Km' with the peptide substrates.  No significant differences were found in the activation energy for the hydrolysis of the various substrates by the different protease preparations. -- The cod proteases were less stable to heating and retained less activity at extremes of pH (less than 3 and greater than 6.5) than porcine pepsin.  The activities of cod protease 1 and 2 with hemoglobin as the substrate were doubled in the presence of 25 mM NaCI while cod protease 3 and porcine pepsin were not stimulated by salt.  The cod proteases did not cross react with antibodies raised against porcine pepsin.  Antibodies were also obtained against the purified proteases T16 and T25 from psychrotrophic pseudomonads.  Anti-T16 IgG precipitated cod protease 1 and porcine pepsin at similar IgG/enzyme ratios but did not affect cod proteases 2 and 3.  Anti-T25 IgG precipitated all the proteases, cod protease 2 and porcine pepsin being precipitated at similar IgG/enzyme ratios.  The subunit molecular weights of all the proteases were in the range of 35-37 kdal as estimated by SDS-PAGE and amino acid composition.  The amino acid compositions of the cod proteases differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin and chymosin differ from each other.  Cod protease 1 was the most different of the cod proteases from the mammalian proteases, while cod protease 3 was more like chymosin.  Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest.  The hydrophobicity indices of cod protease 2 and 3 were intermediate between that of porcine pepsin A and chymosin.


identifier: Simpson, Benjamin Kofi <https://research.library.mun.ca/view/creator_az/Simpson=3ABenjamin_Kofi=3A=3A.html>   (1984) Isolation, characterization, and some applications of trypsin from Greenland cod (Gadus ogac).  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: Trypsin E. C. 3. 4. 21: 4, was isolated from the pyloric ceca or the intestines of the Greenland cod (Gadus ogac) and purified by the successive steps of ammonium sulfate fractionation, acetone precipitation, and affinity chromatography using soybean trypsin inhibitor  coupled to CNBr-activatod Sepharose 4B.  Some of the physical and catalytic properties of the Greenland cod trypsin were compared with those of commercially available bovine pancreatic trypsin.  The Greenland cod trypsin was shown to be homogeneous by analytical polyacrylamide gel electrophoresis and also by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.  Although certain properties of Greenland cod trypsin were similar to those of bovine trypsin there were also some significant differences between the two trypsins. -- Greenland cod trypsin and bovine trypsin were alike with respect to various criteria.  The pH activity profile of Greenland cod trypsin was similar to that of bovine trypsin.  Likewise the amino acid composition of Greenland, cod trypsin revealed that it was rich in potential acidic amino acid residues as has been reported for trypsin from bovine and other sources.  The Greenland cod trypsin was similar to bovine trypsin in being able to hydrolyze ester and amide linkages involving the carboxyl group of arginine.  The two trypsins were both inhibited by phenyl methyl sulfonyl fluoride, trasylol and soybean trypsin inhibitor and also by the thiol reagents.  2-mercaptoethanol and dithioerythritol, and were both effective in preventing milk oxidation induced by copper.  The molecular weight of Greenland cod trypsin, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was similar to values reported for tripsin from bovine and other sources. -- The Greenland cod trypsin differed from bovine trypsin in the following respects : the Greenland cod trypsin was most stable at alkaline pH, unlike bovine tripsin which was stable at acid pH; the Greenland cod trypsin was heat abile while bovine trypsin was heat stable.  The temperature coefficients and activation energies for the hydrolysis of amide, ester and protein subtrates were considerably lower for Greenland cod trypsin than bovine trypsin.  The apparent Michaelis-Menten constants (Km') and molecular activities for the hydrolysis of substrates were considerably higher for the cod enzyme than the bovine enzyme.  Based on the amino acid compositions, the calculated average hydrophobicity of Greenland cod trypsin was considerably lower than that of bovine trypsin and the cod enzyme contained fewer cysteine residues than bovine trypsin.  Greenland cod trypsin activity was depressed to a greaterextent by thiol reagents than that of bovine trypsin.  Finally, the peptide maps of the two trypsins resulting from the cleavage by papain and cyanogen bromide were different.


identifier: Dillon, Ronda Marie <https://research.library.mun.ca/view/creator_az/Dillon=3ARonda_Marie=3A=3A.html>   (1992) Isolation, detection, and partial characterization of Listeria in smoked seafood.  Masters thesis, Memorial University of Newfoundland.

abstract: Over a one-year period, 258 samples of smoked fish products were obtained from retail outlets in Newfoundland and processed for Listeria using the listeria isolation protocol adopted by Health and Welfare Canada. Direct plating was also carried out. Listeria spp. were isolated from 43 of 258  (16.7%) samples, with hot smoked products yielding 25.4% (36/142) of the isolates, and cold smoked products yielding 6% (7/116). Cod had the highest rate of Listeria contamination at 46.7%. Of the 43 Listeria spp. isolated, 18 (41.9%) were L. innocua, 13 (30.2%) were L. welshimeri, and 12 (27.7%) were L. monocytogenes. Eleven isolates of L. monocytogenes were serotype 1/2, and one was 4b. There was an indication that Listeria spp., particularly L. monocytogenes, occurred more commonly during the cooler months. All Listeria isolates were detected after 48h primary enrichment. The secondary enrichment step failed to enhance the isolation rate. Direct plating yielded only 26% of the total isolates. PALCAM, a listeria selective agar, proved more efficient in recovering Listeria spp. from smoked fish products. -- Multilocus enzyme electrophoresis is an epidemiological tool applied in this study to Listeria  strains isolated from smoked seafoods to determine if the strains were from a common contamination source. The 57 strains analyzed resulted in 57 distinct electrophoretic types (ETs) indicating many sources of contamination. Major divisions among the four different Listeria spp. (L. innocua, L. monocytogenes, L. welshimeri, L. ivanovii) occurred at genetic distances of 0.72 to 0.85. All 14 enzymes were polymorphic and had a mean genetic diversity of  0.683. The L. monocytogenes strains were not of a single clone nor were they of the same ETs as the two human isolates. This indicates the great need for better sanitation practices in the food processing environment. -- Experiments were conducted to study the effects of the cold smoking process and storage on the organism in artificially inoculated cod fillets. In three trials, cod fillets were surface inoculated with L. monocytogenes, cold smoked, vacuum-packaged, stored at 4°C for 3 weeks or at -20°C for 3  months, and quantitatively processed for Listeria using the three-tube most probable number method (MPN). The initial inoculation concentrations were 10⁵ MPN/g in trial 1, 10² MPN/g in trial 2, and 10⁰ MPN/g in trial 3. The organism, relatively stable during the smoking process, increased with  storage at 4°C, but decreased in cell number at -20°C. Growth was very slow in trial 3 possibly because of the low inoculum level. Hence, L. monocytogenes contamination in smoked seafoods, not cooked prior to consumption, may pose a  health risk to the consumer.


identifier: McEachren, Todd Mervyn <https://research.library.mun.ca/view/creator_az/McEachren=3ATodd_Mervyn=3A=3A.html>   (1996) Interaction of C-reactive protein with pulmonary surfactant.  Masters thesis, Memorial University of Newfoundland.

abstract: The influence of the acute inflammatory phase protein human C-reactive protein (CRP) on the adsorption of porcine pulmonary surfactant from a subphase into the air-water interface with and without dynamic surface compression has been investigated. CRP was shown to detract from the ability of surfactant to rapidly adsorb to the air-water interface at a molar ratio of 0.03 : 1, protein to phospholipid (weight ratio, 0.5 : 1). On a weight basis, CRP was found to  be more effective than fibrinogen or globulin at reducing the adsorption rate of surfactant. The effect of CRP required the presence of calcium, and was reversed by the addition of phosphocholine, in a concentration dependent manner. The inhibition of surfactant adsorption by CRP was effectively eliminated by the addition of phosphocholine at a molar ratio of 300 : 1, phosphocholine : CRP, but it was not diminished by the addition of identical molar ratios of o-phosphoethanolamine or DL-α-glycerophosphate at the same molar ratios. These data suggest that the potent inhibition of surfactant adsorption by CRP is primarily a result of a specific interaction between CRP and the phosphocholine headgroup of surfactant lipids in the subphase and that it can be reversed by the water-soluble CRP ligand, phosphocholine. Experiments were performed to determine the effect of the addition of the complement protein C1q on the adsorption of porcine lipid extract pulmonary surfactant from the subphase to the air-water interface with and without dynamic compression. C1q, at a weight ratio of 10% (C1q : PL), when added to lipid extract surfactant, which is void of SP-A, increased the adsorption rate of the lipid extract surfactant to approach the rate at which whole surfactant, which contains 5-7% SP-A, by weight. C1q at this  weight ratio did not detract from the ability of lipid extract surfactant from attaining a minimum surface tension under dynamic compression. Albumin, when added to lipid extract surfactant at the identical weight ratio at which C1q was a added, detracted from the ability of lipid extract  surfactant to adsorb to the air-water interface. Albumin, also detracted from the ability of this mixture in attaining  a minimum surface tension under dynamic compression. This suggests that C1q, which shares quaternary structural homology with SP-A, increases the adsorption of lipid extract surfactant by a similar molecular interaction as that of SP-A. Serum CRP levels, expressed μg/ml and as percentage of total protein, were elevated in patients with ARDS and those patients at risk of ARDS compared to normals.  A weak correlation exists between the serum CRP in ICU patients, expressed as percentage of total protein and the APACHE II score. This suggests that CRP levels may be useful  in the clinical evaluation of patients in the ICU.


identifier: Drover, Victor A. B. <https://research.library.mun.ca/view/creator_az/Drover=3AVictor_A=2E_B=2E=3A=3A.html>   (1995) Sequence and evolution of apolipoprotein A-I in three salmonids.  Masters thesis, Memorial University of Newfoundland.

abstract: A cDNA library was constructed from brown trout liver tissue and one clone, BTLB27, containing the processed mRNA transcript of apolipoprotein A-I was isolated and sequenced.  This sequence was then compared to all other known apoA-I cDNA/genomic DNA sequences and the phylogeny of the represented species inferred.  The sequences isolated from the fish species grouped outside both avian and mammalian sequences while the avian sequence was an outgroup to the mammals.  The phylogenetic tree also revealed that rodents diverged from the mammals before lagomorphs, carnivores, artiodactyls, and primates.  Using the cDNA/genomic DNA comparisons, the insertion sites of apolipoprotein A-I intruns 11 and 111 were predicted and amplified using the polymerase chain reaction and subsequently sequenced.  We find that there are two loci for apolipoprotein A-I, one of which has undergone major evolutionary changes.  Also, phylogenetic inference using intron II sequences support the findings from the cDNA sequences.  Molecular clocks were constructed from the phylogenetic data and the accepted fossil record to estimate the time of various evolutionary events.  We find that the accepted time for the divergence of rat and mouse is much too recent.  Estimates of the divergence times of three salmonid species as well as a genome duplication event which preceded salmonid speciation are also greater than current accepted values, although the differences are not as great as that observed for the rodent lineage.


identifier: Pal, Sebely <https://research.library.mun.ca/view/creator_az/Pal=3ASebely=3A=3A.html>   (1991) The effect of polyunsaturated fatty acids on cholesterol metabolism.  Masters thesis, Memorial University of Newfoundland.

abstract: Atherosclerosis is characterized by deposition of cholesterol and esterified cholesterol in cells of the vascular intima.  These lipid laden cells are called foam cells due to their microscopic appearance and arise primarily from smooth muscle cells and monocyte macrophages.  It is believed that the transformation of these cells into foam cells is a key step in the initiation of atherosclerosis.  Therefore, to better understand the process of atherogenesis it is important to investigate the mechanisms that may regulate cholesterol esterification and hence the formation of foam cells. -- Polyunsaturated fatty acids (PUFAs) in the diet can substantially reduce the incidence of coronary heart disease.  However, dietary ω-3 polyunsaturated fatty acids found primarily in marine oils have been shown to be better in reducing the risk of atherosclerosis than other polyunsaturated fatty acids.  Unfortunately, it is unclear how ω-3 PUFAs are involved in delaying the atherogenic process.  It is possible that these fatty acids may be antiatherogenic by inhibiting cholesterol esterification or increasing cholesterol removal from cells thereby inhibiting the initial step of foam cell formation. -- The major objective of this study was to investigate the impact of different polyunsaturated fatty acid enrichment of nonhepatic tissues on cholesterol esterification and cholesterol efflux.  Human fibroblasts and macrophages were enriched with either linoleic acid (18:3, ω-6) or eicosapentaenoic acid (20:5, ω-3) by supplementing the culture medium.  The incorporation of radiolabelled oleoyl CoA into cholesterol esters was reduced by 44% when human fibroblasts were enriched with ω-3 polyunsaturated fatty acids compared with cells enriched with ω-6 polyunsaturated fatty acids.  Also, macrophages enriched with ω-3 PUFAs had significantly lower (52%) total cholesterol content than those enriched with ω-6 PUFAs.  Cholesterol efflux was measured in cells enriched with ω-3 PUFAs and ω-6 PUFAs.  In the presence of HDL, the rate of efflux of radiolabelled cholesterol from cells enriched with ω-3 PUFAs was substantially faster (2x) than efflux from cells enriched with ω-6 PUFA enriched cells.  The data suggests that ω-3 PUFAs may be antiatherogenic by inhibiting cholesterol esterification and by accelerating cholesterol efflux from nonhepatic cells.


identifier: Quarcoo, Ezekiel Tawia <https://research.library.mun.ca/view/creator_az/Quarcoo=3AEzekiel_Tawia=3A=3A.html>   (1983) Glutamine synthesis and synthetase in guinea pig kidney.  Masters thesis, Memorial University of Newfoundland.

abstract: It is now generally believed that kidneys of many species increase ammonia excretion (through the action of glutaminases) in the urine, in response to an acid challenge, and that the physiological significance of such enhanced ammoniagenesis is to primarily conserve extra-cellular fluid cations, especially Na⁺. The source material for urine ammonia has been identified as glutamine. However,  the physiological stimulus for renal ammonia excretion, the relative importance of enzymatic and physicochemical factors in the mechanism of ammonia excretion, and the rate-limiting  metabolic step(s) which is (are) modified during acidosis, are still not clear. -- In an attempt to unravel some of these mysteries, studies were conducted on renal synthesis of glutamine, and glutamine synthetase. Glutamine synthetase  has the potential to regulate ammoniagenesis by removing ammonia. It is clear, therefore, that results of studies performed on a system which contains high activities of both  the ammonia producing enzyme (glutaminase), and the ammonia consuming enzyme (glutamine synthetase), would be difficult to interprete in terms of net ammonia metabolism at any given time. A system with a relatively high activity of one or the other enzyme was necessary. -- Earlier studies had implicated glutamine synthetase in the regulatory events of renal ammoniagenesis in the rat. The guina pig was chosen as  a suitable experimental animal model for the studies reported, because the guinea pig kidney has a relatively high activity of glutamine synthetase, and a low activity of  glutaminase. -- These studies showed that the guinea pig kidney cortex tubule synthesizes glutamine rapidly at pH 7.4 (503+78μmoles/30 min/g dry wt), when incubated with glutamate and ammonium chloride. Furthermore, the high rates  of glutamine synthesis were reduced by about 50% at pH 7.1, regardless of whether the lower pH was produced by decreasiag the HC0₃ concentration (metabolic acidosis) or increasing the PC0₂ (respiratory acidosis). The inhibition of glutamine synthesis was reversible. -- Subcellular fractionation studies on guinea pig kidney cortex indicated an exclusive oytosolic localization of glutamine synthetase.  Such localization was not altered when the pH of the homogenizing medium was changed, since glutamine synthetase almost consistently appeared in the soluble fraction of spun  homogenates, using sucrose or KC1 media.


identifier: McCloy, Ursula <https://research.library.mun.ca/view/creator_az/McCloy=3AUrsula=3A=3A.html>   (1996) Growth, thiamin status, erythrocyte fatty acid composition, and visual acuity in fullterm infants fed breastmilk, formula, or evaporated milk.  Masters thesis, Memorial University of Newfoundland.

abstract: The effects of feeding breastmilk (BM), commercial formulas (F), or evaporated milk formula (EM) on growth, thiamin status, erythrocyte phosphatidylethanolamine (PE) fatty acid composition and visual acuity in 100 healthy full term infants were determined. Growth, thiamin status, and erythrocyte PE fatty acid composition was determined at birth, 3, and 6 months of age. Thiamin status was assessed by determining the erythrocyte transketolase activity (TKA) and the fatty acid composition of milk and blood was determined by gas-liquid-chromatography. Preferential looking acuity was assessed at 3 and 6 months of age using the acuity card procedure. -- There were no significant differences in weight, length, or head circumference between  any feeding groups. However, daily weight gain between three  and six months was significantly lower for the BM group (weight gain: ﻌ ± SD, BM 15.5 ± 4.0 g, F= 20.4 ± 5.8 g, EM =  20.2 ± 6.8 g, p < 0.05). As well, breastfed infants had slightly lower head circumference growth velocity between three and six months of age. -- There were no differences in  the thiamin pyrophosphate effect between groups, however, BM  infants had significantly lower transketolase activity than the F group which correlated with energy but not thiamin intake. -- The breastmilk obtained from mothers in the study  contained on average (% total fatty acids by weight), 12.1 %  linoleic acid (18:2(n-6)), 2.1% linolenic (18:3(n-3)), and 0.2% docosahexaenoic acid (22:6(n-3)). Infants in the F group consumed either Similac or Enfalac. Similac contained 30.5% 18:2(n-6), and 4.9% 18:3(n-3). Enfalac contained 17.1%  18:2(n-6) and 1.8% 18:3(n-3). EM contained (% total fatty acids) 2.1% 18:2(n-6), and 0.8% 18:3(n-3). Only breastmilk contained 22:6(n-3). -- In the circulation, F fed and EM fed  infants had lower arachidonic acid (20:4(n-6)) at 3 months (p < 0.05) than BM fed infants (ﻌ ± SD, 20.3±2.8, BM, 18.3 ±  2.7, F, 18.7 ± 2.7, EM, % total fatty acids) but not at 6 months, and EM fed infants had lower adrenic acid (22:4(n-6)) at both 3 and 6 months. 22:6(n-3) was the highest in the  BM group at both 3 (6.0 ± 1.7,BM, 3.1 ± 0.8, F, 4.1 ±0.9, EM, % total fatty acids) and 6 months of age (5.3 ± 1.6, BM,  2.9 ± 0.8, F, and 4.2 ± 1.3, EM, % total fatty acids) followed by EM then F. Visual acuity was higher in the BM group than EM (3.86 ± 0.29 cycles/degree vs 3.29 ± 0.41, 3 mos, 9.03 ± 0.29 vs 7.54 ± 0.25,6 mos, p < 0.05) with intermediate values in the F group (NS compared to EM and BM). Differences seen in visual acuity may be due to the low  18:3(n-3) in EM of 0.3 % of energy and are not reflective of  22:6(n-3) in circulation. -- From the results of the present  study it appears that EM formulas may not meet the essential  fatty acid requirements for optimal visual acuity, however, it may be adequate for thiamin and optimum growth. The differences seen in the BM group in growth and TKA can be related to lower energy intakes and do not indicate any deficiencies.


identifier: Nakhla, N. Anthony <https://research.library.mun.ca/view/creator_az/Nakhla=3AN=2E_Anthony=3A=3A.html>   (1987) Structural elucidation and synthesis of the glycoconjugate of the o-antigen isolated from the lipopolysaccharide of the gram-negative bacteria Yersinia ruckeri.  Masters thesis, Memorial University of Newfoundland.

abstract: In process


identifier: Maher, Maurica <https://research.library.mun.ca/view/creator_az/Maher=3AMaurica=3A=3A.html>   (1991) Studies of the Pancreatotoxicity of 1-cyano-2-hydroxy-3-butene.  Masters thesis, Memorial University of Newfoundland.

abstract: A racemic mixture of (R)- and (S)-1-cyano-2-hydroxy-3-butene  (CHB) was synthesized using 1,3-butadiene as a starting compound. The effects of CHB on pancreatic non-protein thiol and glutathione S-transferase activity were investigated in adult male rats when administered orally in olive oil at a dose of 200 mg/kg. At 4 hours after dosing, pancreatic non-protein thiol content was depleted but rebounded at 24 hours  to 3 times control values. At 120 hours after dosing, pancreatic non-protein thiol levels were still elevated over   control levels. Hepatic non-protein thiol levels were greater than control levels at 4 hours after dosing, but were not significantly different from control values at 24 and 120 hours after dosing. The pattern of pancreatic non-protein thiol changes with synthetic CHB resembles that reported with the natural (S)- isomer of CHB. -- Glutathione  S-transferase activity was decreased at 4 hours following CHB treatment in the rat pancreas, and elevated at 24 hours.  Hepatic glutathione S-transferase activity was not altered by CHB treatment. -- The effect of increasing intraduodenal doses of CHB (25-200 mg/kg) on rat pancreatic juice secretion and bile secretion was investigated in conscious rats. CHB was administered intraduodenally and caused a stimulation in pancreatic juice secretion followed by a return to control values at the lower doses. At higher doses, CHB causes the same early stimulation, but is followed by a depression of pancreatic juice flow. All doses  of CHB administered resulted in a dose-dependent decrease of  protein concentration in pancreatic juice. -- CHB caused the  pancreata of treated rats to become oedematous, a condition also seen in certain forms of acute pancreatitis. The severity of the oedema was dose-dependent and could be visually detected. The exudate collected from these pancreata had protein concentrations comparable to plasma. -- Radioactive CHB ([¹⁴C]) was synthesized and administered to rats intraduodenally that were also fitted with biliary fistulae. CHB was found to be excreted over 8 hours in the bile and urine, with higher amounts excreted in the urine. Little [¹⁴C] was detected in the pancreas and kidneys, but did accumulate in the adipose tissue (7.7%) and in the liver  (5.5%). -- These results indicate that synthetic CHB is a powerful and selective pancreatotoxin and the present data are not incompatible with a glutathione-mediated mechanism, as is known to occur with other unsaturated nitriles.


identifier: Ng, Tzi Bun <https://research.library.mun.ca/view/creator_az/Ng=3ATzi_Bun=3A=3A.html>   (1979) Pituitary hormones in teleost fish with emphasis on gonadotropins.  Doctoral (PhD) thesis, Memorial University of Newfoundland.

abstract: This investigation on fish gonadotropins provides considerable evidence to support the concept of duality of gonadotropins in teleosts proposed by Campbell and Idler in 1976.  Vitellogenic hormones were isolated from pituitaries of American plaice (Hippoglossoides platessoides) and winter flounder (PseudopTeuronectes americanus) and chum salmon (Oncorhynchus keta), devoid of oocyte maturational and androgenic activities, and they could be distinguished from the maturational hormones which were active in stimulating oocyte maturation and androgen production.  The bioassays used to isolate the vitellogenic and maturational hormones utilized the hypophysectomized flounder as the assay animal, and this study represents one of the few investigations utilizing homologous bioassays to isolate fish gonadotropins. -- Vitellogenic and maturational hormones are different in their amino acid compositions, carbohydrate contents, immunological activities and chromatographic behavior on Concanavalin A-Sepharose.  An antiserum to vitellogenic hormone was capable of inhibiting incorporation of vitellogenin into the ovary, whereas an antiserum to maturational hormone depressed the plasma concentrations of estradiol and vitellogenin, indicating that the two gonadotropins play different roles in fish reproduction.  Chromatography of maturational hormone on Concanavalin A-Sepharose did not lead to isolation of additional material unadsorbed on the immobilized lectin.  All available evidence thus suggest that the vitellogenic hormone and the maturational hormone are different entities. -- Thyroid stimulating hormone and gonadotropin were shown to be separate hormones.  Two thyroid stimulating hormones could be isolated from pituitaries of both American plaice and winter flounder.  The thyroid stimulating hormone unadsorbed on Concanavalin A-Sepharose stimulated mainly the production of triiodothyronine in hypophysectomized flounder while the thyroid stimulating hormone adsorbed on the immobilized lectin enhanced mainly the production of thyroxine.


identifier: Yang, Cheng Yong <https://research.library.mun.ca/view/creator_az/Yang=3ACheng_Yong=3A=3A.html>   (1991) Studies on rat c-reactive protein : binding to isolated rat hepatocytes and clearance from circulation.  Masters thesis, Memorial University of Newfoundland.

abstract: The clearance mechanism of C-reactive proteins (CRP) was examined in this study.  In vitro binding assays using isolated rat hepatocytes were followed by clearance studies in vivo and using perfused rat liver. -- It was shown that rat asialo CRP was rapidly cleared from the circulation (half-life about 4 min), whereas rat and rabbit CRP remained in the circulation for much longer periods of time (half-life 7.8 h for rat CRP).  The clearance of rat asialo CRP was shown to be mediated through the hepatic asialoglycoprotein receptor, and this clearance was not affected by the phosphorylcholine binding domain of rat asialo CRP.  Results of the clearance studies also suggested that, in addition to liver, lungs might also be involved in the clearance of CRP. -- It was observed that large amounts of rat asialo CRP, rat CRP and rabbit CRP bound to isolated rat hepatocytes, and the binding was shown to be mediated predominantly through the phosphorylcholine binding domain of these proteins.  In addition, rat asialo CRP was shown to bind to the hepatic asialoglycoprotein receptor on the hepatocytes, despite the avid phosphorylcholine binding domain-mediated binding of this protein.  It was demonstrated that the binding of large amounts of rat asialo CRP, rat CRP and rabbit CRP to the hepatocytes through the phosphorylcholine binding domain resulted from a disruption of the hepatocyte surface.


identifier: Zijenah, Lynn Sodai <https://research.library.mun.ca/view/creator_az/Zijenah=3ALynn_Sodai=3A=3A.html>   (1986) Syntheses and conformation of synthetic peptide substrates of protocollagen lysyl hydroxylase.  Masters thesis, Memorial University of Newfoundland.

abstract: Hydroxylation of specific lysyl residues by lysyl hydroxylase is an important posttranslational modification process in collagen biosynthesis. The main objective of this  work was to investigate the conformational requirement for the enzymic reaction. -- Eight lysine-containing peptides which had amino acid sequences comparable to amino acid sequences around hydroxylysine or lysine in collagen were synthesized by solution-phase techniques. The peptides varied in length from three to seven amino acid residues. The structures of these peptides were investigated through circular dichroism (CD) and infrared (IR) spectroscopic methods. -- Lysyl hydroxylase was partially purified from chicken embryos using the established procedures. Seven of the synthetic peptides were tested for their ability to act as substrates of partially purified lysyl hydroxylase. The hydroxylation reaction was assayed by a technique involving measurement of ¹⁴CO₂ released stoichiometrically from 2-[1-¹⁴C]oxoglutarate and/or by a specific chemical procedure for  hydroxylysine. -- Five peptides with the -Lys-Gly- sequence were hydroxylated to varying degrees, the degree of hydroxylation increasing with increasing chain length. Examination of these hydroxylated peptides by CD and IR spectral measurements revealed that the tripeptides NαtBocIleLysGlyOH and NαtBocAlaLysGlyOH adopt a γ-turn in which lysine occupies the second position of this structure.  The tetrapeptide (NαtBocAlaLysGlySerOH) adopts both a β- and  γ-turn and is more hydroxylated than the precursor tripeptide. This increase in the degree of hydroxylation may  be attributed to the presence of the β-turn which may stabilize the γ-turn formed by the AlaLysGlyOH segment. The hexapeptide (NαtBocLeuHyPGlyAlaLysGlyOH) adopts a consecutive β- and γ-turn and is more hydroxylated than the tetrapeptide. This increase in hydroxylation may be attributable to the Gly³-Ala⁴ segment which may increase the  binding of the enzyme to the substrate thereby enhancing hydroxylation. The heptapeptide (NαtBocLeuHyPGlyAlaLysGlySerOH) is hydroxylated more than the precursor hexapeptide. CD and model building studies have shown that NαtBocLeuHyPGlyAlaLysGlySerOH adopts two consecutive β-turns and a γ-turn. The second β-turn which is  similar to that found in the tetrapeptide (NαtBocAlaLysGlySerOH) may be responsible for the increase in hydroxylation in comparison with the hexapeptide. -- All the hydroxylated peptides have one structural feature in common, namely the γ-turn with lysine in the second position. In contrast, two peptides (NαtBocAlaGlyLysOH and NαtBocAlaGlyLysHyPOH) which have the Gly-Lys sequence were not hydroxylated. Interestingly, both peptides adopt a γ-turn but the lysine is found in the third position of this structure. These data indicate that lysyl hydroxylase recognizes specific secondary structure(s) in its substrates. The nature of the amino acid around lysine and the chain length of the peptide may be the critical determinants in the synthesis of hydroxylysine by lysyl hydroxylase.


identifier: Rehse, Peter Hugo <https://research.library.mun.ca/view/creator_az/Rehse=3APeter_Hugo=3A=3A.html>   (1985) Synthesis of and conformational studies on proline-containing peptides : ion-binding by linear tetrapeptides.  Masters thesis, Memorial University of Newfoundland.

abstract: The relationship between peptide backbone topology and ion-binding was investigated through proton and carbon-13 NMR, CD and IR spectroscopy. A literature review on calcium-binding proteins and peptides indicated that a structure characterized by two overlapping urns may be involved. To  test this hypothesis two peptides which could potentially form the above structure were synthesized. -- The peptides NαtBocProDAlaAlaNHCH₃ and its glycine analogue NαtBocProGlyAlaNHCH₃ were characterized in the uncomplexed state by all of the above mentioned techniques in a variety of solvents. Proton NMR temperature-dependence studies in DMSO-d₆ and NH coupling constants, along with CD and IR spectroscopy indicated that both peptides in solution were made up of a Type II β-turn followed by an overlapping Type I β-turn. Carbon-13 NMR, IR and CD spectroscopy indicated that the DAla-peptide was, as expected, more stable than the Gly-peptide. -- The binding of metal ions to the peptides was monitored using CD spectroscopy. Both peptides were found to be ion-specific. While both were capable of binding  calcium ion and incapable of binding sodium and lithium to any significant extent, they differed in their ability to bind magnesium. While the NαtBocProDAlaAlaNHCH₃ peptide bound magnesium weakly at a level comparable to its binding to the monovalent ions, the glycine analogue bound magnesium to a significant extent. The Gly-peptide also required a lower concentration of calcium ion to reach binding saturation. This was attributed to the greater flexibilty of  glycine and hence, its enhanced ability to "fit" the metal ion. -- The conformational change of the peptides upon calcium ion titration was followed by proton and carbon-13 NMR. The changes in chemical shifts of the carbonyl resonances and the amide proton resonances indicated that all four peptide carbonyls coordinated to the calcium ion resulting in a breaking of the intramolecular hydrogen bonds  of the uncomplexed species. Analyses of the CD binding-curves indicated that at low concentrations of calcium to peptide a 2:1 peptide:ion complex was formed, while at higher concentrations a 1:1 complex was predominant. The 2:1, peptide:ion complex is able to fill all eight calcium coordination sites with the peptide carbonyls while the 1:1 complex requires either the perchlorate anion or water molecule to fill the remaining coordination sites.


identifier: Manu-Tawiah, Wireko <https://research.library.mun.ca/view/creator_az/Manu-Tawiah=3AWireko=3A=3A.html>   (1988) Studies on the biomass production of Pleurotus ostreatus using peat extract as substrate.  Masters thesis, Memorial University of Newfoundland.

abstract: Pleurotus ostreatus mushroom mycelium was grown in submerged culture with acid extract from peat as a basic substrate. The proximate chemical composition of the peat extract was determined. The total reducing sugars constituted about 50% of the total carbohydrate (TCH) concentration. The total nitrogen content was low at about 0.6 g/L of the pet extract. The total ash was about 4.5%, of which 1.2% was P₂O₅ and 0.87% K₂O. -- Different concentrations of water-diluted peat extract were tested in shake flask experiments in an attempt to overcome the effect of growth inhibitors apparently present in non-diluted peat extracts. The best results were obtained with a ratio of one part peat extract diluted with one part water. Several operating variables were studied to optimize the growth of the mycelial biomass of P. ostreatus. The best results produced approximately 5 g/L of dry biomass with a yield of 60% and an efficiency of 33%. The results were obtained in 192 hours at an inoculum ratio of 5.0% (v/v), and incubation temperature of 28 ± 1°C, an initial pH of 5.0 ± 0.1 and an agitation speed of 150 rpm.  The addition of 1.5% glucose to the diluted peat extract medium increased the growth to approximately 14 g/L of dry biomass with a yield of 70% and an efficiency of 45%, indicating that the diluted peat extract was deficient in carbon. -- The nutritional requirements of the P. ostreatus mycelium grown in peat extract were studied to enhance the growth of the mycelial biomass. The fungus grew better on hexose sugars (glucose and mannose) than on pentose sugars (xylose and arabinose) and required additional nitrogen, phosphorus and potassium for growth. Growth was also better on organic sources of nitrogen than on ammonium salts of inorganic acids. The addition of 5.0 g/L yeast extract and 4.0 g/L KH₂PO₄ to the peat extract media produced approximately 7 g/L dry biomass with a yield of 73% and an efficiency of 46%. -- Agitation and aeration were optimized and the dissolved oxygen concentration was monitored in a 2 Liter fermenter to study the growth of the P. ostreatus mycelium biomass in the peat extract. The best results produced approximately 9 g/L of biomass with a yield of 72% and a productivity of 70 mg/L/hour. These results were obtained in 96 hours at an agitation speed of 200 rpm, an aeration rate of 1.0 vvm, an inoculum ratio of 5.0% (v/v), an incubation temperature of 28 ± 1°C and a pH of 5.0 ± 0.1. Higher agitation speeds and aeration rates produced lower growth and a dispersed mycelial growth form. The morphology of the P. ostreatus growth form was also affected by the pH and the media composition. -- The chemical composition of the P. ostreatus mycelium biomass was analyzed and compared with that of the fruiting body produced on solid peat to study the nutritional quality of the product. The mycelium contained approximately 40% crude protein, 8% ash, which contained K, P, Ca and Mg as major minerals, 36% TCH, 6% fiber and 3% total fat. The protein contained 17 amino acids, including all the essential amino acids, and was high in threonine, lysine, tryptophane, arginine, leucine and phenyalanine, but was deficient in methionine when compared with egg protein. Three essential fatty acids (palmitic, oleic and linoleic acids) were identified in the biomass.


identifier: Diniz, Fabio Mendonca <https://research.library.mun.ca/view/creator_az/Diniz=3AFabio_Mendonca=3A=3A.html>   (1996) Studies on the enzymatic hydrolysis of dogfish (Squalus acanthias) muscle protein.  Masters thesis, Memorial University of Newfoundland.

abstract: Response surface methodology (RSM) was used to study the combined effects of pH, temperature and enzyme - substrate ratio (E/S) on the degree of hydrolysis (DH) of dogfish muscle protein. The effects of the factor variables which affect hydrolysis were described using a three-level Box-Behnken factorial design giving a mathematical model equation that showed the influence of each variable and their interactions. The optimization of the enzymatic process was performed with the RSM statistical technique having the recovery of soluble nitrogen (NR) from the original substrate as the process response. The optimum values for enzyme / substrate ratio, temperature and pH were  found to be 3.7 % (w/w), 55.3 °C, and 8.3, respectively, resulting in a maximum NR of 77.68 %. The coefficient of determination (R²ad) was greater than 95 % at the 0.1 % probability level, and a lack-of-fit test revealed a non-significant value for the NR model equation, indicating that  the regression equation was adequate for predicting the nitrogen recovery under any combination of values of the variables. The shark protein hydrolysate produced under these conditions contained a high crude protein concentration (> 85 %, dry weight basis), and its high nutritional value was indicated by the presence of all essential amino acids, and by high protein efficiency ratio (PER) values. The functional, physical and rheological properties of the optimized spray dried protein hydrolysate was compared to the protein hydrolysate obtained by autolytic degradation, and a control, freeze dried muscle. The Alcalase®-assisted hydrolysis, in general, improved functional properties such as solubility and dispersibility, indicating that enzymatic hydrolysis under the experimental conditions described in this study can produce a new source protein ingredient with superior protein functionality. Physical and rheological product characteristics were also subjected to significant changes. The off-white protein hydrolysate had a low viscosity in aqueous dispersions even at high protein concentration.