Some of the factors affecting the form and size of crystals produced by the solvent exchange method are described for lindane. The effect of different crystal forms on the availability of this toxicant to the ambrosia beetle Trypodendron lineatum (Oliv.) was evaluated from gross functional effects. The beetles were brought into contact for known periods of time with deposits of a prescribed dosage of lindane crystals of two forms. Mortality was used as an index of the availability of the different deposits. The loss in weight with time was followed for deposits of different dosages of lindane crystals of the two forms, as a possible factor contributing to differences in residual toxicity. With dosages corresponding to those used for preventing damaging attacks on logs by this ambrosia beetle, viz. 180 mg./ft.² , no difference in effect was demonstrated between crystals of the two forms. No striking difference in rate of residue loss was demonstrated when deposits "aged" in a draught free cupboard. The minimum and maximum temperature recorded in the cupboard during the storage period was 14.5° and 23.5°C. The concept of availability of insecticidal deposits and the factors affecting it are discussed. / Science, Faculty of / Zoology, Department of / Graduate

A group of five goldfish, Carassius auratus, treated ten and sixteen days with a 1: 3,980,000 concentration of synthetic sodium thyroxine during the winter, had their oxygen consumption significantly raised. Five goldfish treated with .03 per cent thiourea for the same lengths of time during the same period had their oxygen consumption significantly depressed. Two series of similar treatments of three, seven, fourteen and twenty-one days duration throughout the summer did not statistically alter the oxygen consumption and ammonia excretion. Treatments with a 1: 1500,000 concentration of methyl testosterone for three, seven, fourteen and twenty-one days statistically elevated the oxygen consumption and ammonia excretion. The thiourea, synthetic sodium thyroxine and methyl testosterone treatments for three, seven, fourteen and twenty-one days did not appreciably alter the total lipid and moisture content of the goldfish tissue. These treatments do not appear to alter the oxygen carbon dioxide tension at death nor the length of time to asphyxiate the goldfish. / Science, Faculty of / Zoology, Department of / Graduate

An investigation was undertaken to study the kinetics and possible cellular control mechanisms for imipramine HCl metabolism in the isolated perfused rat liver. The isotope ¹⁴C-imipramine was used and quantification was done by liquid scintillation counting. Analysis for imipramine (IMI), desmethylimipramine (DMI), free hydroxy (OH), glucuronide (G) and N-oxide (N-0) metabolites was done on the perfusate, bile and liver. The rate of IMI metabolism was found to be dependent on two major enzymatic routes, N-demethylation (formation of DMI) and aromatic hydroxylation (formation of G, OH) of imipramine and one minor enzymatic route, N-oxidation (N-O). The rate of aromatic hydroxylation of IMI was found to be inhibited after thirty minutes, with IMI concentration 2 X 10 ⁻⁵M. This inhibition of aromatic hydroxylation could not be detected if the perfusate half-life for IMI (t½=18.5 minutes) was the only parameter monitored. After incubation periods of fifteen, thirty and sixty 80 per cent and the remainder of IMI was in the perfusate. The dose of IMI was varied (0.5 X 10⁻⁵ M, 1 X 10⁻⁵ M and 2 X 10⁻⁵ M) for metabolism by the perfused rat liver. The incubation time was kept constant at fifteen minutes. The rate of imipramine metabolism (formation of DMI and GOH) followed first order kinetics when the dose of IMI was 0.5 X 10⁻⁵ M or 1 X 10⁻⁵ M. Increasing the dose of IMI to 2 X 10⁻⁵ M slightly suppressed the formation of DMI and the formation of GOH followed zero order kinetics. It was found that the endogenous DMI formed from IMI metabolism inhibited the formation of GOH after fifteen minutes and thirty minutes of IMI metabolism as shown by the following results. DMI (1.65, 3.33, 6.66 or 13.32 X 10⁻⁶ M) was preincubated prior to addition of IMI. DMI (1.65 or 3.33 X 10⁻⁶ M) was found to specifically inhibit aromatic hydroxylation of IMI. Higher concentration of DMI (6.66 or 13.32 X 10⁻⁶ M) inhibited the formation of GOH and DMI. Ethyl alcohol (1 mM) preincubated prior to addition of 1 X 10⁻⁵ M of IMI specifically inhibited DMI formation. No inhibition of GOH occurred. Ethyl alcohol (1 mM) caused inhibition of formation of DMI from IMI metabolism when the dose of IMI was 2 X 10⁻⁵ M. The incubation time for IMI metabolism was fifteen and sixty minutes. With this decrease of DMI formation, the formation of GOH increased after fifteen or sixty minutes of incubation time. From these experiments it was concluded that suppression of aromatic hydroxylation of imipramine was due to the formation of endogenous DMI formed from IMI metabolism. Optimal conditions were found to study possible cellular control mechanisms for IMI metabolism in the isolated perfused rat liver. The dose of IMI was 1 X 10⁻⁵ M and the incubation time was fifteen minutes. Dibutyryl cyclic AMP (2 X 10⁻⁶ M) caused inhibition of IMI metabolism. DMI formation was inhibited 28 per cent while GOH formation was inhibited 29 per cent. NADPH (1.1 X 10⁻⁶ M) or NADH (1.3 X 10⁻⁶ M) was found to inhibit imipramine metabolism. GOH and DMI were both inhibited. Succinic acid (1.6 X 10⁻³ M) was found to inhibit DMI formation but not GOH. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

INTRODUCTION: Blood lipids, more precisely triglycerides (TG), are important fuel sources that are highly regulated since an exaggerate amount can lead to cardiovascular diseases. TG breakdown after a meal is mainly controlled by an enzyme expressed in adipose tissue called lipoprotein lipase (LPL). Recent evidence in animals report that adipose tissue LPL is inhibited after an exposure to an environment with reduced oxygen content, leading to a raised level of plasmatic TG. The objective of this thesis was to characterize the effects of an acute exposure to hypoxia on the plasmatic lipolytic activity level and on postprandial TG levels in humans. It was hypothesized that postprandial TG level and plasmatic lipolytic activity, a proxy of LPL activity, would be negatively affected by hypoxia. METHODS: Postprandial TG, non-esterified fatty acid (NEFA), glucose levels, and postheparin plasmatic lipolytic activity were measured on healthy young men (n=7) exposed for 6 h to either control (FiO2=0.2093) or hypoxia (FiO2=0.1200) in a randomized crossover fashion. RESULTS: Exposure to acute hypoxia led to a close to significant (p = .06) increase in postprandial plasmatic TG level and significant postprandial NEFA levels. Postprandial glucose levels were not affected by acute exposure to hypoxia. A significant increase in postheparin plasmatic lipolytic activity was observed after acute hypoxia exposure as compared to the control condition. CONCLUSION: Acute hypoxia in healthy men tend to negatively affects postprandial TG level while increasing plasmatic lipolytic activity. These results lend support to the increased blood lipid levels reported in individuals exposed to lower partial pressure of oxygen during sojourn at high altitude.

Induction of phenylpropanoid metabolism in many plants is associated with the induction of plant defence responses. Among these are the accumulation of phenylpropanoid-derived phytoalexins, increase in lignification around infected sites, and the accumulation of wall-bound phenolic compounds. I show in this work, that H11 hybrid cell suspension cultures when treated with either of three elicitors respond with an increase in phenylpropanoid metabolism. Activation proceeds rapidly from PAL and 4CL mRNA accumulation, to a massive increase in extractable PAL enzyme activity and finally there is accumulation of specific phenolic compounds in the cell extracts, culture filtrates, and cell walls. In addition, elicitor treatment causes cells to turn brown, indicative of phenolic compound accumulation. As in other plants, induction is dependent on culture age, is dose dependent, and the kinetics of induction is the same with all three elicitors. Based on the previously established mode of action of PGA lyase as an elicitor, it is concluded that in poplar, as in other plants, defence responses can be induced by elicitors from both fungal and plant cell wall origin. These results illustrate the successful use of plant suspension cultures as a simplified system to study inducible defence responses. In addition, and consistent with the ubiquitous nature of phenolics in poplar, phenylpropanoid metabolism may play an important role in plant defence responses in this species. / Science, Faculty of / Botany, Department of / Graduate

I. Previous studies on biogenesis of simple phenols in plants have been restricted to hydroquinone. Among the other simple phenols, catechol is of particular interest because of its potential role as a ring-cleavage substrate. Tracer studies on the biogenesis of catechol in Gaultheria leaf discs showed that it was formed from salicylic acid by oxidative decarboxylation. Salicylate decarboxylating activity could be detected in buffered extracts of very young leaves. II. Among the numerous caffeic acid esters presently known in plants, only 3-0-caffeoylquinic acid (chlorogenic acid) has been studied in detail. Rosmarinic acid (alpha-O-caffeoyl-3, 4-dihydroxyphenyllactic acid) has been reported to occur in a number of plants but nothing was known of its biosynthesis or metabolic role. Tracer studies demonstrated that in Mentha the caffeic acid moiety was formed from phenylalanine via cinnamic and para-coumaric acids. In contrast, the structurally similar 3, 4-dihydroxyphenyllactic acid moiety was formed from tyrosine and 3, 4-dihydroxyphenylalanine. There was no evidence of the participation of a para-coumaroyl ester intermediate. Time-course studies and use of labelled rosmarinic acid showed that endogenous rosmarinic acid was turning over slowly. The caffeoyl moiety, however, does not appear to be contributing to the formation of insoluble polymers, as has been suggested for chlorogenic acid in other plants. III. Bacteria and fungi readily degrade aromatic compounds to carbon dioxide. Despite the large quantities of aromatic compounds formed in plants, little attention has been paid to the ability of plant tissues to degrade aromatic rings. No reported studies have used completely sterile plants and techniques. This has left open the possibility that the microflora associated with the plant might be carrying out the observed reactions. The ability of sterile plant tissue cultures to degrade aromatic ring-¹⁴C compounds to carbon dioxide was studied. It was established that a number of tissues (Ruta, Triticum, Phaseolus, Melilotus) have the ability to cleave the aromatic ring of phenylalanine. Melilotus tissue could also degrade cinnamic acid-ring-¹⁴C suggesting that a dihydroxy phenolic acid may be the ring-cleavage substrate. Neither Ruta nor Melilotus tissues were able to degrade benzoic acid or salicylic acid-ring-¹⁴C. Tryptophan benzene ring-¹⁴C was shown to be degraded to carbon dioxide by both Ruta and Melilotus. In summary, the ability of plants to cleave the benzene ring of aromatic compounds when free of micro-organisms was thus established. / Science, Faculty of / Botany, Department of / Graduate

It is known that the functional activity of the nervous system is associated with ammonia formation and that the administration of ammonium salts to experimental animals produces convulsions. Mechanisms, therefore, that control ammonia metabolism in the brain are of importance for brain cell function. The presence of ammonia utilizing mechanisms in the brain maintains the low free cerebral ammonia levels found in vivo. There is, however, a rapid formation of ammonia in the brain on the death of the animal and a further liberation of ammonia takes place when isolated brain cortex is incubated aerobically in the absence of glucose. Studies of these and other aspects of ammonia metabolism form the subject matter of this thesis. The estimation of ammonia in these studies is based on a modification of the diffusion technique of Conway. Ammonia and amino acid analyses have been carried out using the Beckman amino acid analyzer. The rapid rate of cerebral ammonia formation that takes place when the brain is removed from the animal is partially arrested by trichloracetic acid (TCA), presumably by the inactivation of cerebral enzymes. Our results rule out the possibility that glutamine, glutamate, taurine and ATP are significant contributors to the initial or pre-incubation levels of ammonia and the evidence favors the involvement of TCA-insoluble components as precursors of such ammonia. In the presence of glucose the pre-incubation levels of amino acids of cerebral cortex slices of the rat are maintained during subsequent aerobic incubation at 37°C for one hour. In the absence of glucose, however, we have found marked changes in the pre-incubation levels of amino acids of cerebral cortex slices under these experimental conditions. A considerable rise of ammonia also occurs in the absence of glucose and this can be largely accounted for by a net loss of –NH₂-groups of the amino acid pools of brain slices. The significant fall in the cerebral levels of glutamate and glutamine under these conditions indicates that for short periods of incubation (one hour), these amino acids may serve as major sources of ammonia formation by respiring brain cortex slices. Our findings of a marked suppression of ammonia formation by cerebral cortex slices incubated for one hour either anaerobically, or aerobically, in a glucose-free medium in the presence of amytal, D-glutamate or α-methylglutamate, implicate the oxidative deamination of cerebral glutamate as a major mechanism for ammonia liberation. D-glutamate also acts by inhibiting the hydrolysis of glutamine. In the presence of glucose aerobic incubation with 2, 4-dinitrophenol, iodoacetate, malonate, hydroxylamine or D-glutamate, increases the rate of ammonia formation by cerebral cortex slices. This is doubtless due to diminished activity of cerebral glutamine synthetase required for ammonia fixation to occur. We find that the level of ammonia in the brain tissue itself is not markedly affected by the presence of absence of glucose. The increased quantity of ammonia formed by cerebral cortex slices in the absence of glucose is found largely in the incubation medium. This fact points to the formation of ammonia in specific compartment(s), and the retention of ammonia within such compartment(s) up to a certain level. Above this level there is efflux of ammonia into the incubation medium. Such a conclusion helps to explain the apparent high concentration ratio (tissue:medium) of NH₄⁺ obtained either aerobically (viz. 42) or anaerobically (viz. 12) at the end of one hour incubation in the presence of glucose. There is some accumulation of NH₄⁺ ions in rat brain cortex slices against a concentration gradient. Our finding that ouabain stimulates ammonia formation in respiring cerebral cortex slices is in accord with the fact that ouabain inhibits the utilization-of NH₄⁺ for the biosynthesis of glutamine, presumably by affecting the transport of NH₄⁺ to the site of glutamine synthesis. Ouabain has no effect on the cerebral glutaminase of the rat. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Bacillus subtilis and Micrococcus lysodeikticus grew poorly when subjected to conditions of limiting iron nutrition after inocula were washed. Growth of both organisms was stimulated by ferrichrome and various phenolic acids. The chelating agents ethylene-diamine tetraacetic acid and nitrilotriacetic acid stimulated growth of B. subtilis but not of M. lysodeikticus . The effect of these compounds as antagonists to the action of the peptide antibiotic albomycin depended on growth conditions. Phenolic acid production by B. subtilis required relatively high levels of a carbon source, with glucose serving as the most effective substrate of those tested. B. subti lis WB746 produced only 2,3-dihydroxybenzoic acid whereas strain B1471 produced an unidentified phenolic acid early in log phase and 2,3-dihydroxy-benzoylglycine in the late log and early stationary phases of growth, under iron deficient conditions. Mutant strains of B. subtilis produced phenolic acids in the absence or presence of iron in the growth medium. DHB synthesizing enzymes were repressed by growth of B. subtilis in the presence of iron, ferrichrome or the aromatic amino acids. Active dihydroxybenzoic acid synthetase was not affected by these compounds. The DHB synthetase system from B. subtilis was partially purified by DEAE-cellulose column chromatography and sucrose gradient centrifugation; the result suggested the existence of a multienzyme complex. Iron uptake by B. subtilis and typhimurium was shown to be energy-dependent and repressible by growth in the presence of adequate iron. The iron uptake capacity of M. lysodei kticus appeared to be inducible, with a chelating agent or an Fe: chelate complex serving as the inducer. Ferrichrome and dihydroxybenzoic acid appeared to serve as iron transport factors for all three organisms, whereas ethylenediaminetetraacetic acid inhibited iron uptake. 3-F1uorobenzoic acid, a dihydroxybenzoic acid analog, had little effect on growth, but did reduce both phenolic acid production by, and iron uptake capacity of, Bacillus subtilis. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In greenhouse and controlled environment experiments, the influences of temperature, P nutrition-, and foliar sprays of 3 growth retarding chemicals on the growth, yield and mineral composition of Pisum sativum L. cv. Dark Skin Perfection were investigated. The utilization of P under 4 air and soil temperature regimes within the physiological range was also studied. The dwarfing effect of high temperature was related to that due to relatively high concentrations of growth retarding chemicals. Banded P fertilizer, applied at rates of up to 352 lb. per acre, increased plant growth, pea yield and the uptake of N, P, K, Ca and Mg. P increased the total contents of all 5 minerals in all 3 tissues (vine, pod and pea seed), but had multiple effects on mineral concentrations. Efficiency of P in producing pea yield increase was maximum at the 44 lb. per acre rate. The high air temperature of 30° decreased growth, pea yield, and total mineral uptake, compared with a temperature of 21°. The high soil temperature of 18° increased these 3 groups of variables, as compared with a temperature of 10°. Increases in mineral concentration at the high air temperature were largely due to "concentration effects'' resulting from smaller plants. Increases due to the high soil temperature were absolute because they occurred even in bigger plants. Increase in mineral uptake at the high soil temperature was not due to increased root growth, but was a result of increased metabolic activity. The effect of soil temperature on total absorption was greater than on translocation into the pea seed. (2-chloroethyl) trimethylammonium chloride (Cycocel) at 1 ppm was the most effective in terms of growth and yield stimulation. 2,4-dichlorobenzyl tributylphosphonium chloride (Phosfon) at 100 ppm was the most effective with respect to growth retardation, but markedly decreased pea yield. N-N-dimethylamino succinamic acid (B-Nine) at concentrations of 1 and 100 ppm was ineffective in altering growth pattern. Effects of the growth retarding chemicals on mineral uptake largely reflected plant size differences, and were not absolute effects. Cycocel and Phosfon at low concentrations are promising for use in arresting excessive vegetative growth and its attendant problems, and in increasing pea yield without deformative effects. The effects of relatively high concentrations of Cycocel and Phosfon were similar to those of high temperatures with respect to plant dwarfing, changes in mineral composition and alteration in the levels of Glucose, G-l-P, G-6-P, F-6-P, Fl,6-P, ADP and ATP. It appears that high concentrations of growth retarding chemicals and high temperatures depress plant growth by reducing the utilization of ATP in the phosphorylation of sugars, in the glycolytic sequence. The nearest-optimal air and soil temperature regime for plant growth and mineral uptake was the 21/13/18° day/night/soil. For uniformity in the nomenclature of plant growth regulators, it is suggested that growth retarding chemicals be called "RETARDINS". / Land and Food Systems, Faculty of / Graduate

Suspensions of C. perfringens, when grown on a peptone-free, semi-defined medium, have been shown to remain resistant to autolysis for extended periods of time. The stability of these suspensions has been compared with that of cells grown on complex media. Extracts of cells grown on this semi-defined medium were found to contain all of the enzymes of the Embden-Meyerhof pathway of glycolysis, in addition to lactic acid dehydrogenase and the pyruvate-clastic system, but no evidence of glucose-6-phosphate dehydrogenase activity could be demonstrated. Evidence has been presented for the implication of the Embden-Meyerhof pathway as the major pathway of glucose degradation by this organism. Resting suspensions of C. perfringens were shown to transport radioactive glucose and mannose, but not other carbohydrates by a common mechanism and accumulate glucose to concentrations several hundred times those found in the external medium. The transport system was found to be an enzymatic, energy-dependent, temperature-sensitive, and highly specific mechanism which was saturated at high substrate concentrations. The carbohydrate was found to be accumulated as an equilibrium mixture of phosphorylated hexoses. The phosphorylation mechanism involved in accumulation was demonstrated to be other than the soluble hexo-kinase. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate