Effect of continuous infusion of a low dose of GnRH antagonist on serum LH and testosterone concentrations, spermatogenesis and semen quality in the rhesus monkey (Macaca mulatta)

Treatment of 4 adult male rhesus monkeys for 8-12 months with 100-400 micrograms of a GnRH antagonist/day by means of using osmotic minipumps led to suppressed serum concentrations of LH and testosterone followed by various degrees of recovery toward pretreatment values. The serum LH response to a challenge of native GnRH was reduced by 30-75% during antagonist treatment. The serum testosterone response to GnRH was exaggerated above the response in the pretreatment period, suggesting hypersensitivity of the testis to gonadotrophin. Antagonist administration under these conditions did not alter body weight or abolish ejaculatory response. Antagonist infusion caused a 96% decrease in sperm counts. Spermatozoa recovered during the final month of antagonist treatment showed a reduced ability to penetrate denuded hamster ova. Testicular biopsies performed at the end of antagonist treatment revealed persistent spermatogenesis. However, the cellularity of the seminiferous tubules was decreased below that of pretreatment biopsies. The results of this study suggest that the amount of testosterone needed to maintain normal spermatogenesis is greater than that needed to maintain electroejaculatory response in monkeys.     

Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity

A three-dimensional model of yeast alcohol dehydrogenase, based on the homologous horse liver enzyme, was used to compare the substrate binding pockets of the three isozymes (I, II, and III) from Saccharomyces cerevisiae and the enzyme from Schizosaccharomyces pombe. Isozyme I and the S. pombe enzyme have methionine at position 294 (numbered as in the liver enzyme, corresponding to 270 in yeast), whereas isozymes II and III have leucine. Otherwise the active sites of the S. cerevisiae enzymes are the same. All four wild-type enzymes were produced from the cloned genes. In addition, oligonucleotide-directed mutagenesis was used to change Met-294 in alcohol dehydrogenase I to leucine. The mechanisms for all five enzymes were predominantly ordered with ethanol (but partially random with butanol) at pH 7.3 and 30 degrees C. The wild-type alcohol dehydrogenases and the leucine mutant had similar kinetic constants, except that isozyme II had 10-20-fold smaller Michaelis and inhibition constants for ethanol. Thus, residue 294 is not responsible for this difference. Apparently, substitutions outside of the substrate binding pocket indirectly affect the interactions of the alcohol dehydrogenases with ethanol. Nevertheless, the substitution of methionine with leucine in the substrate binding site of alcohol dehydrogenase I produced a 7-10-fold increase in reactivity (V/Km) with butanol, pentanol, and hexanol. The higher activity is due to tighter binding of the longer chain alcohols and to more rapid hydrogen transfer.     

Lipid metabolism in various regions of squid giant nerve fiber

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.     

Intra-tree foraging behavior of Ceratitis capitata flies in relation to host fruit density and quality

We examined the intra-tree foraging behavior of individually-released, wild-population Mediterranean fruit flies (medflies), Ceratitis capitata (Wiedemann), on field-caged host trees bearing each of three different densities (0, 3, or 12 per tree) of non-infested host fruit (kumquat) or each of two levels of fruit quality (12 non-infested fruit or 12 fruit infested with eggs and covered with host marking pheromone). With increasing density of non-infested fruit, medflies tended to remain longer in trees, visit more fruit before leaving, oviposit more often, accept a proportionately smaller number of fruit visited, and emigrate sooner after the last egg was laid (i.e. have a shorter Giving-Up-Time). Medflies spent much less time, oviposited much less often, and exhibited a longer Giving-Up-Time on trees harboring pheromone-marked fruit than non-infested fruit. Variation in temperature within the range at which experiments were conducted (25–36°C) had little detectable influence on foraging behavior. We compare our findings with published findings on the intra-tree foraging behavior of another tephritid fly, Rhagoletis pomonella (Walsh), and with current foraging behavior theory. We discuss implications of our findings with respect to medfly management strategies, particularly fruit stripping in eradication programs and use of synthetic marking pheromone for control.

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Résumé Nous avons étudié le comportement de prospection dans un arbre, de femelles d'une population sauvage de C. capitata, libérées individuellement à l'intérieur de cages contenant des Eriobotrya japonica (kumquat), portant chacun 3 densités différentes de fruits no contaminés (0, 3, 12 par arbre) et chacun 2 niveaux de qualité de fruits: 12 fruits non infestés ou 12 fruits contaminés par des oeufs et recouverts de phéromone de marquage de l'hôte. C. capitata avait terndance à rester plus longtemps dans les arbres, à visiter plus de fruits avant le quitter, à pondre plus souvent, à accepter proportionnellement un nombre plus réduit de fruits déjà visités, à émigrer plus tôt après la ponte du dernier oeuf (c'est-à-dire à présenter un temps d'abandon plus bref), quand la densité des fruits non contaminés augmentait. C. capitata a dépensé beaucoup moins de temps, pondu beaucoup moins souvent, et présenté un temps d'abandon plus long sur les arbres portant des fruits marqués par la phéromone que sur ceux ayant des fruits non contaminés. Les variations de température dans la gamme de cells où les observations ont eu lieu (23–36°C) n'ont eu qu'une faible influence décelable sur le comportement de prospection. Nous avons comparé nos résultats avec ceux publiés sur la prospection à l'intérieur de l'arbre par une autre téphritide (Rhagoletis pomonella) et avec la théorie dominante sur le comportement de prospection. Nous discutons les conséquences de nos résultats sur les stratégies de lutte contre C. capitata, en particulier l'élimination des fruits dans les plans d'erradication et l'utilisation de phéromone synthétique de marquage.

     

Modification of expression of the malignant phenotype in radiation-initiated cells

Carcinogenesis is a multistage process consisting minimally of initiation and promotion/progression stages. Radiation and many environmental xenobiotics are potent initiating agents. We have shown that initiation of carcinogenesis in vivo by these agents is a common cellular event. In the irradiated thyroid (5 Gy) at least one in 20 cells is initiated. Initiation by both radiation and chemicals has also been shown to be a common cellular event in the mammary gland. Initiation therefore is most likely not the sole rate-limiting event in the carcinogenic process. The propensity of the initiated cell to express the malignant phenotype is modulated by many factors, including environmental chemicals and physiological and genetic factors. Scopal and abscopal physiological factors can either enhance or suppress the progression of initiated cells to a frank tumour. For example, prolactin enhances the rate of progression of radiation and chemically initiated mammary tumours while glucocorticoids suppress this progression. TSH enhances the progression of radiation-initiated thyroid tumours while a scopal factor associated with unirradiated thyroid cells suppresses progression of this tumour type.     

Isolation and culture of cells derived from human cerebral microvessels

Summary Microvessels were isolated from non-neoplastic human cerebral cortical fragments resected for treatment of intractable seizure disorder. The microvessels were incubated in modified Lewis medium with 20 or 30% fetal bovine serum. Within 1–2 weeks, two cell populations emerged from the isolates. One type of cells had polygonal morphology, showed density-dependent contact inhibition at confluence in vitro, showed lectin-binding characteristics of endothelium (but only moderate positivity for factor VIII antigen), demonstrated induction of -glutamyl trans-peptidase when exposed to astrocyte-conditioned media, and responded to insulin by a pronounced increase in DNA synthesis. The other variety of cells grew in vitro more slowly in irregular strands separated by clear zones, showed ultrastructural features of smooth muscle, and isoelectric focusing of cell proteins revealed the presence of smooth-musclespecific -isoactin. Both types of cells could be serially subcultured. The ability to isolate and grow the two cell types, tentatively identified as human cerebral microvascular endothelium and smooth muscle, may facilitate studies of human blood-brain barrier function as well as the pathogenesis of cerebral microangiopathies unique to the human brain.Funded by Canadian Heart Foundation, Heart and Stroke Foundation of Ontario and UCLA Biomedical Research Support Grant     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.