The temperature-dependence of the loss of latency of lysosomal enzymes

1. When Triton-filled lysosomes from rat liver are incubated for up to 50min at 37 degrees C, pH7.4, in 0.25m-sucrose, no loss of latency of N-acetyl-beta-glucosaminidase or p-nitrophenyl phosphatase occurs unless the incubated lysosomes are cooled to approx. 15 degrees C. 2. It is suggested that a phase change takes place in the incubated lysosomal membranes on cooling; it starts at approx. 15 degrees C and probably is not complete at 0 degrees C. 3. Incubation of the lysosomes causes an increased potential for loss of latency of the lysosomal enzymes. This potential is not fully expressed at elevated temperature (e.g. 37 degrees C), but is expressed on cooling. 4. The increase at elevated temperature in potential for loss of latency exhibits biphasic kinetics, with an initial rapid phase followed by a slower phase, which is linear with respect to time. The extra loss of latency resulting from the rapid phase in proportional to the temperature of the incubation. 5. Arrhenius plots of the increase is potential for loss of latency during the slow phase for N-acetyl-beta-glucosaminidase and p-nitrophenyl phosphatase exhibit marked deviations from linearity beginning at approx. 15 degrees C. This suggests that the increase in potential for loss of latency is affected by a phase change that occurs around this temperature. 6. Activation energies for the increase in potential for loss of latency at and above 22 degrees C are 53.1+/-5.4kJ/mol (12.7+/-1.3kcal/mol) for N-acetyl-beta-glucosaminidase and 45.2+/-7.5kJ/mol (10.8+/-1.8kcal/mol) for p-nitrophenyl phosphatase. It is postulated that these energies reflect enzymic action, the products of which cause loss of latency to occur on cooling.     

The nature of the changes in liver mitochondrial function induced by glucagon treatment of rats. The effects of intramitochondrial volume,aging and benzyl alcohol

(1) The effects of changes in the intramitochondrial volume, benzyl alcohol treatment and calcium-induced mitochondrial aging on the behaviour of liver mitochondria from control and glucagon-treated rats are reported. (2) The stimulatory effects of glucagon on mitochondrial respiration, pyruvate metabolism and citrulline synthesis could be mimicked by hypo-osmotic treatment of control mitochondria and reversed by calcium-induced aging of mitochondria or by treatment with 20 mM benzyl alcohol. Hypo-osmotic treatment increased the matrix volume whilst aging but not benzyl alcohol decreased this parameter. (3) Liver mitochondria from glucagon and adrenaline-treated rats were shown to be less susceptible to damage by exposure to calcium than control mitochondria and frequently showed slightly (15%) elevated intramitochondrial volumes. (4) Aging, benzyl alcohol and hypo-osmotic media increased the susceptibility of mitochondria to damage caused by exposure to calcium. (5) Glucagon-treated mitochondria were less leaky to adenine nucleotides than control mitochondria. (6) These results suggest that glucagon may exert its action on a wide variety of mitochondrial parameters through a change in the disposition of the inner mitochondrial membrane, possibly by stabilisation against endogenous phospholipase A₂ activity. This effect may be mimicked by an increase in the matrix volume or reversed by calcium-dependent mitochondrial aging.     

An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.     

Control of hepatic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase during the foetal/neonatal transition, suckling and weaning in the rat.

(1) We assayed active and total (i.e. active plus succinylated) 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase in mitochondria isolated from foetal, neonatal, suckling or weaned rats. (2) HMG-CoA synthase was substantially succinylated and inactivated in mitochondria isolated from term-foetal, (1-h-old, 6-h-old, 1-day-old) neonatal, suckling and high carbohydrate/low-fat (hc)-weaned rats. Succinylation of HMG-CoA synthase was very low in mitochondria isolated from the livers of foetal, 30-min-old neonatal and high-fat/carbohydrate-free (hf)-weaned rats. (3) There was a negative correlation between active HMG-CoA synthase and succinyl-CoA content in mitochondria isolated from term-foetal, suckling and hc-weaned rats. (4) Differences in active enzyme could not be entirely accounted for by differences in succinylation and inactivation of the synthase. Immunoassay confirmed that the absolute amounts of mitochondrial HMG-CoA synthase increased during the foetal/neonatal transition and decreased with hc weaning. The levels remained elevated with hf weaning. (5) From these data we propose that mitochondrial HMG-CoA synthase is controlled by two different mechanisms in young rats. Regulation by succinylation provides a mechanism for rapid modification of existing enzyme in response to changing metabolic states. Changes in the absolute amounts of HMG-CoA synthase provide a more long-term control in response to nutritional changes.     

Benomyl Tolerance of Ten Fungi Antagonistic to Plant-parasitic Nematodes

Ten strains of fungi were tested for tolerance to the fungicide benomyl. Verticillium chlamydosporium strain 2 did not grow in the presence of benomyl; Drechraeria coniospora strains 1 and 2 and Chaetomium sp. tolerated only 0.1 μg benomyl/ml medium; Acremonium bacillisporum, an unidentified fungus, and Phoma chrysanthemicola uniformly grew at 1 μg/ml, but some hyphae grew at higher benomyl concentrations; Fusarium sp. tolerated 475 μg/ml, but some hyphae grew on medium amended with 1,000 μg/ml; Verticillium lecanii and V. chlamydosporium strain 1 routinely tolerated 1,000 μg/ml. Fungi generally grew more slowly at higher than at lower benomyl concentrations. Strains with elevated tolerance to benomyl were selected from Acremonium bacillisporum, Drechmeria coniospora, Fusarium sp., and an unidentified fungus. These strains retained the increased tolerance after repeated transfers on unamended medium.     

Composition of Orchid Scents Attracting Euglossine Bees

Fragrance composition of flowers from 101 plant species, especially orchids, were analyzed. Several compounds, including allo-aromadendrene, β-bourbonene, α-copaene, α-cubebene, 1,2-dimethoxybenzene, 1,3,5-trimethoxybenzene, epoxygeranyl acetate, 7,11-epoxymegastigma-5(6)-en-9-one, two γ-lactones, germacradienol, germacrene D, humulene, methoxy-phenyl-ethyl acetate, myrcene epoxide, sabinene, styrene, and undecatriene were detected for the first time in orchids. Fragrance composition of flowers pollinated by male euglossine bee species (perfume flowers) of four plant families are compared and contrasted with those of orchid species with other pollination systems. Melittophilous, but highly specialized orchids, produce fragrances rich in different sesquiterpenes and other rare compounds. In the species that are exclusively pollinated by fragrance-collecting male euglossine bees, the fragrances are primary attractants that serve both as an attractant and as a reward. The unusually intensely smelling flowers mostly produce esters and monoterpenes. The fragrances of euglossophilous flowers of the three plant families investigated are composed of nearly the same sets of chemical compounds, suggesting convergent evolution. Typically, euglossophilous plant species produce large amounts of few fragrant substances while melittophilous species often produce rare compounds in small amounts.     

Evidence for Glucocorticoid Target Cells in the Rat Optic Nerve. Hormone Binding and Glycerolphosphate Dehydrogenase Induction

Abstract: Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol-3-phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid-inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. Correlative immunocytochemical studies found GPDH-positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high-affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately twofold in degenerating nerves. Finally, in vivo accumulation of [³H]DEX and [³H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo , a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.