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.     

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.     

Turnover of endogenous, microinjected, and sequestered protein in Xenopus oocytes

Pulse-labeled oocyte proteins were found to have a maximum average half-life of 73 h. In general, larger peptides underwent degradation at a faster rate than smaller peptides. In this respect, oocytes are similar to most other cells. Microinjected ¹²⁵I-labeled bovine serum albumin (BSA) was degraded over a 40 h period with a half-life of 20–30 h, regardless of the method of protein labeling, culture medium employed, size of oocyte microinjected, or hormonal history of the oocyte. The last two results, if applicable to oocyte proteins in general, imply that protein catabolism is constant throughout the later stages of oogenesis and that growth is primarily regulated by a stimulation of anabolism. Individual proteins microinjected into oocytes undergo rates of degradation consistent with turnover rates obtained in other systems. Sequestered ¹²⁵I-labeled BSA is only partially (40%) degraded, which indicates that, unlike microinjected ¹²⁵I-labeled BSA, it has access to a cytoplasmic compartment (yolk platelets?) within which it is relatively stable.     

CO2 Saturation and Trophic Shift Induced by Microbial Metabolic Processes in a River-Dominated Ocean Margin (Tropical Shallow Lagoon,Chilika, India)

An effort has been made for the first time in Asia's largest brackish water lagoon, Chilika, to investigate the spatio-temporal variability in primary productivity (PP), bacterial productivity (BP), bacterial abundance (BA), bacterial respiration (BR) and bacterial growth efficiency (BGE) in relation to partial pressure of CO₂ (pCO₂) and CO₂ air–water flux and the resultant trophic switchover. Annually, PP ranged between 24 and 376 µg C L^?1 d^?1 with significantly low values throughout the monsoon (MN), caused by light limitation due to inputs of riverine suspended matter. On the contrary, BP and BR ranged from 11.5 to 186.3 µg C L^?1 d^?1 and from 14.1 to 389.4 µg C L^?1 d^?1, respectively, with exceptionally higher values during MN. A wide spatial and temporal variation in the lagoon trophic status was apparent from BP/PP (0.05–6.4) and PP/BR (0.10–18.2) ratios. The seasonal shift in net pelagic production from autotrophy to heterotrophy due to terrestrial organic matter inputs via rivers, enhanced the bacterial metabolism during the MN, as evident from the high pCO₂ (10,134 µatm) and CO₂ air–water flux (714 mm m^?2 d^?1). Large variability in BGE and BP/PP ratios especially during MN led to high bacteria-mediated carbon fluxes which was evident from significantly high bacterial carbon demand (BCD >100% of PP) during this season. This suggested that the net amount of organic carbon (either dissolved or particulate form) synthesized by primary producers in the lagoon was not sufficient to satisfy the bacterial carbon requirements. Lagoon sustained low to moderate autotrophic–heterotrophic coupling with annual mean BCD of 231% relative to the primary production, which depicted that bacterioplankton are the mainstay of the lagoon biogeochemical cycles and principal players that bring changes in trophic status. Study disclosed that the high CO₂ supersaturation and oxygen undersaturation during MN was attributed to the increased heterotrophic respiration (in excess of PP) fuelled by allochthonous organic matter. On a spatial scale, lagoon sectors such as south sector, central sector and outer channel recorded “net autotrophic,” while the northern sector showed “net heterotrophic” throughout the study period.     

Heterocyclic ketones as inhibitors of histone deacetylase

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.