Sex-specific metabolic changes in the annual reproductive cycle of a freshwater catfish

Sex-specific enzymatic and other biochemical changes were studied in the annual reproductive cycle of the freshwater catfish, Clarias batrachus. Citrate synthase (CS) activity of brain, liver and skeletal muscle was maximum in spawning and minimum in postspawning showing a sharp decline in aerobic capacity after spawning. Similar CS activity in remaining phases of the annual reproductive cycle reflects similar energy need during regressed, preparatory and prespawning phases. Glucose 6-phosphate dehydrogenase (G6-PDH) activity declined in spawning and postspawning indicating a possible decrease in lipid and nucleic acid syntheses. The subsequent increase in G6-PDH activity with onset of resting and maintenance of the increased level throughout preparatory and prespawning phases shows restoration of biosynthetic activity. Higher activity of G6-PDH in female than male may be to satisfy a greater biosynthetic need of female reproduction and breeding. The decreased RNA content of tissues showed reduction in protein synthesis capacity during spawning and subsequent increase through postspawning until resting phase. The RNA content of brain and liver was higher in female than male during preparatory and prespawning, which may be associated with higher protein synthesis requirement of female for preparation of reproductive activities. The requirement based sex related changes in metabolism of catfish may be enzyme, tissue or reproductive phase-specific.     

Amino acid content in several brain regions of the active and hibernating frog, Rana esculenta

1. Total free amino acid contents in the optic lobe and diencephalon increased significantly during hibernation. 2. Free glutamate + glutamine showed significant increases in the cerebral hemisphere, optic lobe, medulla oblongata and diencephalon. 3. Free aspartate + asparagine showed significant increases in the cerebral hemisphere, optic lobe, diencephalon and olfactory lobe. 4. GABA showed a significant change only in the medulla oblongata. 5. Total protein amino acid level in the cerebellum and olfactory lobe decreased significantly during hibernation and most of the amino acids decreased significantly in these regions. 6. The amino acid metabolism during amphibian hibernation differs from that of the mammal.     

Variations in the uptake of 3H-dopamine during the estrous cycle

The in vitro uptake of ³H-Dopamine (³H-DA) into nuclei-free homogenates from brain parts was studied during each phase of the rat estrous cycle. Across four time points (12:00, 2:00, 4:00 and 6:00 p.m.) each phase exhibits a unique pattern of uptake in the diencephalon. Differences in the level of uptake between phases are most marked at 2:00 p.m. with diestrus animals displaying the highest rate of uptake. Metestrus animals show the lowest rate of uptake (less than 50% of the diestrus rate), while estrus and proestrus show intermediate rates. At 12:00, 2:00 and 4:00 p.m. during proestrus, the average rate of uptake is significantly less than the average uptake rate of any other phase. At 6:00 p.m. proestrus, the rate of uptake is significantly elevated over the values found at the previous three time points. Kinetic analysis of the diencephalic uptake system reveals hourly changes in the kinetic properties of the uptake system (Km and Vmax) during all phases. In non-diencephalic regions (“brain minus diencephalon”), few variations in uptake are observed. The variations in the rate of uptake in the diencephalon are thought to reflect changes in the functional activity of the DA neurons. Thus, during proestrus the reduction of ³H-DA uptake at 12:00, 2:00 and 4:00 p.m. suggests an increase in DA receptor activation.     

Scaling effects on metabolism of a teleost

Scaling effects on citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), RNA. RNA/DNA ratio and protein contents of brain, liver and skeletal muscle were studied in a teleost, Clarias batrachus. The activity of white skeletal muscle CS decreased significantly as a function of increasing body mass of the fish. It shows that the fulfilment of energy demand in white skeletal muscle is not dependent on aerobic metabolism. The activity of liver G6-PDH decreased with the increasing body mass showing reduction in NADPH generation for lipogenic activity. However, increase in G6-PDH activity showed enhancement in reductive synthesis in skeletal muscle of the larger-sized individuals. A positive scaling of RNA, RNA/DNA ratio and protein contents reflects changes in macromolecular turnover for ATP-supplying enzymes and proteins.     

The influence of hibernation patterns on the critical enzymes of lipogenesis and lipolysis in prairie dogs

White-tailed prairie dogs (Cynomys leucurus) are spontaneous hibernators that enter torpor each fall, whereas black-tailed prairie dogs (C. ludovicianus) hibernate facultatively only when food- or water-stressed during the winter. The body masses of both species greatly increase during the fall feeding period, with most of this gain in the form of depot fat. Body fat is utilized during winter fasting and/or hibernation. We measured the activities of fatty acid synthase (FAS), ATP-citrate lyase (ACL), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), and hormone-sensitive lipase (HSL) in the tissues of both C.leucurus (hibernating and euthermic) and C. ludovicianus (euthermic only) under controlled conditions. The activities of FAS, ACL, and G6PDH in the liver all decreased during hibernation. The activities of ME and G6PDH in white adipose tissue (WAT) were also reduced during hibernation. Euthermic C. leucurus and euthermic C. ludovicianus differed only in brown adipose (BAT) ACL and WAT G6PDH activities. No significant differences in HSL activities were found between these two species or between euthermic and hibernating animals. These results suggest that this seasonal body fat cycle is due, at least in part, to seasonal variations in the activities of FAS, ME, ACL, and G6PDH that affect the rate of fatty acid synthesis. This study also demonstrates that spontaneous hibernators do not have a greater capacity to synthesize fatty acids during the fall than facultative hibernators, as previously suggested.     

Cytochemical assay of glucose-6-phosphate dehydrogenase in koilocytes

New cervical smears were obtained from 24 patients with a cytologic diagnosis of typical condyloma for a cytochemical assay of glucose-6-phosphate dehydrogenase (G6PDH) activity in the koilocytes that are pathognomonic of this lesion. The smears were air dried and were processed according to Nachlas' modified technique. The controls used were smears from normal cases (which show no G6PDH activity), from dysplasias (which show high levels) and from carcinomas (which show very high G6PDH levels). In the cases of typical condyloma studied, the level of G6PDH was null in 16 (66.7%), very low in 2 (8.3%) and low in 6 (25.0%). If this assay for G6PDH gives the total enzymatic activity of the cell, showing low enzymatic levels in condylomas and high enzymatic levels in dysplasias and carcinomas, an increase in G6PDH activity could indicate the transition of an intraepithelial lesion from condyloma to cervical intraepithelial neoplasia.     

Seasonal variations in the renal cortical (Na+ + K+)-ATPase and Mg2+-ATPase of a hibernator, the ground squirrel (Spermophilus richardsonii).

1. The specific activity of renal cortical (Na+ + K+)-ATPase of the Richardson ground squirrel is markedly reduced during hibernation, in contrast to the specific activity of the accompanying Mg2+-ATPase which is markedly increased. 2. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is unchanged by hibernation. 3. Both the non-linear thermal dependence of (Na+ + K+)-ATPase and the linear thermal dependence of Mg2+-ATPase are also unchanged by hibernation. 4. The energy of activation of both enzymes is unchanged during hibernation, or by comparison with that determined in awake controls. 5. There is no evidence for inherent "cold resistance" in these enzyme preparations compared to similar preparations from the non-hibernating rabbit. This parameter does not change during hibernation. 6. Both the rate and amount of specific [3H]-ouabain binding to the renal cortical preparations of (Na+ + K+)-ATPase decrease during hibernation. This decrease matches the fall in enzyme activity so that the ratio of pumping sites/unit of enzyme activity shows no seasonal variations. 7. These findings suggest that the amount of renal cortical (Na+ + K+)-ATPase enzyme falls during hibernation, but that the enzyme which remains functions with the same thermodynamic efficiency and identical biochemical characteristics of that found in the awake summer controls.     

Variations in the levels of cyclic adenosine 3':5'-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3':5'-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn²⁺ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K_m (1 μm) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn²⁺ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.     

Activités de quelques enzymes associés à la conidiogenèse du Neurospora crassa

Summary Enzyme activities have been measured and compared at several stages of the development of Neurospora crassa i.e. from free conidia (inoculum) to conidiated mycelia grown on sucrose versus acetate (poor versus highly conidiogenous) media.Glucose-6-phosphate dehydrogenase (G-6-PDH) and NADP nucleotidase (NADPase) show inverse activity-time curves, both on sucrose and acetate media. G-6-PDH has its higher activity at the preconidiating stage while NADPase progressively increases to reach its maximal value in the mature conidia.Malate dehydrogenase (MDH) has a higher activity in extracts from acetate compared to sucrose cultures; in both conditions, maximal MDH activity corresponds with the initiation of conidiation. Malate synthetase (MS) has a delayed activity which is much higher in acetate extracts.Succinate dehydrogenase (SDH) is more active in sucrose than acetate extract with a sharp activity peak just preceding conidiation proper.Isozymes of G-6-PDH and MDH, as well as total soluble proteins from extracts of sucrose versus acetate cultures have been compared after their separation on polyacrylamide columns.     

Starvation-induced impairment of metabolism in a freshwater catfish

Starvation induced changes in citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), DNA, RNA, RNA/DNA ratio and protein were studied in the freshwater catfish Clarias batrachus. Starvation gradually decreased the activity of CS, G6-PDH and LDH in brain, liver and skeletal muscle of the freshwater catfish. The maximum reduction in these enzyme activities upto 35-45% was observed after 35 days of fasting. This shows substantial decline in aerobic and biosynthetic capacity during starvation period. DNA, RNA, RNA/DNA ratio and protein contents were also reduced from 40-67% which reflects reduction in an overall capacity of the protein synthesis. Starvation-induced macromolecular changes indicate impairment of metabolism in fish.     

Enzyme histochemical studies in testes and seminal vesicles of the Pied flycatcher (Ficedula hypoleuca) during the breeding season

Enzymatic conditions in testes and seminal vesicles of the Pied flycatcher during the breeding season were studied by histochemical techniques. The following enzyme activities were studied: glucose-6-phosphatase dehydrogenase (G-6-PDH), NADPH₂-diaphorase, glutamic dehydrogenase (GDH), NADH₂-diaphorase, adenosine triphosphatase (ATPase) and acid phosphatase. Phospholipids and PAS-positive substances were also studied. Towards the end of the nest-building period, a time when the males showed maximal sexual activities, some enzyme activity changes were observed. There was an increase of the GDH activity in the sperm bundles, an increase of the ATPase activity in the interstitium and tunica propria of the tubuli seminiferi. The phospholipid content increased in all spermatogenetic cells and the PAS-positive material increased in the Sertoli cells and spermatozoa. The enzymic activities indicated a steroid production in the Leydig cells.
The epithelium of the seminal vesicles did not show any variations in enzyme activities as long as the Leydig cells persisted. Most enzymes showed a very high activity during this time, indicating a secretory function of the epithelium. The amount of phospholipids increased at the end of the nest-building period. Parallel with the degeneration of the epithelium the enzymic activities, as well as the phospholipid content, were lowered markedly.     

Adjustment of metabolite composition in the haemolymph to seasonal variations in the land snail <Emphasis Type="Italic">Helix pomatia</Emphasis>

In temperate regions, land snails are subjected to subzero temperatures in winter and hot temperatures often associated to drought in summer. The response to these environmental factors is usually a state of inactivity, hibernation and aestivation, respectively, in a temperature and humidity buffered refuge, accompanied by physiological adjustments to resist cold or heat stress. We investigated how environmental factors in the microhabitat and body condition influence the metabolite composition of haemolymph of the endangered species Helix pomatia. We used UPLC and GC–MS techniques and analyzed annual biochemical variations in a multivariate model. Hibernation and activity months differed in metabolite composition. Snails used photoperiod as cue for seasonal climatic variations to initiate a physiological state and were also highly sensitive to temperature variations, therefore constantly adjusting their physiological processes. Galactose levels gave evidence for the persistence of metabolic activity with energy expenditure during hibernation and for high reproductive activity in June. Triglycerides accumulated prior to hibernation might act as cryoprotectants or energy reserves. During the last month of hibernation snails activated physiological processes related to arousal. During activity, protein metabolism was reflected by high amino acid level. An exceptional aestivation period was observed in April giving evidence for heat stress responses, like the protection of cells from dehydration by polyols and saccharides, the membrane stabilization by cholesterol and enhanced metabolism using the anaerobic succinic acid pathway to sustain costly stress responses. In conclusion, physiological adjustments to environmental variations in Helix pomatia involve water loss regulation, cryoprotectant or heatprotectant accumulation.     

Changes in activity of the regulatory glycolytic enzymes and of the pyruvate-dehydrogenase complex during the development of Xenopus laevis

In this study we investigated the variations of the maximal activities of the rate-controlling glycolytic enzymes (i.e., hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK) and of the pyruvate-dehydrogenase complex (PDHc) during the early embryogenesis of Xenopus laevis (from cleavage through hatching). All the enzymatic assays, using different coupled reactions, were performed spectrophotometrically on cytosolic and mitochondrial fractions. The maximal HK activity increases markedly from neurulation onwards, PFK activity presents a peak around gastrulation, PK activity remains relatively constant throughout the period studied and the highest PDHc activity is observed during cleavage. The specific activities display the same temporal pattern. Furthermore, in the sequence of reactions by which glucose is degraded to form acetyl-CoA, the maximal activities of PFK and PK are not limiting while those of HK and PDHc could be rate-limiting at relatively late developmental stages (hatching).     

Neuroanatomical Distribution and Variations across the Reproductive Cycle of Aromatase Activity and Aromatase-Immunoreactive Cells in the Pied Flycatcher (Ficedula hypoleuca)

The anatomical distribution and seasonal variations in aromatase activity and in the number of aromatase-immunoreactive cells were studied in the brain of free-living male pied flycatchers (Ficedula hypoleuca). A high aromatase activity was detected in the telencephalon and diencephalon but low to negligible levels were present in the optic lobes, cerebellum, and brain stem. In the diencephalon, most aromatase-immunoreactive cells were confined to three nuclei implicated in the control of reproductive behaviors: the medial preoptic nucleus, the nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. In the telencephalon, the immunopositive cells were clustered in the medial part of the neostriatum and in the hippocampus as previously described in another songbird species, the zebra finch. No immunoreactive cells could be observed in the song control nuclei. A marked drop in aromatase activity was detected in the anterior and posterior diencephalon in the early summer when the behavior of the birds had switched from defending a territory to helping the female in feeding the nestlings. This enzymatic change is presumably controlled by the drop in plasma testosterone levels observed at that stage of the reproductive cycle. No change in enzyme activity, however, was seen at that time in other brain areas. The number of aromatase-immunoreactive cells also decreased at that time in the caudal part of the medial preoptic nucleus but not in the ventromedial nucleus of the hypothalamus (an increase was even observed), suggesting that differential mechanisms control the enzyme concentration and enzyme activity in the hypothalamus. Taken together, these data suggest that changes in diencephalic aromatase activity contribute to the control of seasonal variations in reproductive behavior of male pied flycatchers but the role of the telencephalic aromatase in the control of behavior remains unclear at present.     

Effect of X irradiation on adenosine triphosphate and glucose-6-phosphate dehydrogenase in the CaNT mouse tumor

The levels of adenosine triphosphate (ATP) in the transplantable CaNT murine tumor grown in CBA mice at various times following 5, 10, and 15 Gy X rays (100 kVp) were increased within 45 min. Maximal ATP levels occurred at 2.5 h following the 10 Gy dose (3.8 times that of unirradiated controls), returning almost to control levels by 13 h after irradiation. The specific activity of glucose-6-phosphate dehydrogenase (G-6-PDH) after 10 Gy increased about 1.5-fold 1 h after irradiation, returning to control levels by 48 h. It is suggested that the increased ATP following irradiation might play a major role in energy provision when cellular repair processes are able to operate. The increased G-6-PDH activity after irradiation may reflect enhanced metabolism associated with cellular repair mechanisms.     

Histochemistry of oxidative enzymes in the neuroglia in course of myelination.

The histoenzymic pattern of oxidative enzymes (G-6-PDH, G-PDH, ICDH, SDH, HBDH, NADH-2:tetrazolium dehydrogenase) was investigated in the developing neuroglia of rabbit brains, with special regard to the period of myelinogenesis. The obtained results lead to following conclusions: (1) During the early period of postnatal development there is maximal oxidative enzyme activity in ependymal cells, somewhat less reactive are the undifferentiated matrix cells and the differentiating cells of the mantle layer. No distinction can be made between the response of spongio- and neuroblasts. (2) Distinctly increased oxidoreductase activity, as compared to the early period of postnatal development, is demonstrated by the differentiating cells of myelination gliosis, no prevalence being demonstrable for enzymes of the particular metabolic pathways (pentose shunt, glycolysis or Krebs cycle). (3) G-6-PDH, G-PDH and oxidoreductases acting within the citric acid cycle are demonstrable only in single cells of the interfascicular oligodendroglia of adult rabbit brains, while almost all cells exhibit appreciable activity of HBDH and NADH-2 tetrazolium dehydrogenase.     

Ontogeny of the Murine Glucose-6-Phosphatase System

A deficiency in microsomal glucose-6-phosphatase (G6Pase) activity causes glycogen storage disease type 1 (GSD-1), a clinically and biochemically heterogeneous group of diseases. It has been suggested that catalysis by G6Pase involves multiple components, with defects in the G6Pase catalytic unit causing GSD-1a and defects in the putative substrate and product translocases causing GSD-1b, 1c, and 1d. However, this model is open to debate. To elucidate the G6Pase system, we have examinedG6PasemRNA expression, G6Pase activity, and glucose 6-phosphate (G6P) transport activity in the murine liver and kidney during normal development. In the liver,G6PasemRNA and enzymatic activity were detected at 18 days gestation and increased markedly at parturition, before leveling off to adult levels. In the kidney,G6PasemRNA and enzymatic activity appeared at 19 days gestation and peaked at weaning, suggesting that kidney G6Pase may have a different metabolic role.In situhybridization analysis demonstrated that, in addition to the liver and kidney, the intestine expressedG6Pase.Despite the expression ofG6Pasein the embryonic liver, microsomal G6P transport activity was not detectable until birth, peaking at about age 4 weeks. Our study strongly supports the multicomponent model for the G6Pase system.     

Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots

The pivotal role of glucose-6-phosphate dehydrogenase (G-6-PDH)-mediated nitric oxide (NO) production in the tolerance to oxidative stress induced by 100 mM NaCl in red kidney bean (Phaseolus vulgaris) roots was investigated. The results show that the G-6-PDH activity was enhanced rapidly in the presence of NaCl and reached a maximum at 100 mM. Western blot analysis indicated that the increase of G-6-PDH activity in the red kidney bean roots under 100 mM NaCl was mainly due to the increased content of the G-6-PDH protein. NO production and nitrate reductase (NR) activity were also induced by 100 mM NaCl. The NO production was reduced by NaN(3) (an NR inhibitor), but not affected by N(omega)-nitro-L-arginine (L-NNA) (an NOS inhibitor). Application of 2.5 mM Na(3)PO(4), an inhibitor of G-6-PDH, blocked the increase of G-6-PDH and NR activity, as well as NO production in red kidney bean roots under 100 mM NaCl. The activities of antioxidant enzymes in red kidney bean roots increased in the presence of 100 mM NaCl or sodium nitroprusside (SNP), an NO donor. The increased activities of all antioxidant enzymes tested at 100 mM NaCl were completely inhibited by 2.5 mM Na(3)PO(4). Based on these results, we conclude that G-6-PDH plays a pivotal role in NR-dependent NO production, and in establishing tolerance of red kidney bean roots to salt stress.     

Protein oxidation and proteolysis during aging and oxidative stress

Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.     

Enzymatic activity in the lung cells to the intrapulmonary administration of antigen

Histochemical study of the activity of the redox and hydrolytic enzymes in the immunocompetent cells of the lungs of guinea pigs following intratracheal immunization with complete typhoid antigen demonstrated that following a single immunization at the initial periods elevation of the activity of AP, LDH, NAD- and NADP-diaphorases was seen in the macrophages; later GDH, G-6-PDH and AP was activated in the plasma cells. Double immunization was followed by an earlier and more intensive increase of the enzymatic activity in the cells under study.