The significance of cAMP induced alterations in the cellular structure of Phycomyces.

Effect of cyclic AMP (cAMP) on Phycomyces blakesleeanus was studied by growing sporangiospores on glucose-asparagine agar or liquid medium containing three different levels of cAMP (10, 20 and 40 micronM) in addition to the control (no cAMP added). The response of Phycomyces to the exogenous cAMP concentration in the medium is as follows: (1) the time required for germ tube emergence is reduced; (2) the diameter of the mycelium is increased (sometimes more than 10 times) and frequency of branching is also increased; (3) the cell wall of the mycelium is thickened (in some cases more than 5 times); (4) the glycogen in the cytoplasm is decreased as visualized in thin sections and also demonstrated in biochemical quantitation; and (5) the distribution of intercalated membranous particles (Imp) on plasma membrane is altered and this can be easily detected in freeze-fractured replica. Such a change in Imp is seen in the formation of small clusters of aggregated particles on the plasmic half (PF) and craters on the complementary exoplasmic half (EF) of the plasma membrane. Although the mechanism of cAMP action requires further exploration, it is possible that the addition of cAMP to the culture medium leads to degradation of glycogen and enhancement of chitin synthesis since the cell wall is largely composed of chitin. The alteration in Imp may be related to a change in the activity of chitin synthetase which is a plasma membrane-bound enzyme.     

Effect of substrates and coenzymes on the molecular symmetry-related properties of horse liver and yeast alcohol dehydrogenases

Thermal inactivation of horse liver alcohol dehydrogenase (LADH) exhibits the following biphasic kinetics A = Afast.e-Kfast.t + Aslow.e-Kslow.t Where A is the per cent residual activity at time t,Afast and Aslow are amplitudes (expressed as % of initial activity) and kfast and kslow first-order rate constants of the fast and slow phases, respectively. For apoenzyme, Afast = Aslow = 50% of the initial activity under all conditions of temperature and pH. On the addition of a substrate or coenzyme ligand, there is a ligand concentration-dependent increase in per cent Aslow and a decrease in kslow. At sufficiently high ligand concentration, the entire time-course of inactivation can be described as a single exponential decay. The variations of per cent Aslow and of kslow with ligand concentration are consistent with the existence of two binding sites of different ligand affinities. Inactivation of LADH by excess EDTA also exhibits a similar biphasic kinetics with Afast = Aslow = 50% of the initial activity. Addition of ethanol or NAD+ brings about a concentration-dependent decrease in kfast and kslow without affecting amplitudes of the two phases. The NAD+ concentration-dependence of this decrease is consistent with a single dissociation constant for the coenzyme. Inactivation of yeast alcohol dehydrogenase by heat or excess EDTA can be represented as a single exponential decay of activity under all conditions of temperature and pH in the absence as well as presence of ethanol or NAD+. Implications of these results for the molecular symmetry of the two oligomeric enzymes in solution are discussed.     

Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores

The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 micrograms and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 micrograms. Despite such a large secretory response, the catecholamine content of the K+-stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with alpha-methyl-p-tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, L-3,4-dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal medulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by alpha-methyl-p-tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.     

Effect of radiation on some haematological parameters and its modification by vitamin E in chicks.

White leghorn male chicks of 1 and 7 day age groups were studied for acute (2.25 Gy) gamma radiation (with or without vit. E pretreatment) induced haematological changes in the peripheral blood at days 1, 3, 5, 7, 14 and 28 postirradiation. A continuous decrease in the erythrocyte numbers was observed in the animals irradiated without vit. E treatment. The changes in haematocrit, haemoglobin, MCV, MCH and MCHC values were in line with the erythrocytic changes reflecting radiation induced damage to the erythroid elements. Animals pretreated with vit. E show lesser depression in the erythrocytic component at all the stages indicating its radio-protective influence. The significant increase in the immature RBC's in the peripheral blood in vit. E treated animals after irradiation, implies enhanced erythropoiesis.     

Ligational behavior of N-substituted acid hydrazides towards transition metals and potentiation of their microbiocidal activity.

New complexes of Cu(II), Ni(II), and Co(II) with 3-benzoyl-1-[2-N-(substituted-2'-thienyl methylmethylene/methylene)] prop-2-ene-1-oic acid hydrazides have been synthesized and characterized by elemental analysis, molecular weight determination, molar conductance, and magnetic moment and spectroscopic techniques. Conductance measurements indicate the nonionic nature of the complexes. From the spectroscopic studies, it has been concluded that the N-substituted acid hydrazides act as tridentate ligands forming an O-N-S conjugate system and coordinating with metal ions through oxygen of carbonyl group, nitrogen of azomethine, and sulphur of thiophene moiety. Octahedral geometry has been proposed for all the complexes. The ligands and their complexes were tested for in vitro growth inhibitory activity against phytopathogenic fungi viz. Alternaria alternata, Colletotrichum capsicum, Fusarium oxysporum, and Rhizoctonia solani at 28 degrees C; and bacteria viz. gram positive Bacillus subtilis and gram negative Escherichia coli at 37 degrees C by a two-fold serial dilution technique. In some cases an increase in the biocidal activity of the ligands as a consequence of coordination with metal ions was observed in terms of minimum inhibitory concentration (MIC) values. The trend of growth inhibition in the complexes was found to be in the order: Cu greater than Ni greater than Co.     

Identification of new allosteric sites and modulators of AChE through computational and experimental tools

Allosteric sites on proteins are targeted for designing more selective inhibitors of enzyme activity and to discover new functions. Acetylcholinesterase (AChE), which is most widely known for the hydrolysis of the neurotransmitter acetylcholine, has a peripheral allosteric subsite responsible for amyloidosis in Alzheimer’s disease through interaction with amyloid β-peptide. However, AChE plays other non-hydrolytic functions. Here, we identify and characterise using computational tools two new allosteric sites in AChE, which have allowed us to identify allosteric inhibitors by virtual screening guided by structure-based and fragment hotspot strategies. The identified compounds were also screened for in vitro inhibition of AChE and three were observed to be active. Further experimental (kinetic) and computational (molecular dynamics) studies have been performed to verify the allosteric activity. These new compounds may be valuable pharmacological tools in the study of non-cholinergic functions of AChE.     

Therapeutic potential of meso 2,3-dimercaptosuccinic acid or 2,3-dimercaptopropane 1-sulfonate in chronic arsenic intoxication in rats

The therapeutic efficacy of two thiol chelators, meso 2,3-dimercaptosuccinic acid (DMSA) or 2,3-dimercaptopropane sulfonate (DMPS) in treating chronic arsenic intoxication was investigated in male rats. Both the chelators were effective in promoting urinary arsenic excretion and restoring arsenic induced inhibition of blood -aminolevulinic acid dehydratase activity and hepatic glutathione level. Elevation of urinary -aminolevulinic acid excretion and arsenic concentration in blood, liver and kidneys were reduced significantly by both the chelators. Histopathological lesions induced by arsenic were also effectively reduced by the above chelators. DMSA being more effective than DMPS. The results suggest DMSA and DMPS to be effective antidotes for treating chronic arsenic toxicity in experimental animals.     

Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy.For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats. (Mol Cell Biochem151: 165–172, 1995)