Ligational behavior of a novel biologically active donor toward Cu(II) and Ni(II) ions and potentiation of its antibacterial activity by chelation with metal ions

The chelating behavior of a new multidentate ligand with tuberculostatic activity toward Cu(II) and Ni(II) ions has been studied. This ligand 3-(2-carboxyhydrazine)phenylimino-2-oximobutane(H2C POB) is found to chelate the above metal ions in both its keto and enol forms. The probable structures of all the complexes and the location of the bonding sites have been established through magnetic and spectroscopic (infrared, electronic) studies. The Cu(II) complex of the enol form exhibits subnormal magnetic moment at room temperature, indicating the probable existence of some sort of super exchange phenomenon in the system. The ligand itself and a few of its Cu(II) complexes have been found to exert powerful in vitro antibacterial activity toward some tuberculosis mycobacteria, such as Mycobacterium flae, Mycobacterium smegmatis, and Mycobacterium H37Rv.     

Induction of high-density-lipoprotein receptors in rat corpus luteum by human choriogonadotropin. Evidence of protein synthesis de novo.

The present studies investigated the specific binding of 125I-labelled high-density lipoprotein (125I-HDL) to plasma membranes. Golgi, rough endoplasmic reticulum and mitochondria/lysosomes, prepared from ovaries of rats injected with human choriogonadotropin (hCG) or 0.9% NaCl. Treatment in vivo with hCG resulted in 2-3-fold induction of 125I-HDL binding activity in all the subcellular organelles. The specific binding of HDL to various subcellular organelles was dependent on the amount of protein, lipoprotein concentration and incubation time. Equilibrium-binding studies revealed comparable Kd values (13-22 micrograms of HDL protein/ml) for HDL binding in all the subcellular organelles tested. Treatment with cycloheximide (2.0 mg/kg body wt.) before hCG administration abolished the induction of HDL receptors, suggesting the involvement of a protein-synthesis-dependent process in receptor induction. Analysis of equilibrium dissociation constants (Kd) for 125I-HDL binding in membranes from hCG-, cycloheximide-and saline-treated animals suggests that the increase in binding was due to an increase in the number of binding sites rather than a change in the affinity. Additionally, pretreatment with tunicamycin, an inhibitor of N-linked glycosylation, had no effect on hCG-mediated receptor induction, suggesting that glycosylation of the receptor may not be necessary for the interaction of HDL with its receptors.     

Phosphorylation of liver plasma membrane-bound calmodulin

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [gamma-32P]ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3':5'AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.     

Influence of age and splanchnic nerve on the action of melatonin in the adrenomedullary catecholamine content and blood glucose level in the avian group

Summary A single intraperitoneal (IP) melatonin injection (0.5 mg/100 g body wt.) caused an increase in norepinephrine (NE) fluorescence and elevation of NE content in newly-hatched pigeons (Columba livia), but a reduction of NE fluorescence and depletion of NE content in the adrenal medulla of newly-hatched crows (Corvus splendens) after 0.5 h of treatment. In contrast, in adults melatonin caused increase in NE fluorescence and elevation of NE content only in the parakeet (Psittacula krameri).Half an hour of IP melatonin treatment (0.5 mg/100 g body wt.) induced release of epinephrine (E) from the adrenal medulla of newly-hatched pigeon and parakeet. In contrast, in the adults melatonin caused more than a two-fold increase in E in the pigeon, and a significant increase in the crow.Single IP melatonin injection (0.5 mg/100 g body wt.) caused hypoglycemia in the newly-hatched parakeet and adult pigeon, and hyperglycemia in newly-hatched pigeon after 0.5 h of treatment. Melatonin failed to regulate glucose homoeostasis in newly-hatched and adult crow.Splanchnic denervation of the left adrenal gland was performed in the adult pigeon. The right adrenal served as the innervated gland. Melatonin-induced modulation of catecholamines following a single IP injection (0.5 mg/100 g body wt.) revealed significant increases in NE fluorescence and NE content at 4 and 12 h after treatment in the denervated gland only, which gradually approached normal levels 9 days after treatment. In contrast, E content showed more than a two-fold increase over the control value in both the innervated and denervated glands 0.5 and 24 h after treatment. At 9 days after treatment, E content showed significant depletion in the innervated gland.The results of this study indicate that melatonin modulates catechol hormone content in avian adrenal medulla, and also regulates glucose homoeostasis (except in the crow). The splanchnic nerve plays a vital role in the synthesis of NE but has no effect on E.     

Optically detected magnetic resonance study of tyrosine residues in point-mutated bacteriophage T4 lysozyme

Two spectroscopically distinct types of tyrosine (Tyr) residues in triply point mutated bacteriophage T4 lysozyme, which contains no tryptophan (Trp), have been detected by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Their triplet states are characterized by similar E but different D values. The Tyr site which exhibits the lower D value and has the red-shifted phosphorescence origin is quenched by energy transfer to Trp and has D and E values comparable to previously studied Tyr residues. The blue-shifted Tyr site, which is not quenched by Trp, exhibits a larger D value that has been found previously. Calculation of energy-transfer efficiencies of Tyr-Trp pairs based on the crystal structure of the native enzyme provides a possible assignment of Tyr sites to the two different spectral types.     

Enhancement of cytotoxicity of modeccin by nigericin in modeccin-resistant mutant cell lines

We have isolated a Chinese hamster ovary cell mutant (DMPR-2) simultaneously resistant to diphtheria toxin and modeccin. In addition to the increased resistance to these two toxins used in the selection, this mutant is more resistant to Pseudomonas toxin and hypersensitive to ricin than the parental cell line. In contrast to the wild-type cells in which nigericin protects cells from modeccin, the cytotoxicity of modeccin in the DMPR-2 mutant is enhanced by nigericin. We have also studied the effects of nigericin and NH4Cl on the cytotoxicity of modeccin in a modeccin-resistant mutant of HeLa cells (ModRI). The cytotoxicity of modeccin is enhanced by nigericin in ModRI mutant cells, in contrast to the protection of modeccin cytotoxicity by nigericin in the parental HeLa cells. Our results suggest that modeccin can reach the cytosol of mammalian cells by two distinct routes; the major route requires endosomal acidification and the minor route is activated by nigericin.     

Effect of erythropoietin on the lipid composition of red blood cell membrane

Effect of Ep on [14C]acetate incorporation into different lipid fractions of RBC membranes in starved and phenylhydrazine-treated rats was studied. The incorporation was increased into both neutral and phospholipid fractions on Ep treatment to starved or phenylhydrazine-treated rats. A slight decrease in the ratio of neutral lipid to phospholipid was observed under the influence of Ep in starved rats (23%) or in phenylhydrazine-treated rats (36%). Incorporation of radioactivities into different phospholipid fractions of RBC membrane increased on Ep treatment to starved rats, whereas, the relative percentages of these phospholipids (except LPC) remained more or less unchanged under similar conditions. Phenylhydrazine treatment increased the relative percentage of PC and concomitantly decreased the percentage of Sph. Percentage composition of both these two phospholipids showed a tendency to return to their normal levels on administration of Ep to phenylhydrazine-treated rats. Ep decreased the sigma saturated/sigma unsaturated ratio of fatty acids in PE, PS, and PC of RBC membrane in starved rats. On the other hand, no significant change was observed in this ratio of fatty acids in the phospholipids except Sph of RBC membrane in the presence of phenylhydrazine and Ep. In Sph, the ratio went down under similar conditions.     

Lack of expression of RP4-specified beta-lactamase in Azospirillum brasilense

Plasmid RP4, which normally confers resistance to ampicillin (Apr), tetracycline (Tcr), and kanamycin (Kmr) to its hosts, failed to express enhanced Apr when transferred from Escherichia coli to Azospirillum brasilense which has its own intrinsic beta-lactamase. Even in a beta-lactamase-deficient mutant, A. brasilense RG-D16, no increase in beta-lactamase or significant Apr appeared following transfer of RP4. However, A. brasilense RG (RP4) and A. brasilense RG-D16 (RP4) did exhibit Tcr Kmr. When RP4 was transferred back from A. brasilense to E. coli all three drug resistances and beta-lactamase activity were fully expressed.     

Breeding by protoplast fusion for glucoamylase production

Summary Protoplast fusion was carried out between two strains ofAspergillus niger 8-2, a fast growing culture and poor producer of glucoamylase enzyme andA.niger 8-7, a slow growing culture and good producer of the enzyme. The nonconidiating fused mass in presence of benomyl, produced fast growing segregants showing various combinations of the two parental gene markers. Some of the segregants produced up to 68% more glucoamylase than the better yielding parent 8-7.