Replacement of the invariant lysine 77 by arginine in yeast iso-1-cytochrome c results in enhanced and normal activities in vitro and in vivo

Oligonucleotide-directed mutagenesis of the yeast Saccharomyces cerevisiae was used to generate an abnormal iso-1-cytochrome c having an Arg-77 replacement of the normal Lys-77; this Lys-77 residue is evolutionarily conserved in most eukaryotic cytochromes c and is trimethylated in fungal and plant cytochromes c. Examination of strains having a single chromosomal copy of the gene encoding the Arg-77 protein indicated that the altered protein was synthesized at the normal rate and that it had normal or near normal activity in vivo. Examination of enzymatic activities in vitro with cytochrome b2, cytochrome c peroxidase, and cytochrome c oxidase indicated that the altered iso-1-cytochrome c has equal or enhanced catalytic efficiencies. Thus, replacement of the evolutionarily conserved residue Lys-77 produces no or only minor effects both in vivo and in vitro.     

Generation of nitro radical anions of some 5-nitrofurans, 2- and 5-nitroimidazoles by norepinephrine, dopamine, and serotonin. A possible mechanism for neurotoxicity caused by nitroheterocyclic drugs

Catecholamine neurotransmitters such as norepinephrine, dopamine, and related catecholamine derivatives reduce nitroheterocyclic drugs such as nitrofurantoin, nifurtimox, nifuroxime, nitrofurazone, misonidazole, and metronidazole in slightly alkaline solutions. Drugs which contain 5-nitrofurans are reduced at lower pH than drugs which contain 2- and 5-nitroimidazoles. 5-Nitroimidazole derivatives such as metronidazole and ronidazole are known to be more difficult to reduce than 2-nitroimidazole derivatives, due to their lower redox potential. Catecholamines, when reducing nitro drugs, undergo concomitant oxidation to form semiquinone radicals. Both semiquinone radicals and nitro anion radicals formed in a reaction of nitro drug and catecholamine derivative were detected by electron spin resonance spectroscopy. Oxygen consumption studies in solutions containing nitro drug and catecholamine derivative showed that nitro anion radicals formed under aerobic conditions reduce oxygen to form the superoxide radical and hydrogen peroxide. Quinones formed in the reaction of catecholamine and nitro drug were detected by optical spectroscopy. Biosynthetic precursors and some metabolic products of catecholamines were also used in these studies, and they all exhibited reactions similar to catecholamines. Bovine chromaffin granules which synthesize and store catecholamines produced the nitrofurantoin anion radical when intact granules were treated with nitrofurantoin. These radicals formed inside the granules were observed by ESR spectroscopy. The formation of nitrofurantoin radical, semiquinone radicals of catecholamines, and oxygen-derived radicals by chromaffin granules is proposed to cause damage to adrenal medulla, and this process may lead to neurotoxicity.     

Chromaffin cell calcium channel kinetics measured isotopically through fast calcium, strontium, and barium fluxes

Fast Ca2+ uptake into K+-depolarized cultured bovine adrenal chromaffin cells has been isotopically measured in a time scale of 1-10 s. Depolarized cells retained as much as 80-fold 45Ca2+ taken up by resting cells; Ca2+ was not taken up by fibroblasts or endothelial-like cells. Because Ca2+ entry was inhibited by inorganic (La3+, Co2+, Mg2+) and organic (nifedipine) Ca2+ channel antagonists and enhanced by the Ca2+ channel activator Bay-K-8644, it seems clear that Ca2+ gains access to the chromaffin cell cytosol mainly through specific voltage-dependent Ca2+ channels. Ca2+ uptake evoked by 59 mM K+ was linear during the first 5 s of stimulation and continued to rise at a much slower rate up to 60 s. The rate of Ca2+ entry became steeper as the external [Ca2+] increased; initial rates of Ca2+ uptake varied from 0.06 fmol/cells . s at 0.125 mM Ca2+ to 2.85 fmol/cell . s at 7.5 mM Ca2+. The early 90Sr2+ uptake was linear but faster than Ca2+ uptake and later on was also saturated; 133Ba2+ was taken up still at a much faster rate and was linear for the entire depolarization period (2-60 s). Increased [K+] gradually depolarized chromaffin cells; Ca2+ and Sr2+ uptakes were not apparent below 30 mM K+ but were linear for 30 to 60 mM K+. In contrast, substantial Ba2+ uptake was seen even in K+-free solutions; and in 5.9 mM K+, Ba2+ uptake was as high as Ca2+ uptake obtained in 60 mM K+. Five to ten-second pulses of 45Ca2+, 90Sr2+, or 133Ba2+ given at different times after pre-depolarization of chromaffin cells served to analyze the kinetics of inactivation of the rates of entry of each divalent cation. Inactivation of Ca2+ uptake was faster than Sr2+, and Ba2+ uptake inactivated very little. Neither voltage changes nor Ca2+ ions passing through the channels seems to cause their inactivation; however, experiments aimed to manipulate the levels of internal Ca2+ using the cell-permeable chelator Quin-2 or the ionophore A23187 strongly suggest that intracellular Ca2+ levels determine the rates of inactivation of these channels.     

Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera

An octadecapeptide capable of inducing pigment dispersion in the chromatophores of the fiddler crab Uca pugilator has been isolated from lyophilized heads of the lubber grasshopper Romalea microptera. This pigment-dispersing factor (PDF) was purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase high performance liquid chromatography. Automated gas-phase sequencing, followed by the identification of the carboxyl-terminal amide, established the primary structure of this PDF as Asn-Ser-Glu-Ile-Ile-Asn-Ser-Leu-Leu-Gly-Leu-Pro-Lys-Leu-Leu-Asn-Asp-Ala- NH2. This structure was confirmed by chemical synthesis and by demonstrating that the synthetic and native PDF displayed identical chromatographic behavior and biological activity. The Romalea PDF is structurally related to the crustacean pigment-dispersing hormones (PDHs), which are also octadecapeptides. The sequence of grasshopper PDF shows 78% homology with beta-PDH (from the crabs U. pugilator and Cancer magister) and 50% homology with alpha-PDH (from the prawn Pandalus borealis). This study provides the first direct chemical evidence for the structural relatedness of insect PDF to the crustacean PDHs, thus identifying them as an authentic family of arthropod peptides.     

Resolution of genetic and uterine environmental effects in a family study of new dermatoglyphic measure: sole pattern ridge counts

The heritability of sole pattern ridge counts was examined in two family studies of endogamous castes from peninsular India. The phenotypes included ridge counts for each of the eight configurational areas separately, all areas combined, and only distal areas combined. Differences in heritability estimates were found between populations as well as among the individual configurational areas. Although some ridge counts do not show familial resemblance, others appear to be moderately heritable. Estimates of h2 range from 0.36 to 0.63 in one family series and from 0.22 to 0.51 in the other. In addition, significant uterine environmental effects were detected in one family series but not in the other.     

Propagation of Morus indica L. (Mulberry) by encapsulated shoot buds

Axillary buds of mulberry (Morus indica L) were encapsulated in alginate and agar to produce individual beads. The beads could be stored at 4°C for 45 days without loss of viability. Amongst the encapsulating agents tested, sodium alginate was found to be a better matrix. Encapsulated buds regenerated complete plantlets on an appropriate medium. This technique would provide an easy and novel propagation system for the elite as well as difficult-to-root species of mulberry.Abbreviations BAP benzylaminopurine - MS Murashige and Skoog (1962).     

2,3-Bisphosphoglycerate protects mitochondrial and cytosolic proteins from proteolytic inactivation

2,3-bisphosphoglycerate at physiological concentration similar to that found in many tissues protects effectively ornithine transcarbamoylase (OTC) from proteolytic inactivation by broken lysosomes. 2,3-bisphosphoglycerate protects also many other mitochondrial and cytosolic proteins, such as glutamate dehydrogenase (GDH) an glyceraldehyde-3-phosphate dehydrogenase (GAPDH), from proteolysis by broken lysosomes and other proteases. It is, thus, suggested that 2,3-bisphosphoglycerate may play an important role in the control of the degradative rates of some proteins, which may explain its high concentration in certain cells.     

Protein and lipid disturbances in rat liver microsomal membranes after bile duct ligation

An analysis of proteins, phospholipids and cholesterol from liver microsomal membranes was performed in normal and post-cholestatic rats. Bile duct ligated rats showed a progressive decrease of these membrane constituents. Minor changes in peptide analysis, a marked decrease of phosphatidylcholine and phosphatidylinositol, disappearance of phosphatidylethanolamine and sphingomyelin, and a clear increment of phosphatidylserine was observed in post-cholestatic as compared to normal group. It was concluded that extra-hepatic cholestasis produces structural changes on the liver microsomes, particularly on phospholipid profile.     

Effects of chronic allopurinol therapy on purine metabolism in Duchenne muscular dystrophy

Adenine, adenosine, inosine, hypoxanthine, xanthosine, xanthine, guanine and guanosine blood levels in 11 Duchenne muscular dystrophy patients treated with allopurinol, 10 untreated patients and 8 healthy controls, were determined by HPLC. Serum ADA, PNP and 5'-NT were also determined. Untreated patients showed lower adenine (p less than 0.001) and higher adenosine, xanthine, ADA and PNP levels (p less than 0.01) than controls. Treated patients had lower adenine and higher xanthine levels (p less than 0.001), but higher hypoxanthine, xanthosine and guanine levels (p less than 0.001), than controls, with normal ADA and PNP. The changes observed in ADA and PNP levels suggest an involvement of these enzymes in accelerated degradation of purines in Duchenne dystrophy.     

Cardiac glycosides stimulate phospholipase C activity in rat pinealocytes

Ouabain and related cardiac glycosides stimulate phospholipase C activity 5-fold in rat pinealocytes. The combined treatment of ouabain and norepinephrine, which also stimulates phospholipase C, produces an additive effect. The effects of either ouabain or norepinephrine are blocked by EGTA. However, there are notable differences. The stimulatory effect of ouabain is lost when extracellular Na+ is reduced to 20 mM and is not blocked by prazosin. In contrast, the stimulatory effect of norepinephrine is not blocked when extracellular Na+ is reduced to 20 mM but is blocked by prazosin. Ouabain appears to increase phospholipase C activity through a mechanism involving inhibition of Na+,K+-ATPase, and an accumulation of intracellular Na+ and Ca2+, not involving alpha 1-adrenoceptors. These findings raise the possibility that activation of phospholipase C might be a more general effect of cardiac glycosides.