Mechanism of action of ryanodine on cardiac sarcoplasmic reticulum

Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This initial depression was followed by a later marked stimulation of calcium uptake. These effects were noted when calcium uptake was measured in the presence or absence of oxalate. The requirement for preincubation with ryanodine was highly dependent on ryanodine concentration and temperature. The mechanism of action of ryanodine clearly was not an effect on oxalate entry or calcium oxalate precipitation because the effects were also observed in the absence of oxalate. Ryanodine also had no effect on passive calcium efflux from actively loaded vesicles. Because ryanodine had no effect on Ca2+-ATPase activity under defined conditions of an ATP-regenerating system and no calcium gradient, we suggest ryanodine does not change the stoichiometry of the pump. Our results are consistent with the hypothesis that ryanodine closes a calcium channel in a subpopulation of the vesicles.     

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

The relationship between first and second chromosome segregation ratios from Drosophila melanogaster males bearing Segregation Distorter

Summary Drosophila melanogaster males heterozygous for the second chromosome locus Segregation Distorter preferentially transmit this chromosome to their progeny due to a dysfunctioning of SD ⁺-bearing sperm. SD males with a normal sex chromosome constitution produce more females than males among SD ⁺ progeny. This report shows that this unequal recovery of sexes is enhanced from XY/Y; SD/SD ⁺ males and enhanced still further from XY/O; SD/SD ⁺ males. It is argued that the probability that a SD ⁺-bearing sperm will dysfunction is related to its sex chromsome complement, with the relative probabilities of dysfunction ranked O> Y> X> XY. It is shown that a modified probit analysis accounts for the relationship between sex ratio and second chromosome segregation frequency for all paternal genotypes. Finally, SD/SD ⁺ males show no increase in sex chromosome nondisjunction with respect to a control.R. E. Denell was supported by U.S.P.H.S. Training Grant No. GM00337 and by a U.S.P.H.S. Postdoctoral Fellowship; George L. Gabor Miklos was supported by A.E.C. Contract No. AT (04-3)-34 PA150.     

The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Changes of inositol phosphates during decapitation-induced lateral bud break of Malus domestica

An increase in phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) occurred during bud break induced by decapitation. Inositol-1-phosphate [Ins(l)P₁], inositol-1,4-bisphosphate [Ins(1,4)P₂], and inositol-1,4,5-triphosphate [Ins(1,4,5)P₃] were found in apple buds and increased progressively following decapitation. Ins(1)P₁ and Ins(1,4)P₂ peaked 48 h after decapitation and Ins(1,4,5)P₃ peaked 72 h after decapitation during the metabolic transition when buds emerged from dormancy. Ins(1,4)P₂ and Ins(1,4,5)P₃ levels declined there after. The lateral buds on shoots with intact terminals and decapitated shoots treated with indole-3-acetic acid (IAA) in the terminals tip remained dormant and there were no significant changes in phospholipid and inositol phosphate contents.     

Molecular characterization and expression of a tobacco histone H1 cDNA

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steadystate mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.     

Normal levels of zinc and sulfhydryls in morphologically abnormal populations of spermatozoa from moderately zinc-deficient rats

Zinc is required for spermatogenesis in mammals and is concentrated in the dense outer fibers of the sperm tail, where it is associated with cysteine-rich protein. To investigate the effects of marginal zinc deficiency upon dense fiber formation and upon sperm quality in general, weanling Sprague-Dawley rats were administered a commercial low-zinc diet, supplemented with phytate, for approximately 60 days, and were compared with controls fed the same diet plus 50 ppm zinc in their drinking water. The following characteristics of the zinc-deficient rats were significantly lower than in the controls: body weight, testis weight, epididymis weight, seminal vesicle weight, sperm content of the cauda epididy-midis, sperm motility, testis zinc, and hair zinc. By contrast, the levels of sperm zinc and sperm sulfhydryls were the same in the zinc-deficient and control rats. The zinc-deficient rats displayed a highly variable spectrum of sperm defects, which included decapitation, disorganized and redundant tail elements, and superfluous cytoplasm. However, abortive dense fiber development was only rarely observed. Apparently, even when availability of zinc is limited and reduced sperm production ensues, elaboration of dense fibers rich in zinc and sulfhydryls continues to be obligatory for the completion of spermiogenesis.     

Measurement of facilitated calcium diffusion by a soluble calcium-binding protein

The flux of calcium through an aqueous compartment was determined in a flow-dialysis cell in which two dialysis membranes separated the middle aqueous compartment from two outer compartments. The contribution of convection to the total calcium flux was large but could be removed by addition of 1% agar. The flux of calcium through the gelled aqueous compartment agreed with theoretical expectations. The self-diffusion coefficient for calcium from these results was calculated to be 0.81 X 10(-5) cm2 X s-1. Carp parvalbumin significantly enhanced the calcium flux at 2.3 X 10(-6)M free calcium. The calcium flux increased linearly with parvalbumin concentration. These observations are consistent with the hypothesis that the overall unidirectional calcium flux J is the sum of free calcium diffusion and protein-calcium diffusion: J = D[Ca] + D'[CaPr]. The value of D', the self-diffusion coefficient for parvalbumin, was calculated from the flux data to be 13.7 X 10(-7) cm2 X s-1.