The effects of Ca2+ and Sr2+ on Ca2+-sensitive biochemical changes in human erythrocytes and their membranes.

1. The Ca2+-dependency of K+ efflux, microvesiculation and breakdown of polyphosphoinositides and of ankyrin have been measured in intact human erythrocytes exposed to ionophore A23187 and HEDTA [N'-(2-hydroxyethyl)ethylenediamine NNN'-triacetate]-Ca2+ buffers. Half-maximal responses were observed at pCa values of 6.4, 4.1, 5.0 and 4.8 respectively. 2. The Ca2+ dependencies of K+ efflux and breakdown of polyphosphoinositides and ankyrin measured in erythrocyte ghosts without addition of ionophore showed almost identical values with those seen in whole cells treated with ionophore. 3. We conclude that ionophore A23187 is able to cause rapid equilibration of extracellular and intracellular [Ca2+] in intact cells and that in the presence of a suitable Ca2+ buffer, ionophore A23187 can be used to precisely fix the intracellular concentration of Ca2+ in erythrocytes. 4. The relatively high concentration of Ca2+ required to produce microvesiculation in intact cells may indicate that microvesiculation could be at least partly dependent on a direct interaction of Ca2+ with phospholipid. 5. Results obtained with Sr2+ paralleled those with Ca2+, although higher Sr2+ concentrations were required to achieve the same effects as Ca2+. Mg2+ produced none of the changes seen with Ca2+ or Sr2+.     

Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide.

1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.     

Fine structure and metabolism of multiply innervated fast muscle fibres in teleost fish

Summary Both the fast and slow muscle fibres of advanced teleost fish are multiply innervated. The fraction of slow-fibre volume occupied by mitochondria is 31.3%, 25.5% and 24.6%, respectively, for the myotomal muscles of brook trout (Salvelinus fontinalis), crucian carp (Carassius carassius), and plaice (Pleuronectes platessa), respectively. The corresponding figures for the fast muscles of these species are 9.3%, 4.6% and 2.0%, respectively. Cytochrome-oxidase and citrate-synthetase activities in the fast muscles of 9 species of teleost range from 0.20–0.93 moles substrate utilised, g wet weight muscle^-1 min^-1 (at 15° C) or around 4–17% of that of the corresponding slow fibres. Ultrastructural analyses reveal a marked heterogeneity within the fast-fibre population. For example, the fraction of fibres with <1% or >10% mitochondria is 0,4,42% and 36, 12 and 0%, respectively, for trout, carp and plaice. In general, small fibres (<500 m²) have the highest and large fibres (>1,500 m²) the lowest mitochondrial densities. The complexity of mitochondrial cristae is reduced in fast compared to slow fibres.Hexokinase activities range from 0.4–2.5 in slow and from 0.08–0.7 moles, g wet weight^-1 min^-1 in fast muscles, indicating a wide variation in their capacity for aerobic glucose utilisation. Phosphofructokinase activities are 1.2 to 3.6 times higher in fast than slow muscles indicating a greater glycolytic potential. Lactate dehydrogenase activities are not correlated with either the predicted anaerobic scopes for activity or the anoxic tolerances of the species studied. The results indicate a considerable variation in the aerobic capacities and principal fuels supporting activity among the fast muscles of different species. Brook trout and crucian carp are known to recruit fast fibres at low swimming speeds. For these species the aerobic potential of the fast muscle is probably sufficient to meet the energy requirements of slow swimming.     

Ultrastructural events and kinetics of microcyst germination in the myxomycete Didymium iridis

Microcysts of the myxomycete Didymium iridis were induced to excyst by transfer to 5mM potassium phosphate buffer. After 1 h in suspension, 90% of the microcysts had germinated into myxamoebae distinguishable by phase contrast microscopy and staining with Lugol's iodine. Both pH and osmolarity affected the kinetics of excystment. The rate and extent of excystment were decreased by cycloheximide but remained unaffected by actinomycin D, suggesting a requirement for protein synthesis but not RNA synthesis. Initially, the outer wall layers separated from the inner layer, which gradually expanded and loosened. The protoplast rehydrated and reverted to a vegetative morphology. Excysting cells were characterized by nucleolar inclusions, changes in the nuclear envelope and plasma membrane, appearance of ringed cisternal elements and microbodies in the cytoplasm, and formation of a densely fibrous zone adjacent to the site of emergence. Excysting populations have been classified into characteristic stages: mature, initiated, swollen, and pre-emergent microcysts.Florida Agricultural Experiment Station Journal Series No. 2407     

Dehalogenation of dichloromethane by cell extracts of hyphomicrobium DM2

A facultatively methylotrophic bacterium was isolated from enrichment cultures containing dichloromethane as the sole carbon source. It was identified as a Hyphomicrobium species. The organism grew exponentially in batch cultures with 10 mM dichloromethane at a specific growth rate of 0.07 h^-1. The release of Cl^- from dichloromethane and the disapperance of substrate paralleled growth. Resting dichloromethane-grown cells, in the presence of potassium sulphite as a trapping agent, converted cichloromethane methane quantitatively to formaldehyde. The conversion of dichloromethane to formaldehyde by cell extracts was stricly dependent on glutathione. Other thiols were inactive. Glutathione was not consumed in the course of the reaction. The specific activity of the enzymic dehalogenation of dichloromethane amounted to 3.8 mkat/kg protein in extracts of dichloromethane-grown cells and to less than 0.1 mkat/kg protein in extracts from cells grown on methanol.     

Adrenergic control of hypothalamic function during osmotic stress in the mallard duck (Anas platyrhynchos)

Summary The role of the paraventricular nucleus (PVN) and biogenic amines (BA) in regulating the level of corticoids in the serum of osmotically stressed mallard ducks (Anas platyrhynchos) was analyzed employing three experimental approaches: 1) pharmacologic alteration of central BA levels, 2) microscopic evaluation of BA distribution, and 3) placement of electrolytic lesions into the PVN. Reserpine and -methyl-p-tyrosine (mpt), agents that decrease the amount of BA's in the central nervous system, produced a fivefold increase in the concentration of serum corticoids. Conversely, pargyline and amphetamine, agents that increase the functional pool of BA's, prevented the rise in serum corticoid concentration normally observed in birds challenged with an intraperitoneal injection of hypertonic saline. When the topographic distribution of BA's was analyzed in the brains of osmotically stressed and nonstressed ducks distinct changes in the intensity of catecholamine (CA) fluorescence were observed in only one location, the PVN of the hypothalamus. Additionally, electrolytic lesions stereotaxically placed in the PVN blocked the osmotic stress-induced rise in serum corticoid concentration. These data therefore indicate that the PVN in the mallard duck plays some role in regulating the observed stress-induced rise in serum corticoid concentration, and that this regulatory function is probably inhibited by catecholamines.This research was supported by research grant No. GB 33321 from the National Science Foundation. We wish to express our sincere thanks to Mr. Howard Funk, research director, Colorado Division of Wildlife, for the use of the State's animal facilitiesThis research was submitted as partial fulfillment for the degree of Doctor of Philosophy, Department of Physiology and Biophysics, Colorado State University, Ft. Collins, CO 80521     

Host factor for coliphage Q beta RNA replication: presence in procaryotes and association with the 30S ribosomal subunit in Escherichia coli.

Summary The Host Factor required for in vitro coliphage Q RNA replication, a heat-stable RNA binding protein present in uninfectedEscherichia coli, has been detected by both immunological and functional tests inAcinetobacter calcoaceticus, Klebsiella pneumoniae, Pseudomonas aeruginosa andPseudomonas putida. It was not detectable by these criteria inBacillus stearothermophilus, Bacillus subtilis, Caulobacter crescentus, Micrococcus lysodeikticus, Rhodopseudomonas capsulata orSaccharomyces cerevisiae. InEscherichia coli the Host Factor protein has been shown to be associated with ribosomes. It is demostrated here that this association is specific for the 30S ribosomal subunit.