Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.     

Correlation between serum ionised calcium and serum albumin concentrations in two hospital populations.

One quarter of 172 patients from two hospitals with no obvious disturbances of calcium homeostasis and with total serum calcium concentrations that were normal after adjustment for albumin concentration had low serum ionised calcium concentrations. The low values were not due to changes in pH but were associated with hypoalbuminaemia. Significant positive regressions of ionised calcium on albumin concentration were observed in patients from both hospitals and also in 48 healthy laboratory staff. Because the regressions did not differ between patients and healthy subjects the low ionised calcium values associated with hypoalbuminaemia are unlikely to have been of pathological importance. These findings indicate that interpreting serum ionised calcium concentrations in patients with a reduced serum albumin concentration on the basis of a reference range determined in subjects with a normal serum albumin concentration may be clinically misleading.     

Merremia discoidesperma: Its taxonomy and capacity of its seeds for ocean drifting

Merremia discoidesperma (Donn. Sm.) O’Donell (Mary’s-bean) is a rarely collected and inadequately described high climbing woody liana of Chiapas, Mexico; Guatemala; Costa Rica; Hispaniola; and Cuba. There is only one record of this species being cultivated and this from Guanajuato, Mexico in 1894, though seeds are used in folk remedies. These seeds are topographically unlike other known convolvulaceous seeds, thus permitting their positive identification. Because seeds remain buoyant for more than three years in seawater, those produced in the Caribbean and Atlantic drainage region may be transported by the Gulf Stream System as far north as the Norwegian coast, a distance of about 9,500 km. Seeds produced in the Pacific drainage region from Chiapas, Mexico south to Costa Rica may be transported by the North Pacific Equatorial Current as far west as Wotho Atoll in the Marshall Islands, a distance of about 11,000 km. Records of drifting and subsequent stranding of these and other tropical New World disseminules are supported by drift bottle studies and drift debris records in the Atlantic Ocean. In the Pacific Ocean region distribution of stranded Mary’s-bean seeds is supported by a similar distribution of New World pumice. There is an indication in the literature that Mary’s-bean seeds may drift to the Philippines. The record from Wotho Atoll to Norway constitutes the widest drift range of any seed or fruit which has been documented. Other tropical disseminules may drift as far or farther, but their origins cannot be ascertained with certainty. The plant is described, its synonyms listed, and its distribution as well as the distribution of its stranded seeds recorded.     

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.     

Cloning and linkage analysis of Neisseria gonorrhoeae DNA methyltransferases.

We have cloned DNA methyltransferases (MTases) from various strains of Neisseria gonorrhoeae. Each of these clones represents a single specificity, indicating that the multiple gonococcal MTase specificities are encoded by monospecific MTases. The DNAs of five strains (FA5100, F62, MS11, Pgh3-2, and WR302) were digested with NheI, SpeI, or NheI plus SpeI and subjected to pulsed-field gel electrophoresis. The DNA MTase clones were used to probe Southern blots of these pulsed-field gels to determine whether the MTase genes are linked and whether there are strain-to-strain differences. The results indicate that none of these genes are closely linked, but variable hybridization patterns indicate that there exist restriction fragment length polymorphisms between the strains tested. Most of the chromosomal regions containing these restriction fragment length polymorphisms are clustered in regions containing gonococcal genes known or suspected to antigenically vary via genetic recombination.     

A note on the isolation and enumeration of Gram positive cocci from marine and estuarine waters

Ninety-two percent of Gram positive cocci detected in estuarine and marine surface waters were identified as Staphylococcus spp. Micrococcus and Streptococcus spp. were rarely isolated from seawater, except at dump site stations located in the Puerto Rico Trench area of the Atlantic Ocean, where they were present in unusually large numbers. Staphylococci were present in greatest abundance near land, but were also found in open ocean surface waters in low, but relatively constant, numbers. Staphylococcus epidermidis , or strains related to Staph. hominis , were present in all samples. Staphylococci from surface water samples collected at the Puerto Rico Trench dump site degraded a wide range of compounds. It is considered that these cocci were derived from the wastes dumped, as very few were found in unpolluted marine waters and in those cases in which stimulatory factors, such as disposal of wastes, do not play a role, Gram positive cocci will not be recovered unless large volumes of water are filtered. The Gram positive cocci are present in very low numbers in natural seawater of the open ocean and, therefore, are commonly overlooked. The significance of these bacteria in the nutrient cycles of the sea remains to be elucidated. Documentation of a widespread occurrence of Gram positive cocci in North Eastern Atlantic seawater is provided.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) in adipose tissue in vivo by glucocorticoids and insulin.

1. The regulation of the synthesis of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in epididymal adipose tissue, liver and kidney in vivo was studied immunochemically. 2. Phosphoenolpyruvate carboxykinase (GTP) synthesis in adipose tissue is increased by starvation, diabetes and noradrenaline, and decreased by re-feeding and insulin. These changes were also seen in adrenalectomized rats and are qualitatively similar to those observed for the liver enzyme. This indicates the involvement of cyclic AMP as an inducer and insulin as a de-inducer in the regulation of phosphoenolpyruvate carboxykinase (GTP) in both tissues. (Induction and de-induction are defined as selective increase and decrease respectively in the rate of enzyme synthesis, regardless of the mechanism involved.)3. Adrenalectomy had little effect on phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney, but increased the synthesis rate of the adipose-tissue enzyme. Starvation and adrenalectomy had additive effects in increasing the synthesis rate of adipose-tissue phosphoenolpyruvate carboxykinase (GTP). In adrenalectomized diabetic rats glucocorticoids increased phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney while decreasing enzyme synthesis in adipose tissue. De-induction of adipose tissue phosphoenolpyruvate carboxykinase (GTP) is therefore regulated independently by glucocorticoids and insulin. 4. Although liver, kidney and adipose-tissue phosphoenolpyruvate carboxykinases (GTP) are seemingly identical, there is an apparent tissue-specific differentiation in regulatory systems for the enzyme.     

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.