Cell-associated oligosaccharides of Bradyrhizobium spp.

We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species.     

Endotoxic lipid A interaction with human platelets. Structure-function analysis of lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide

We previously reported that human blood platelets are directly stimulated by endotoxic Lipid A via the protein kinase C pathway (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). To study the relationship between the molecular structure of Lipid A and its ability to activate human platelets, we used Lipid A homologs derived from Salmonella minnesota Re595 lipopolysaccharide. Preparations of Lipid A are heterogeneous in regard to the degree of substitution of fatty acids which result in multiple homologs. These were separated by thin-layer chromatography and characterized by fast atom bombardment spectroscopy and related techniques (Johnson R. S., Her, G.-R., Grabarek, J., Hawiger, J., and Reinhold, V. N. (1990) J. Biol. Chem. 265, 8108-8116). The homologs of monophosphoryl Lipid A (MLA) present in fractions TLC-8 (heptaacyl MLA ion, m/z 1953), TLC-7 (three hexaacyl species with predominant MLA ion m/z 1715), and TLC-6 (four pentaacyl homologs with predominant MLA ion, m/z 1505) induced secretion of [14C]serotonin and aggregation of platelets. Lipid A homologs in fractions TLC-5 (three tetraacyl MLA ions, m/z 1323, 1307, and 1279), TLC-4 (one major triacyl MLA ion, m/z 1097), TLC-3 (tetraacyl MLA ion, m/z 1278), TLC-2 (a diphosphoryl hexaacyl Lipid A ion, m/z 1795, and several ions of low abundance), and TLC-1 (two ions, m/z 1097 and 666) were not active in regard to human platelet aggregation and [14C]serotonin secretion. The most active homolog was heptaacyl MLA ion, m/z 1953, present in TLC-8, while homologs present in TLC-7 and TLC-6 were 5 and 10 times less active, respectively. Rapid phosphorylation of a human platelet protein of Mr 40,000-47,000 (P47), a substrate for protein kinase C activation, preceded secretion of serotonin when platelets were triggered by the most active heptaacyl MLA ion, m/z 1953. These events were time-dependent, with half-maximal response of phosphorylation of P47 at 30 s and [14C]serotonin secretion at 45 s. A marked difference in the degree of phosphorylation of P47 was observed with heptaacyl MLA homolog present in TLC-8 inducing complete phosphorylation (97%), whereas less acylated Lipid A homologs present in TLC-1 caused marginal phosphorylation (20%). These results indicate that the degree of acylation of monophosphoryl Lipid A determines its functional properties toward human platelets in regard to secretion of [14C]serotonin, aggregation, and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for Mediated HCO(3) Transport in Isolated Pea Mesophyll Protoplasts

The kinetics of ¹⁴C fixation, and inorganic C (C_inorg) accumulation, have been followed in isolated pea mesophyll protoplasts. NaH¹⁴CO₃ was supplied to the protoplasts in media the pH of which was varied between 7 and 8.     

Protonation and light synergistically convert plasmalemma sugar carrier system in mesophyll protoplasts to its fully activated form

The course of sugar fluxes into and out of protoplasts isolated from the mesophyll of Pisum sativum L. has been followed over brief time intervals (minutes). Light strongly stimulated net sugar influx at pH 8 as well as at pH 5.5. The proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited initial influx in the light, both at pH 8.0 and at pH 5.5. CCCP was without effect in the dark at either pH. All these results applied both to sucrose and to the nonmetabolizable glucose analog 3-O-methyl-d-glucose.When protoplasts at pH 5.5 were transferred from light to darkness, "stored" light driving force maintained uptake in the dark at the full light rate for the first 7 minutes. At pH 8, however, even 4 minutes after transfer to dark, uptake was well below the light rate. Initial uptake rates over a range of external concentrations were derived from progress curves obtained in the light and in the dark, both at pH 5.5 and at 7.7. When initial rate was plotted against concentration, simple Michaelis-Menten kinetics were observed only under the condition pH 5.5, light. In the dark at both pH values, and in the light at pH 7.7, complex curves with intermediate plateaus were obtained, strongly resembling curves reported for systems where mixed negative and positive cooperativity is operating.The same "K(m) for protons" was observed in the dark and in the light (10(-7) molar). Switching protoplasts in the dark from pH 8 to 5.5 failed to drive sugar transport by imposed protonmotive force, as judged by lack of sensitivity to CCCP. Switching protoplasts which had taken up sugar in the dark at pH 5.5 to pH 7 induced net efflux of sugar. Flux analysis showed that this effect was entirely due to the prompt fall in influx.It is concluded from the kinetic experiments that protonation alone is not sufficient to convert the sugar transport system to its fully activated high affinity form. A further light-dependent factor which acts synergistically with protonation is required.     

Degradation of pyrimidine bases in Clostridium sticklandii

Resting cells of Clostridium sticklandii took up thymine or uracil, when grown in a medium containing 40 mM serine and 20 mM thymine or uracil. The uptake was much lower, when the cells had been grown in a complex medium. Cell-free extracts from cells grown in the complex medium reduced the two bases to the dihydro compounds and decomposed dihydrothymine to -ureidoisobutyrate, as indicated by thin-layer chromatography. Uptake and degradation were stimulated by both NADH and NADPH. Further breakdown did not occur, as ¹⁴CO₂ was not evolved from C-2-labelled thymine or uracil. The rates of pyrimidine uptake and breakdown of C. sticklandii were lower than those reported for C. sporogenes (Hilton et al., 1975).     

Distribution patterns of formaldehyde-induced fluorescence of hypothalamic monoamines

Summary The adventitial cells surrounding the spermatheca of the reproductive system of Sonorella santaritana (Mollusca: Gastropoda) appear to have an unusual system of vesicles. Electron micrographs of the membranes forming these vesicles show that they have multiple openings to the cell's exterior and that each opening has a pore complex. In addition, secondary vesicles appear to be generated by the primary vesicles. Evidence is presented suggesting that these vesicles represent a previously unreported membrane transport system.Southwestern Medical School and Veterous Administration Hospital Dallas, Texas, USA     

PEtab—Interoperable specification of parameter estimation problems in systems biology

Reproducibility and reusability of the results of data-based modeling studies are essential. Yet, there has been—so far—no broadly supported format for the specification of parameter estimation problems in systems biology. Here, we introduce PEtab, a format which facilitates the specification of parameter estimation problems using Systems Biology Markup Language (SBML) models and a set of tab-separated value files describing the observation model and experimental data as well as parameters to be estimated. We already implemented PEtab support into eight well-established model simulation and parameter estimation toolboxes with hundreds of users in total. We provide a Python library for validation and modification of a PEtab problem and currently 20 example parameter estimation problems based on recent studies.     

Alcohol Impairs Predation Risk Response and Communication in Zebrafish

The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism.