Low-affinity, high-capacity system of glucose transport in the ruminal bacterium Streptococcus bovis: evidence for a mechanism of facilitated diffusion

The glucose phosphotransferase system (PTS) of Streptococcus bovis could not account for the glucose consumption of exponential cultures, and the kinetics of glucose transport were biphasic. A PTS-deficient mutant lost the high-affinity, low-capacity system but retained its ability to take up glucose at high substrate concentrations. The low-affinity, high-capacity system did not require a proton motive force or ATP and could not be driven by an artificial membrane potential in the presence or absence of sodium. Since low-affinity transport was directly proportional to the external substrate concentration and exhibited counterflow kinetics, it appeared that a facilitated-diffusion mechanism was responsible for glucose transport at high substrate concentrations.     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.     

Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice

Rapid penetration of baby hamster kidney cells was used as a selective pressure for the isolation of pathogenesis mutants of the S.A.AR86 strain of Sindbis virus. Unlike most Sindbis virus strains, S.A.AR86 is virulent in adult as well as neonatal mice. Two classes of mutants were defined. One class was attenuated in adult mice inoculated intracerebrally as well as in neonatal mice inoculated either intracerebrally or subcutaneously. Sequence analysis of the glycoprotein genes of the parent virus and three such mutant strains revealed a single point mutation which resulted in an amino acid change at position 1 in the E2 glycoprotein. The change from a serine in S.A.AR86 to an asparagine in the mutants created a new site for N-linked glycosylation which appeared to be utilized. This mutation did not retard release of infectious particles; however, mutant virions contained the E2 precursor protein (PE2) rather than the E2 glycoprotein itself. The mutants also lost the ability to bind two E2-specific monoclonal antibodies, R6 and R13. A second class of mutants was attenuated in neonatal mice upon subcutaneous inoculation but remained virulent in adults and in neonates when inoculated intracerebrally. Sequence analysis of three such strains revealed the substitution of an arginine residue for a serine at position 114 in the E2 glycoprotein. Reactivity with monoclonal antibodies R6 and R13 was reduced, yet members of this mutant class were more susceptible than S.A.AR86 to neutralization by these antibodies.     

A recombination event that redefines the Huntington disease region

We report both a recombination event that places the Huntington disease gene proximal to the marker D4S98 and an extended linkage-disequilibrium study that uses this marker and confirms the existence of disequilibrium between it and the HD locus. We also report the cloning of other sequences in the region around D4S98, including a new polymorphic marker R10 and conserved sequences that identify a gene in the region of interest.     

Formation of Novel Polysaccharides by Bradyrhizobium japonicum Bacteroids in Soybean Nodules

Certain strains of Bradyrhizobium japonicum form a previously unknown polysaccharide in the root nodules of soybean plants (Glycine max (L.) Merr.). The polysaccharide accumulates inside of the symbiosome membrane—the plant-derived membrane enclosing the bacteroids. In older nodules (60 days after planting), the polysaccharide occupies most of the symbiosome volume and symbiosomes become enlarged so that there is little host cytoplasm in infected cells. The two different groups of B. japonicum which produce different types of polysaccharide in culture produce polysaccharides of similar composition in nodules. Polysaccharide formed by group I strains (e.g., USDA 5 and USDA 123) is composed of rhamnose, galactose, and 2-O-methylglucuronic acid, while polysaccharide formed by group II strains (e.g., USDA 31 and USDA 39) is composed of rhamnose and 4-O-methylglucuronic acid. That the polysaccharide is a bacterial product is indicated by its composition plus the fact that polysaccharide formation is independent of host genotype but is dependent on the bacterial genotype. Polysaccharide formation in nodules is common among strains in serogroups 123, 127, 129, and 31, with 27 of 39 strains (69%) testing positive. Polysaccharide formation in nodules is uncommon among other B. japonicum serogroups, with only 1 strain in 18 (6%) testing positive.     

Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum.

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.     

Analysis ofl-alanine:2-oxoglutarate aminotransferase isozymes in maize

Isozyme analysis ofl-alanine:2-oxoglutarate aminotransferase (ALT) in maize indicates that there are three genes encoding this enzyme activity. Two of the gene products interact with each other to form heterodimers, while the third gene product does not interact with the other two. Another isozyme that appears after gel electrophoresis and ALT staining is shown to be glutamate dehydrogenase-1. Anaerobic treatment does not result in increased ALT levels, indicating that the previously reported increase in alanine levels caused by this treatment may be due to increases in the level of pyruvate, a substrate of ALT.D. A. Russell was partially supported by a graduate student fellowship from the Division of Biology and Biomedical Sciences, Washington University. V. M. Peschke was partially supported by a postdoctoral fellowship from Monsanto. This research was supported by NIH Grant R01 GM34740.     

Interleukin-1 stimulates diglyceride accumulation in the absence of protein kinase C activation

Despite advances in the knowledge of the intracellular signalling in response to extracellular messengers, the mechanism of action of interleukin-1 (IL-1) has remained an enigma. In the present study, we have employed human dermal fibroblasts (Detroit 532 cells) to investigate IL-1 beta-induced changes in intracellular signals. Both recombinant human IL-1 beta and a native preparation purified from human placental tissue were employed. Cyclic AMP levels in cell monolayers were unaltered by IL-1 beta. Also, IL-1 beta did not influence significantly the levels of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate in the membrane, nor the water-soluble inositol phosphates, inositol monophosphate, inositol bisphosphate and inositol trisphosphate, in cells prelabelled with myo-[3H]inositol. In addition, intracellular calcium as measured by Quin2 was unaffected by interleukin-1. However, in cells labelled with [3H]glycerol or [3H]arachidonic acid, IL-1 beta caused an immediate rise in diglyceride (DG) accumulation. As the effects of IL-1 beta have been reported to be mimicked by tumour-promoting phorbol esters, this rise in DG suggested the involvement of protein kinase C (PKC). However, repeated experiments failed to reveal any acute effect of IL-1 beta on the activity of this enzyme. Furthermore, IL-1 beta did not cause the translocation of PKC between the membrane and the cytosol as has been found in response to other extracellular signals. Rather, IL-1 beta appeared to increase the synthesis of PKC in both membrane and cytosol preparations, an effect which could be prevented by coincubation with cycloheximide. These findings suggest that the diglyceride formed in response to IL-1 beta does not activate protein kinase C.