Sinorhizobium fredii and Sinorhizobium meliloti produce structurally conserved lipopolysaccharides and strain-specific K antigens

Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of Sinorhizobium fredii and Sinorhizobium meliloti, which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy-D-manno-2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens of S. fredii and those of S. meliloti is the presence of N-acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS of Sinorhizobium spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction. Sinorhizobium spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others: S. fredii HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of Sinorhizobium spp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.     

Effects of butylated hydroxyanisole and dicoumarol on the toxicity of menadione to rats

The enzyme DT-diaphorase catalyses the 2-electron reduction of quinones. This reaction may facilitate the detoxification of such compounds, since the hydroquinone so formed can be converted into non-toxic conjugates. There is evidence for the involvement of DT-diaphorase in the detoxification of menadione (2-methyl-1,4-naphthoquinone) in a wide range of cells and tissues in vitro, but no information is available on the possible influence of this enzyme on the harmful effects of menadione in vivo. In animals, menadione is selectively toxic to erythrocytes, causing haemolytic anaemia. In the present study, rats were treated with dicoumarol, an inhibitor of DT-diaphorase, or butylated hydroxyanisole (BHA), a substance that increases the activity of this enzyme in vivo. They were then challenged with a toxic dose of menadione. Dicoumarol increased the severity of menadione-induced haemolytic anaemia while BHA decreased it, consistent with a role for DT-diaphorase in the detoxification of menadione in vivo, as previously described in vitro.     

The 5-HT1A receptor antagonist p-MPPI blocks 5-HT1A autoreceptors and increases dorsal raphe unit activity in awake cats

The effects of the putative 5-HT1A receptor antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzam ide (p-MPPI) were examined on the activity of serotonergic dorsal raphe nucleus neurons in freely moving cats. Systemic administration of p-MPPI produced a dose-dependent increase in firing rate. This stimulatory effect of p-MPPI was evident during wakefulness (when serotonergic neurons display a relatively high level of activity), but not during sleep (when serotonergic neurons display little or no spontaneous activity). p-MPPI also blocked the ability of the 5-HT1A receptor agonist 8-hydroxy-(2-di-n-propylamino)tetralin (8-OH-DPAT) to inhibit serotonergic neuronal activity. This antagonism was evident both as a reversal of the neuronal inhibition produced by prior injection of 8-OH-DPAT and as a shift in the potency of 8-OH-DPAT following p-MPPI pretreatment. Overall, these results in behaving animals indicate that p-MPPI acts as an effective 5-HT1A autoreceptor antagonist. The increase in firing rate produced by p-MPPI supports the hypothesis that autoreceptor-mediated feedback inhibition operates under physiological conditions.     

Genomic variation in C. albicans

Candida albicans displays many types of variation which affect a broad spectrum of phenotypes. Among them are antigenic, chromosomal, morphologic, and biochemical variation. The ability to modulate many phenotypes is clearly an important factor in the success of this fungus as a pathogen and variation at the genomic level may be the common denominator among the different systems. Genomic variation in C. albicans has been studied by many researchers and a number of different mechanisms have been identified. Among them are ploidy fluctuations, which allow the organism to cycle from 2n chromosome number to 4n or higher; translocation, which has been demonstrated to involve many different chromosomes and affects many phenotypes including virulence; mitotic recombination, which has been demonstrated to increase resistance to certain drugs; and nondisjunction, which has been shown to have morphological consequences. The number and diversity of these mechanisms combine to make C. albicans a highly successful organism. Although normally a commensal of humans, when invasive, C. albicans can inhabit almost any site in the body. It is not known what governs the transition of C. albicans from a commensal to pathogenic invader, however, variation at the genomic level likely plays a role. One possible consequence of variation is the generation of atypical strains, further expanding the documented phenotypic plasticity of this organism. The exposure of patients to cytotoxic drugs during treatment of such diseases as AIDS or cancer increases the selective pressure and has exacerbated both the frequency and degree of variability observed in C. albicans. The molecular analysis of genomic variation in C. albicans is proving to be a fertile area of research and future investigations can only be expected to add to the mechanisms documented in this review.     

Renin secretion from permeabilized juxtaglomerular cells requires a permeant cation

The cytosolic concentration of chloride correlates directly with renin secretion from renal juxtaglomerular granular (JG) cells. In the present study, the mechanism by which chloride stimulates renin release was investigated in a preparation of permeabilized rat glomeruli with attached JG cells. An isosmotic increase in the concentration of chloride by 129 mM stimulated renin release 16- to 20-fold. Substitution of K+ by the impermeant cation N-methyl-d-glucamine (NMDG) abolished this response, while substitution with Na+ caused marginal inhibition. Substitution with Cs+ had no effect. Addition of sucrose, which permeates the secretory granules poorly, also abolished the stimulation of renin secretion by KCl. The response to KCl was not affected by K+-channel antagonists or by agonists of K+ channels. Chloride channel blockers were also without effect on the secretory response to KCl. When the ATP concentration was lowered from 1 to 0.1 mM renin release was stimulated, while an increase in the ATP concentration from 1 to 5 mM had no effect. Blockers of ATP-sensitive (KATP) channels did not modify the response to chloride. The present data suggest that chloride stimulates renin release after entry of KCl into the renin secretory granules which results in swelling and release of renin.     

Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.     

Membrane dynamics of the water transport protein aquaporin-1 in intact human red cells

Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-alphaAQP1) in the undeformed red cell membrane is 66 +/- 10% and the average lateral diffusion coefficient is (3.1 +/- 0.5) x 10(-11) cm2/s. F-alphaAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-alphaAQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, approximately 50% of the aspirated F-alphaAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-alphaAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6 x 10(-10) cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin-based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.     

Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15 degreesC is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER-Golgi transport.