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Abstract: Solute carriers (SLCs) is the largest group of transporters, embracing transporters for inorganic ions, amino acids, neurotransmitters, sugars, purines and fatty acids among other substrates. We mined the finished assembly of the human genome using Hidden Markov Models (HMMs) obtaining a total of 384 unique SLC sequences. Detailed clustering and phylogenetic analysis of the entire SLC family showed that 15 of the families place into four large phylogenetic clusters with the largest containing eight SLC families, suggesting that many of the distinct families of SLCs have a common evolutionary origin. This study represents the first overall genomic roadmap of the SLCs providing large sequence sets and clarifies the phylogenetic relationships among the families of the second largest group of membrane proteins. [Copyright &y& Elsevier]

Campylobacter concisus is an opportunistic pathogen commonly found in the human oral cavity. It has also been isolated from clinical sources including gastroenteritis cases. Both secreted and cell-associated hemolytic activities were detected in C. concisus strains isolated from children with gastroenteritis. The secreted hemolytic activity of C. concisus strains was labile and was detected in variable levels from fresh-culture filtrates only. In addition, another secreted hemolysin/cytotoxin with a molecular weight <10 kDa was detected in a single C. concisus strain (RCH 12). A C. concisus genomic library, constructed from strain RCH 3 in Escherichia coli XL1-Blue, was screened for hemolytic clones. Subcloning and sequence analysis of selected hemolytic clones identified ORFs for genes that enhance hemolytic activity but do not appear to be related to any known hemolysin genes found in Gram-negative bacteria. In a previous study, a stable cell-associated hemolysin was identified as an outer-membrane phospholipase A (OMPLA) encoded by the pldA gene. In this study, we report cloning of the pldA gene of the clinical strain C. concisus RCH 3 and the complementation of phospholipase A activity in an E. coli pldA mutant. [ABSTRACT FROM AUTHOR]

UDP-xylose (UDP-Xyl) is a sugar donor for the synthesis of glycoproteins, polysaccharides, various metabolites, and oligosaccharides in plants, vertebrates, and fungi. In plants, the biosynthesis of UDP-Xyl from UDP-glucuronic acid (UDP-GlcA) appears to be catalyzed by numerous UDP-glucuronic acid decarboxylase (Uxs) isoforms. For example, six Uxs isoforms in Arabidopsis thaliana (L.) and four in rice have been identified. However, the reason/s for the existence of several isoforms that are necessary for the synthesis of UDP-Xyl remains unknown. Here, we describe a Uxs isoform in Arabidopsis, AtUXS2, encoding an integral membrane protein that appears to be localized to the Golgi apparatus. The enzyme is a dimer and has distinct properties. Unlike the UXS3 isoform, which is shown here to be a soluble protein, the UXS2 isoform is membrane bound. The characteristics of the membrane-bound AtUxs2 and cytosolic AtUxs3 support the hypothesis that unique UDP-GlcA-DCs possessing distinct sub-cellular localizations can spatially regulate specific xylosylation events in plant cells. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) are linear carbohydrate polymers expressed on all cell surfaces, and bind growth factors that recognize specific disaccharide sequences. Such sequences in the GAG chain are not genetically determined but may be assembled by the cell in response to environmental changes. GAGs are strongly hydrophilic and negatively charged molecules that do not bind well to either polystyrene surfaces or to hydrophobic blotting membranes. Cationic detergents were used to derivatize hydrophobic membranes to become hydrophilic and positively charged. Binding of GAGs to derivatized membranes was optimized regarding pH and ionic strength. Five different monoclonal antibodies (Mab) were used to detect sequence epitopes in immobilized GAGs. Parallel samples were stained with Alcian Blue and the staining intensities were quantitated by scanning and densitometry. By calculating the ratio between the antibody staining (epitope) and Alcian Blue staining (mass), the epitope density, i.e. the number of repetitive epitopes per mass, is obtained. The epitope density with each antibody was different with different GAGs. Some epitopes were common in GAG, i.e. highly repetitive epitopes. Some epitopes were rare and possibly expressed only once per GAG molecule, i.e. low degree of repetition. An epitope density profile was obtained when each sample was stained with all antibodies and their epitope densities calculated at the plateau level. The epitope profile is an indirect measure of the sequence variability in GAGs. Determination of epitope density and profile can be used to characterize a GAG population and to discriminate between different populations with similar chemical composition. [ABSTRACT FROM AUTHOR]

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 gains energy by reducing sulfur with H2 to H2S. From this hyperthermophile, a sulfur-reducing complex catalyzing this reaction was purified 13.5-fold. The native complex exhibited a brownish-yellow colour and showed an apparent molecular mass of 520 kDa. SDS/PAGE revealed the presence of nine different major polypeptides with apparent molecular masses of 82, 72, 65, 50, 47, 42, 40, 30 and 24 kDa. The native complex contained 50-55 mol acid-labile sulfur, 50-55 mol iron, 1.6 mol nickel, 1.2 mol copper, 2.8 mol cytochrome b and 0.3 mol cytochrome c (all per mol native complex). The temperature optimum of the H2 : sulfur oxidoreductase complex was 100 +C, which is consistent with the physiological growth optimum of the native organism. The complex is extremely heat stable. During 5 h incubation at 100 +C, no decrease in H2S-forming activity could be observed. [ABSTRACT FROM AUTHOR]

We examined the role of the cellular localization of antigen on the immune response after DNA immunization of mice with three forms of ovalbumin (OVA). DNA encoding OVA which was secreted (sOVA) generated 10- to 100-fold higher IgG responses with 50-and 100-fold higher levels of IgG1 than the cytoplasmic (cOVA) or membrane bound (mOVA) forms. An IgG2a predominance was seen only in cOVA and mOVA immunized mice. Although the antibody response was CD4+ T cell dependent, the differences in the antibody response could not be compensated for by provision of excess CD4+ T cell help in TCR transgenic mice. Together with our hapten-carrier studies, this would indicate that membrane or intracellular localization limits the availability of antigen for B cell priming which affects the magnitude and form of the antibody response. Surprisingly, stronger cytotoxic T lymphocyte (CTL) responses were generated for sOVA or mOVA than for cOVA via intramuscular (i.m.) injection. Since a cytoplasmic antigen should have best access to the canonical class I pathway for antigen presentation, our results indicate that priming of CTL responses after i.m. DNA immunization is probably by cross-presentation of antigen by nontransfected professional antigen-presenting cells. In contrast, intradermal immunization with cOVA produced optimal CTL responses but, as with mOVA, suboptimal antibody responses. This, together with our ex vivo RT-PCR analysis showing similar mRNA levels from all three constructs 7 days post-immunization, argues against the differential CTL response for i.m. injection to be due to dose. [ABSTRACT FROM AUTHOR]

A form of CAT-like activity was found bound present in rat brain synaptosomal membranes which could be recovered in the Triton X-114 phase. The enzyme activity was slightly activated by NaCl, had a pH maximum around 8 and showed a temperature dependence with a Q of 2.28. It was inhibited 100% by 10 M naphthyl vinyl pyridinium but not by 10 M diisopropyl phosphofluoridate. The kinetics of this bound form of CAT were similar to the soluble form of the enzyme. The K was 405±58 μM for choline and 62±8 μM for AcCoA. Five isoelectric forms were found with pH's of 4.55, 6.05, 7.06, 7.36, and 8.00 which is in contrast to the three isoelectric forms found of the soluble enzyme in rat brain. The presence of a CAT-like activity in the plasma membrane was confirmed with experiments performed using intact synaptosomes and intact cells in culture. Acetylcholine, synthesized from radioactive AcCoA by intact rat brain synaptosomes, was recovered in the incubation medium and only in the presence of exogenous choline or when the production of choline was stimulated by oleate via the activation of phospholipase D. This was also seen in experiments with intact pheochromocytoma cell cultures (PC 12) which synthesize acetylcholine that was recoverved in the incubation medium. Acetylcholine formation in the presence of choline and AcCoA was stimulated in cells that had been grown in the presence of nerve growth factor (NGF). The localization of 1% of CAT activity in a transbilayer position in the plasma membrane, could suggest a possible role of this enzymatic form in the regulation of acetylcholine synthesis. [ABSTRACT FROM AUTHOR]

A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN, 5 mM β-mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000 g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72-76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 μg). It required 0.8 mM Ca for half-maximal activation and 5 mM Ca for optimal activation. Mg (5 mM) was ineffective while Zn and Hg were inhibitory. The pH optimum was ranged from 7.5-8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purufied enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases. [ABSTRACT FROM AUTHOR]

The thermophilic fungus Malbranchea sulfurea produces extracellular α-amylase whereas α-glucosidase is mainly cell bound. Extraction of the cell bound enzyme was maximum with one molar NaCl, followed by Triton ×100 and Urea-NaSO extractants. Supplementation of Triton ×100 in growth medium significantly affected the presence of enzymes at various locations. A role for cell bound α-amylase and α-glucosidase has been suggested in rapid starch utilization by the fungus during early growth phase. [ABSTRACT FROM AUTHOR]