Charge-based separation of detergent-resistant membranes of mouse splenic B cells

Current biochemical characterization for cholesterol- and glycolipid-rich membrane microdomains largely depends on analysis of detergent-resistant membranes (DRMs). In the present study, we succeeded in separation of DRMs of similar density-based on their electrical charge using free-flow electrophoresis (FFE). After crosslinking of B cell receptor (BCR), mouse splenic B cells were lysed with 1% Brij-58 and the resulting lysate was subjected to sucrose density gradient ultracentrifugation. The low-density fraction that recovered a part of DRMs containing IgM together with those enriched in GM1a, the Src family protein tyrosine kinase Lyn, and the alpha subunit of inhibitory heterotrimeric GTP-binding protein was further resolved by FFE. FFE separated the former into more cathodally deflected fractions than the latter. In addition, FFE revealed an anodal shift of DRMs containing a transmembrane protein CD38 upon BCR-crosslinking. The results demonstrate the effectiveness of FFE for the charge-based separation of DRMs.     

Thermodynamic analysis of the activation mechanism of the GCSF receptor induced by ligand binding

The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.     

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNF alpha-induced DNA-binding activity of NF-kappa B and NF-kappa B-dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF-kappa B activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNF alpha-induced activation of NF-kappa B and the expression of NF-kappa B-targeted proinflammatory genes such as E-selectin and cyclooxygenase-2. These results suggest that TrxR1 may act as a positive regulator of NF-kappa B and may play an important role in the cellular inflammatory response.     

Detection of 14 alleles derived from the MHC class I<Emphasis Type="Italic"> A</Emphasis> locus in cynomolgus monkeys

A basic understanding of the major histocompatibility complex (MHC) class I, which, together with T-cell receptors, is a key player in antigen recognition by cytotoxic T lymphocytes, is necessary to study the cellular immune response to intracellular pathogens. The MHC has hardly been reported in cynomolgus monkeys (Macaca facicularis), although cynomolgus monkeys have been frequently used as the surrogate animal model. We attempted to determine the nucleotide sequences of the MHC class I A locus of cynomolgus monkeys (Mafa-A) and eventually 34 independent sequences of Mafa-A were obtained from 29 cynomolgus monkeys. These 34 sequences were classified into 14 Mafa-A alleles according to the results of phylogenetic analyses using the neighbor-joining method. One to three Mafa-A alleles were obtained from a single animal. We also tried to establish a multiplex PCR-SSP method for convenient typing of Mafa-A alleles. cDNA from a family of cynomolgus monkeys, which is composed of four sirs and four dams, were examined by multiplex PCR-SSP. The result of multiplex PCR-SSP showed that an individual cynomolgus monkey had two or three Mafa-A alleles, suggesting that the A locus of cynomolgus monkeys might be duplicated.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the α subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, and , and converts chorismate to anthranilate. Two or more AS -subunit genes have been identified and characterized in several land plants. Although subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS -subunit genes (OASA1 and OASA2) in rice (Oryza sativa). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a subunit to achieve maximal enzymatic activity even with NH ₄ ⁺ as the amino donor. The V _max and K _i for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.     

An ER-localized form of PV72, a seed-specific vacuolar sorting receptor, interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves

Putative vacuolar sorting receptors that bind to the vacuolar targeting signals have been found in various plants; pumpkin PV72, pea BP-80 and Arabidopsis AtELP. PV72 is a seed-specific receptor that is predicted to sort seed storage proteins to protein storage vacuoles. Analysis by surface plasmon resonance showed that the lumenal domain of PV72 bound to an NPIR (a typical vacuolar targeting signal)-containing peptide of the precursor of a cysteine proteinase, AtALEU, in the presence of Ca(2+) (K(D) = 0.1 micro M). To elucidate the receptor-dependent transport of vacuolar proteins in plant cells, we produced transgenic Arabidopsis plants that expressed a fusion protein (PV72-HDEL) composed of the lumenal domain of PV72 and an endoplasmic reticulum (ER)-retention signal, HDEL. The expression of PV72-HDEL induced the accumulation of the AtALEU precursor. The accumulation level of the AtALEU precursor was dependent on that of PV72-HDEL. In contrast, it did not induce the accumulation of a precursor of another cysteine proteinase, RD21, which contains no NPIR. Detailed subcellular localization revealed that both the AtALEU precursor and PV72-HDEL accumulated in the ER fraction. We found that most of the AtALEU precursor molecules formed a complex with PV72-HDEL. The AtALEU precursor might be trapped by PV72-HDEL in the ER and not transported to the vacuoles. This in-planta analysis supports the hypothesis that an Arabidopsis homolog of PV72 functions as a sorting receptor for the NPIR-containing proteinase. The overall results suggest that vacuolar sorting receptors for the protein storage vacuoles and the lytic vacuoles share the similar recognition mechanism for a vacuolar targeting signal.     

Distribution of virulence-associated genes in Vibrio mimicus isolates from clinical and environmental origins

Distribution of virulence-associated genes in Vibrio mimicus was studied including the toxin genes ctxA, tdh, st and vmh and the genes necessary for regulation of toxin production, toxR, toxS, toxT, tcpA and tcpP. Approximately half of clinical V. mimicus isolates possessed one or more genes encoding V. cholerae enterotoxic factors such as ctxA, tdh and st. All of the clinical and environmental isolates possessed vmh encoding V. mimicus hemolysin (VMH). The ctxA encoding cholera toxin was detected in only 2 strains, 5% of the clinical isolates. Furthermore, there were very few strains possessing tcpP and toxT needed for the expression of ctxA. These results may suggest that VMH is a more important pathogenic factor than well recognized toxins such as cholera toxin (CT) in V. mimicus infection.     

Identification of peptidomimetic HTLV-I protease inhibitors containing hydroxymethylcarbonyl (HMC) isostere as the transition-state mimic

Towards the development of chemotherapy for the infection by human T-cell leukemia virus type I (HTLV-I), we have established evaluation systems for HTLV-I protease (PR) inhibitors using both recombinant and chemically synthesized HTLV-I PRs. Newly synthesized substrate-based inhibitors containing hydroxymethylcarbonyl (HMC) isostere showed potent anti-HTLV-I PR activity.