Deletion mutation in Drosophila ma-l homologous, putative molybdopterin cofactor sulfurase gene is associated with bovine xanthinuria type II

Defective xanthine dehydrogenase (XDH) activity in humans results in xanthinuria and xanthine calculus accumulation in kidneys. Bovine xanthinuria was demonstrated in a local herd and characterized as xanthinuria type II, similar to the Drosophila ma-l mutations, which lose activities of molybdoenzymes, XDH, and aldehyde oxidase, although sulfite oxidase activity is preserved. Linkage analysis located the disease locus at the centromeric region of bovine chromosome 24, where a ma-l homologous, putative molybdopterin cofactor sulfurase gene (MCSU) has been physically mapped. We found that a deletion mutation at tyrosine 257 in MCSU is tightly associated with bovine xanthinuria type II.     

Interaction with heparin and matrix metalloproteinase 2 cleavage expose a cryptic anti-adhesive site of fibronectin

We recently found that fibronectin (FN) had a functional site [YTIYVIAL sequence in the heparin-binding domain 2 (Hep 2)] that was capable of suppressing the integrin-mediated cell adhesion to extracellular matrix. However, our results also indicated that this anti-adhesive site seemed to be usually buried within the Hep 2 domain structure because of its hydrophobic nature, raising a question as to the physiological significance of the cryptic anti-adhesive activity of FN. The present study demonstrates that the cryptic anti-adhesive activity can be exposed through the physiological processes. A 30-kDa chymotryptic FN fragment derived from Hep 2 domain (Hep 2 fragment), which had no effect on adhesion of MSV-transformed nonproducer 3T3 cell line (KN(7)8) to FN, expressed the anti-adhesive activity after treatment with 6 M urea. Light scattering and circular dichroism measurements showed that the urea treatment induced the conformational change of the Hep 2 fragment from a more compact form to an unfolded one. Incubation of the Hep 2 fragment with heparin also induced similar conformational changes and expression of anti-adhesive activity. Additionally, both the urea and heparin treatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alphaIII14A), with a recognition site near the anti-adhesive site of FN. Specific cleavage of either the Hep 2 fragment or intact FN by matrix metalloproteinase 2 (MMP-2) released a 10-kDa fragment with the anti-adhesive activity, which was shown to have the exposed anti-adhesive site on the amino-terminal region. Thus, the cryptic anti-adhesive activity of FN can be expressed upon conformational change and proteolytic cleavage of Hep 2 domain.     

The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts

The activities to induce TNF-alpha production by a monocytic cell line, THP-1, and ICAM-1 expression and IL-6 production by human gingival fibroblasts were detected in plural membrane lipoproteins of Mycoplasma salivarium. Although SDS-PAGE of the lipoproteins digested by proteinase K did not reveal any protein bands with molecular masses higher than approximately10 kDa, these activities were detected in the front of the gel. A lipoprotein with a molecular mass of 44 kDa (Lp44) was purified. Proteinase K did not affect the ICAM-1 expression-inducing activity of Lp44, but lipoprotein lipase abrogated the activity. These results suggested that the proteinase K-resistant and low molecular mass entity, possibly the N-terminal lipid moiety, played a key role in the expression of the activity. The N-terminal lipid moiety of Lp44 was purified from Lp44 digested with proteinase K by HPLC. Judging from the structure of microbial lipopeptides as well as the amino acid sequence and infrared spectrum of Lp44, the structure of the N-terminal lipid moiety of Lp44 was speculated to be S-(2, 3-bisacyloxypropyl)-cysteine-GDPKHPKSFTEWV-. Its analogue, S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF, was synthesized. The lipopeptide was similar to the N-terminal lipid moiety of Lp44 in the infrared spectrum and the ICAM-1 expression-inducing activity. Thus, this study suggested that the active entity of Lp44 was its N-terminal lipopeptide moiety, the structure of which was very similar to S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF.     

Molecular epidemiological investigation of enterohaemorrhagic Escherichia coli isolates in Japan

We have established several measures for control and prevention of EHEC infection including designation of the disease as notifiable since there was the sudden increase in the incidence of infection with EHEC O157:H7 in Japan in 1996, involving multiple outbreaks. Improvements in methodologies for isolation of these organisms and enhanced laboratory screening have revealed a variety of sources in food and animals. Although there seems to be a bovine reservoir for O157 EHEC in Japan as well as North America and UK, different foods have been linked to EHEC infection including salads, radish sprouts and salmon roe. There is clear evidence that divergent clones of EHEC O157:H7 are prevalent throughout Japan based on laboratory surveillance, however, we still need to better define the role of EHEC serogroups other than Escherichia coli O157 as important causes of human infection.     

FRL, a novel formin-related protein, binds to Rac and regulates cell motility and survival of macrophages

We have isolated a cDNA, frl (formin-related gene in leukocytes), a novel mammalian member of the formin gene family. The frl cDNA encodes a 160-kDa protein, FRL, that possesses FH1, FH2, and FH3 domains that are well conserved among other Formin-related proteins. An FRL protein is mainly localized in the cytosol and is highly expressed in spleen, lymph node, and bone marrow cells. Formin-related genes and proteins have been reported to play crucial roles in morphogenesis, cell polarity, and cytokinesis through interaction with Rho family small GTPases. FRL binds to Rac at its N-terminal region including the FH3 domain and associates with profilin at the FH1 domain. In a macrophage cell line, P388D1, overexpression of a truncated form of FRL containing only the FH3 domain (FH3-FRL) strongly inhibited cell adhesion to fibronectin and migration upon stimulation with a chemokine. Moreover, expression of the truncated FH3-FRL protein resulted in apoptotic cell death of P388D1 cells, suggesting that the truncated FH3-FRL protein may interfere with signals of FRL. Overexpression in the P388D1 cells of full-length FRL or of the truncated protein containing the FH3 and FH1 domains, with simultaneous expression of the truncated FH3-FRL protein, blocked apoptotic cell death and inhibition of cell adhesion and migration. These results suggest that FRL may play a role in the control of reorganization of the actin cytoskeleton in association with Rac and also in the regulation of the signal for cell survival.     

Molecular analysis of two genes, DD9A and B, which are expressed during the postmolt stage in the decapod crustacean Penaeus japonicus

In decapod crustaceans, deposition of calcium carbonate crystals (calcification) in the exoskeleton takes place during the postmolt phase of the molt cycle. In an attempt to identify proteins which regulate the calcification process, the differential display technique was used to identify genes which were specifically expressed in the integument during the postmolt stage in the penaeid prawn Penaeus japonicus. One of the genes thus identified, named DD9A, was expressed in the epithelial cells of the tail fan. DD9A encoded a putative precursor of a secreted protein of 113 amino acids which exhibited sequence similarities to a group of crustacean and insect cuticular proteins, suggesting that DD9A was a protein component of the exoskeleton. Another gene, DD9B, which was also transcribed specifically during the postmolt period was identified based on its sequence similarity to DD9A. Potential roles of the DD9A protein in the calcification of the exoskeleton will be discussed.     

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.     

Detection of Microsporum canis in the skin scrapings and hairs of dogs with dermatophytosis based on sequences of the chitin synthase 1 gene

In the present study, to confirm Microsporum canis infection rapidly, we detected the chitin synthase gene 1 (CHS1) gene of M. canis in the hair and skin samples of four dogs with dermatophytosis. Amplification of the DNAs in the four samples with CHS1 primers yielded fragments of about 620-bp. Nucleotide sequence analysis of the CHS1 gene fragments from samples and a reference strain of M. canis gene showed more than 99% similarity. The method presented in this study can rapidly detect the DNA of M. canis in skin scrapings, and we anticipate that it will be a useful microbiological tool for the diagnosis of M. canis infections in animals and humans.     

Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides

The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose [(GlcNAc)(2)-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3. The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue. The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).