Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI.

BACKGROUND: Convulxin (CVX), a C-type lectin from the venom of Crotalus durissus terrificus, is a potent activator of human platelets, binding predominantly to glycoprotein (GP)VI. Native CVX is an octamer composed of four alphabeta-heterodimers [(alphabeta)(4)]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid (E), at beta chain residue 89, but the physiological relevance of this difference is unknown. OBJECTIVE: We used the Drosophila S2 system to express recombinant CVX (rCVX) heterodimers (alphabeta) and site-directed mutagenesis to evaluate the influence of multimer size and the substitution betaK89E on CVX function. METHODS: By flow cytometry, native CVX and both recombinant forms bind to human platelets in whole blood. By surface plasmon resonance (BIAcore, Piscataway, NJ, USA), the calculated equilibrium dissociation constants (K(D)) were: rCVX alphabeta89K, 11.3 x 10(-8) m; rCVX alphabeta89E, 9 x 10(-8) m; and native CVX, 2.8 x 10(-8) m. RESULTS: Thus, the affinities of the two rCVX forms for human, recombinant GPVI are essentially the same, but the relative affinity of native CVX is about 3-fold higher. The minimum concentration of native CVX that induces maximal human platelet aggregation (70 pm) is roughly 400-fold lower than that of either rCVX (29 nm). CONCLUSIONS: These results are consistent with the hypothesis that the ability of the native CVX octamer to cluster mobile GPVI molecules within the platelet membrane may be the single most important factor that contributes to the efficiency with which CVX is able to induce platelet activation.

Effect of Helicobacter pylori eradication on ongoing mutation of immunoglobulin genes in gastric MALT lymphoma.

Gastric low-grade mucosa-associated lymphoid tissue (low-grade MALT) lymphomas has been associated with Helicobacter pylori (H. pylori) infection. Although infiltrating T cells with specificity for H. pylori are known to stimulate the development of MALT lymphomas, the effect of H. pylori eradication on rearranged immunoglobulin heavy chain (IgH) genes of low-grade gastric MALT lymphomas is unclear. Gastric biopsies from five cases were investigated by cloning and sequence analysis of rearranged IgH genes before and after the treatment for H. pylori. In all cases, IgH genes were mutated from their germline counterpart. The frequency of intraclonal sequence heterogeneity before the eradication of H. pylori varied from 0.25 to 0.49%. Clones obtained from the tumours before the eradication of H. pylori in cases 1 and 2 showed a tendency to display a mutation pattern by positive antigen selection and their monoclonarity disappeared after the eradication. The frequency of intraclonal sequence heterogeneity of the clones obtained from cases 3, 4 and 5 (0.12% in case 3, 0.10% in 4 and 0.18% in 5) after the eradication of H. pylori was lower than that in tumours before the eradication (0.30% in case 3, 0.49% in 4 and not determined in 5). These findings suggest that low-grade gastric MALT lymphomas expand due to the persistent presence of H. pylori in vivo. The characteristic feature of tumour clones obtained from the tumours after the eradication of H. pylori is a very low intraclonal heterogeneity, which may potentially be independent of H. pylori.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity.

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin.

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.

Glycosylative inactivation of chalcomycin and tylosin by a clinically isolated Nocardia asteroides strain.

Studies on the susceptibility of pathogenic Nocardia to macrolide antibiotics, chalcomycin and tylosin, showed that most of the Nocardia species examined were highly resistant to both antibiotics, although N. nova was moderately susceptible. N. asteroides IFM 0339 converted these macrolides into inactive metabolites by glycosylation at 2'-OH or glycosylation and reduction of the 20-formyl group. The structures of the metabolites were determined from NMR and MS data to be 2'-[O-(beta-D-glucopyranosyl)]chalcomycin (2), 2'-[O-(beta-D-glucopyranosyl)]tylosin (5) and 20-dihydro-2'-[O-(beta-D-glucopyranosyl)]tylosin (4).

[Comparison of five guidelines of community-acquired pneumonia].

The aim of this retrospective study was to elucidate the characteristics of five guidelines of community-acquired pneumonia: ATS (1993), ATS (2001), IDSA (1998), IDSA (2000) and the guidelines of the Japan Respiratory Society (2000). One hundred community-acquired pneumonia patients admitted to the International Medical Center of Japan were investigated in accordance with each set of guidelines based on the physical, laboratory, and chest radiography findings on the first day of treatment. According to the ATS (1993) guidelines, 33% of the cases were classified as "severe" pneumonia. On the other hand, according to the ATS (2001) guidelines, only 8% of the cases were classified as "severe" pneumonia. According to the IDSA guidelines, 35% of the patients were classified as "outpatients". Fluoroquinolone appears to be a very important antibiotic drug in the new guidelines of both ATS and IDSA. The scoring system of IDSA suggested a correlation between the patient's score and the pathogenic bacteria. According to the guidelines of the Japan Respiratory Society, 42% of the cases were classified as "severe" pneumonia. There are evident differences between these guidelines, and clinicians need to have a full understanding of their respective characteristics.

[A case of renal cell carcinoma complicated with interstitial pneumonitis, complete A-V block and pleural effusion during interferon-alpha therapy].

A 76-year-old man with postoperative renal cell carcinoma accompanied by multiple lung metastasis was being treated with recombinant interferon-alpha. After administration of 3 MU/day on 3 days/week for 1 month, he complained of headache and tinnitus. During continuous treatment for 3 months, he complained of appetite loss, low-grade fever and dyspnea. He was then referred to our Department of Internal Medicine. Electrocardiography indicated a complete A-V block, and chest radiography (CXR) showed a reticular shadow in both lower lung fields and bilateral pleural effusion. Chest computed tomography (CT) indicated subpleural emphysematous changes, multiple nodules, consolidation shadow with ground glass opacity in both lower lobes, and bilateral pleural effusion. The findings in the bronchoalveolar lavage (BAL) fluid included increases in the numbers of lymphocytes and eosinophils. We reached a diagnosis of interferon-alpha-induced pneumonitis on the basis of the patient's clinical course, and the CXR, chest CT and BAL fluid findings. Treatment with methylprednisolone pulse therapy for 3 days and then administration of prednisolone for 1 month resulted in marked improvement in the complete A-V block and interstitial pneumonitis. At day 7 after discontinuation of prednisolone, the serum level of C-reactive protein increased, and CXR showed bilateral pleural effusion. We therefore believe that the pleural effusion was probably also induced by interferon-alpha. Interferon is an effective drug for chronic hepatitis C and malignant diseases. Many complications have been reported during interferon therapy. However, although these complications, such as interstitial pneumonitis, complete A-V block and pleural effusion, have rarely been reported, careful attention is required during interferon therapy in case any appear.

Neuroprotectins A and B, bicyclohexapeptides protecting chick telencephalic neurons from excitotoxicity. II. Structure determination.

In the course of our search for neuroprotective agents of microbial origin against kainate-induced neurotoxicity, we have succeeded in the isolation of two new bicyclohexapeptides, neuroprotectins A and B, together with a known compound, complestatin, from the fermentation broth of Streptomyces sp. Q27107. They are closely related in structure to complestatin and possess an oxindolylalanine moiety in place of the tryptophan residue present in complestatin.

Diheteropeptin, a novel substance with TGF-beta-like activity, produced by a fungus, Diheterospora chlamydosporia. II. Physico-chemical properties and structure elucidation.

The structure of diheteropeptin (1), a TGF-beta-like active substance from Diheterospora chlamydosporia Q58044, was determined to be a new cyclotetrapeptide, cyclo[2aminoisobutyryl-(S)-phenylalanyl-(R)-prolyl-(2S,8R,9R)-2-am ino-8,9-dihydroxydecanoyl-] by NMR, mass spectrometric and chemical studies.

Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2.

Sphingomonas sp. strain P2, which is capable of utilizing phenanthrene as a sole carbon and energy source, was isolated from petroleum-contaminated soil in Thailand. Gas chromatography-mass spectrometry and (1)H and (13)C nuclear magnetic resonance analyses revealed two novel metabolites from the phenanthrene degradation pathway. One was identified as 5,6-benzocoumarin, which was derived by dioxygenation at the 1- and 2-positions of phenanthrene, and the other was determined to be 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation were identified as 7, 8-benzocoumarin, 1-hydroxy-2-naphthoic acid and coumarin. From these results, it is suggested that strain P2 can degrade phenanthrene via dioxygenation at both 1,2- and 3,4-positions followed by meta-cleavage.

Sandhoff disease in Cyprus: population screening by biochemical and DNA analysis indicates a high frequency of carriers in the Maronite community.

In the last 15 years, four patients with the infantile form of Sandhoff disease were diagnosed in four different families in Cyprus (population 703,000, birth rate 1.7%). Three of these cases came from the Christian Maronite community (less than 1% of the population) and one from the Greek community (84% of the population). This relatively large number of patients prompted us to initiate an epidemiological study in order to establish the frequency of the mutant allele in Cyprus. Carrier detection was initially based on the measurement of beta-hexosaminidase A and B in both leucocytes and serum. Using the enzyme test, 35 carriers were identified among 244 random Maronite samples and 15 among 28 Maronites with a family history of Sandhoff disease, but only one carrier was found out of 115 random samples from the Greek community. In parallel to the biochemical screening, DNA studies were undertaken in one of the three Maronite patients and in a Greek carrier related to the Greek patient. These studies resulted in the identification of two novel mutations, a deletion of A at nt76 and a G to C transversion at position 5 of the 5'-splice site of intron 8, which have been published. We subsequently screened the carriers detected in the biochemical study for these two mutations using PCR-based tests. Of 50 Maronite carriers examined, 42 were found to have the nt76 deletion. Eight Maronite samples, designated carriers from the biochemical results, were negative for both mutations. It is possible that these individuals were incorrectly classified as carriers since their enzyme values are equivocal, although the presence of another mutation has not been excluded. Two Greek Cypriot carriers and two obligate Lebanese carriers were negative for both mutations. We conclude that there is a high frequency of Sandhoff disease carriers in the Maronite community of Cyprus, approximately 1 in 7, and that a single mutation predominates in this population.

Identification of a taurine transport inhibitory substance in sesame seeds.

An ethanol extract from sesame seeds inhibited the taurine uptake in human intestinal epithelial Caco-2 cells. The uptake of such alpha-amino acids as leucine and glutamic acid was not inhibited by the extract, indicating that this inhibition is specific to the taurine uptake. The unknown inhibitor in the sesame extract was purifled by reversed-phase HPLC by monitoring the inhibitory effect on taurine uptake. The isolated substance was identified as lysophosphatidylcholine, linoleoyl (Lyso-PC), by NMR and MS analysis. Lyso-PC inhibited the taurine uptake in a dose-dependent manner with an IC50 value of approximately 200 microM. Although Lyso-PC is known to be a surface active and cell lytic compound, neither damage nor loss of integrity of the Caco2 cell monolayer was apparent after treating with 200 microM Lyso-PC. Inhibition was observed by incubating cells with Lyso-PC for only 1 min prior to the uptake experiments. These results suggest the direct effect of Lyso-PC on the cell membrane to be the main mechanism for this inhibition. Lyso-PC may play a role in the regulation of certain intestinal transporters.

Structures of ADP-ribosylated rifampicin and its metabolite: intermediates of rifampicin-ribosylation by Mycobacterium smegmatis DSM43756.

23-(O-ADP-Ribosyl)rifampicin [RIP-TAs (3, Na+ form), RIP-TAf (4, H+ form)] was obtained as an intermediate in the conversion process of rifampicin (1) to RIP-Mb (2) that is mediated by cell homogenates of Mycobacterium smegmatis DSM43756 or of Escherichia coli carrying a mycobacterial mono(ADP-ribosyl) transferase gene, in the presence of NADH. 23-[O-(5'-Phosphoribosyl)]rifampicin (5, RIP-TAp) was also obtained by the reaction of rifampicin with NADH in the presence of a homogenate of M. smegmatis. The structures of 3, 4, and 5 were determined by means of MS and NMR analyses.

Competitive reverse transcription-polymerase chain reaction assay for quantification of human multidrug resistance 1 (MDR1) gene expression in fresh leukemic cells.

We have analyzed MDR1 gene expression in 69 clinical samples obtained from 64 patients with leukemic hematologic malignancies by using a competitive reverse transcription-polymerase chain reaction assay with a heterologous competitor RNA. To exclude a false-positive result caused by concomitant normal lymphocytes that physiologically express MDR1, in samples we determined a cut-off value of 8 amol MDR1 transcript per microgram of RNA by simultaneous measurement of rhodamine 123 dye efflux either in lymphocyte or gated leukemic cell populations. Consequently, 23 of 69 samples were concluded to be MDR1-positive in leukemic cells per se. The MDR1 expression rate was significantly correlated with factors such as a history of preceding chemotherapy, elder age of the patient, and certain disease types (eg, leukemia progressed from myelodysplastic syndrome). Moreover, the complete response rate after chemotherapy was significantly higher in MDR1-negative patients than in MDR1-positive patients (52% vs 17%, respectively; P = .01). The assay established will enable the quantification of MDR1 gene expression in blood samples from patients with leukemic hematologic malignancies and will be applicable to clinical laboratories as a routine test.