Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination.

Essentials Botrocetin-2 (Bot2) binds to von Willebrand factor (VWF) and induces platelet agglutination. We identified Bot2 residues that are required for binding to VWF and glycoprotein (GP) Ib. We produced a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Mutant Bot2 could be used as a potential anti-thrombotic reagent to block VWF-GPIb interaction. SUMMARY: Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of α and ß subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the ß subunit (Aspß70Ala), or Argß115Ala and Lysß117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspß70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argß115Ala/Lysß117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argß115 and Lysß117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the ß subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the ß subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argß115Glu and Lysß117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction.

Effect of MDR1 C3435T polymorphism on cure rates of Helicobacter pylori infection by triple therapy with lansoprazole, amoxicillin and clarithromycin in relation to CYP 2C19 genotypes and 23S rRNA genotypes of H. pylori.

BACKGROUND: Polymorphism in MDR1 is associated with variation in the plasma level of a proton pump inhibitor. AIM: To investigate whether MDR1 polymorphism is associated with eradication rates of Helicobacter pylori by a triple therapy with lansoprazole, amoxicillin and clarithromycin in relation to CYP2C19 genotype status and bacterial susceptibility to clarithromycin. METHODS: A total of 313 patients infected with H. pylori completed the treatment with lansoprazole 30 mg b.d., clarithromycin 200 mg b.d. and amoxicillin 750 mg b.d. for 1 week. MDR1 C3435T polymorphism and CYP2C19 genotypes of patients and sensitivity of H. pylori to clarithromycin were determined. RESULTS: Logistic regression analysis revealed that the MDR1 polymorphism as well as CYP2C19 genotypes of patients and clarithromycin-resistance of H. pylori were significantly associated with successful eradication. Eradication rates for H. pylori were 82% (83/101: 95% CI = 73-89), 81% (112/139: CI = 73-87), and 67% (44/73: CI = 48-72) in patients with the MDR1 3435 C/C, C/T and T/T genotype, respectively (P = 0.001). CONCLUSIONS: Polymorphism of MDR1 is one of the determinants of successful eradication of H. pylori by the triple therapy with lansoprazole, amoxicillin and clarithromycin, together with CYP2C19 genotype and bacterial susceptibility to clarithromycin.

Vomeroglandin/CRP-Ductin is strongly expressed in the glands associated with the mouse vomeronasal organ: identification and characterization of mouse vomeroglandin.

Vomeroglandin, a subform of mouse CRP-ductin, is a protein strongly expressed in the glands of mouse vomeronasal system. Both the proteins contain several of scavenger receptor cysteine-rich and CUB domains and one ZP domain. This domain arrangement is similar to those of rat Ebnerin, human DMBT1, and rabbit hensin. In situ hybridization analysis shows strong expression of vomeroglandin mRNA in the glands of vomeronasal system. Immunological analyses detect both membrane-bound and secreted forms of vomeroglandin. The secreted protein seems to be localized in the lumen of the vomeronasal organ, playing a certain role in the pheromone perception.

Electric field assisted thermal desorption ionization using an infrared laser.

A new technique has been developed for the formation of gas-phase electrons and ions by electric field assisted thermal desorption ionization at atmospheric pressure. Experiments were carried out using a sharp tungsten wire tip coated with a thin solid sample film which was irradiated by a 10.6 µm infrared laser. By applying a strong electric field on the laser-irradiated tungsten wire tip, abundant ions such as alkali ions, halide ions, and also multiply charged negative ions S(2)O(6)(2-) and S(2)O(8)(2-), were formed. Copyright 1999 John Wiley & Sons, Ltd.

[The maximum safe dosage of the low molecular weight hydroxyethyl starch, HESPANDAR (HES), estimated from HES induced changes in coagulation parameters and clinical bleeding].

The effects of hydroxyethyl starch on the coagulation system have received attention, and safe dosage of high molecular weight hydroxyethyl starch is generally found to be 20 ml.kg-1. Low molecular weight hydroxyethyl starch, HESPANDAR (HES), seems to induce weaker specific effects on blood coagulation than high molecular weight hydroxyethyl starch. The aims of this study are to estimate the maximum safe dosage of HES, and to investigate the etiology for coagulopathy induced by HES. Forty three patients (18 to 75 years old) who were free of bleeding disorders scheduled for abdominal operations received 20, 30, or 40 ml.kg-1 of HES. We measured coagulation parameters of them including platelet, hematocrit, total protein, activated partial thromboplastin time, prothrombin time and Factor 8, and observed the evidence of clinical microbleeding. As the HES was infused, the values of coagulation parameters except Factor 8 changed with blood dilution. Factor 8 decreased more than expected from the values calculated for hemodilution. The evidences of clinical microbleeding were observed when patients had received HES more than 30 ml.kg-1. The decrease of Factor 8 and the observed tendency of clinical microbleeding showed a significant positive relationship (P = 0.0002). We conclude from our results that the maximum safe dosage of HES is about 30 ml.kg-1, and HES may affect blood coagulation by lowering the plasma concentration of Factor 8.

[Management of foreign tourists' diseases at the American Hospital in Neuilly]. / Prise en charge des touristes étrangers malades à l'Hôpital américain de Neuilly.

More and more foreign tourists are being treated at the American Hospital of Paris. The main reasons for this are the presence of a 24 hour medical and dental emergency service, in addition to the availability of MRI, CT scan and comprehensive laboratory facilities. The hospital is multidisciplinary, where short stay hospitalizations are the rule. English is the second most commonly used language. There is a permanent Japanese language service for Japanese patients. Qualitative data is presented concerning the management of patients according to specialty and nationality. American and Japanese patients admitted to the hospital in 1997 are surveyed according to their mean age, reasons for admission and appropriate specialty as well as mean length of stay noted. The greatest number of hospitalizations in decreasing order and according to nationality are as follows: American, Japanese, Moroccan Egyptian and Saudi Arabian. The consultations and hospitalizations of Japanese patients between 1990 and 1997 are compared.

Histological features and prognosis of patients with mucoepidermoid carcinoma of the parotid gland.

Histological features and prognosis of patients with mucoepidermoid carcinoma of the parotid gland were analysed. Tumours from 13 patients were classified according to histological grades and immunoreactivity for HER-2/neu. Surgical resection of the tumour was performed for all patients, and the overall five-year survival rate was 69 per cent. The patients whose histological grades were 1 or 2 showed a 100 per cent five-year survival rate, but no patient with grade 3 survived five years. Also, patients who had tumours that overexpressed HER-2/neu had a lower survival rate (25 per cent) than patients with tumours that had weaker immunostaining (89 per cent). We considered tumours classified as grade 3 plus strong HER-2/neu expression to be 'high malignancy', and compared them with 'low malignancy' tumours that were grade 1 or 2 and had weaker HER-2/neu staining. Patients with high malignancy tumours had shorter recurrence-free intervals and shorter overall survival than patients with low malignancy tumours. The overall survival period of the low malignancy cases was much longer than the recurrence-free interval; unlike that in the high malignancy tumour patients. These results suggest that the combination of histological grades and expression of HER-2/neu may be a useful predictor of the prognosis for mucoepidermoid carcinomata.

Effects of acidic and basic fibroblast growth factors on cell proliferation in pleomorphic adenoma of the parotid gland.

Localization and biochemical characteristics of acidic and basic fibroblast growth factor (aFGF and bFGF) were studied in the normal parotid gland (NPG) and in pleomorphic adenoma (PA). In addition, the effects of these factors on proliferation activity were investigated in cultured PA cells. aFGF and bFGF were detected immunohistochemically in 62% and 58% of NPG and 44% and 58% of PA, respectively, and they localized in the cytoplasm of the ductal segments of the NPG, and of the tubular, trabecular and squamous components of PA. Both aFGF and bFGF, each with a molecular weight of 18 kDa, were identified in PA using heparin-sepharose chromatography and Western blot analysis. Both recombinant human aFGF and bFGF stimulated [3H]-thymidine incorporation by cultured PA cells. These results indicated that aFGF and bFGF, probably produced by neoplastic cells, play important roles in the proliferation of PA of the parotid gland.

Possible pheromone-carrier function of two lipocalin proteins in the vomeronasal organ.

We report the molecular cloning and characterization of two secretory proteins specifically expressed in vomeronasal and posterior glands of the nasal septum, the ducts of which open into the lumen of the vomeronasal organ. These two proteins are members of the lipocalin superfamily, consisting of hydrophobic ligand carriers. We immunohistochemically localized one of the proteins in the mucus covering the vomeronasal sensory epithelium, where the primary reception of pheromone takes place. The immunoreactivity on the vomeronasal sensory epithelium was evident in the neonatal and post-pubertal periods, when the close contact between animals plays critical roles in suckling and sexual behaviors, respectively. These results suggest that small lipophilic molecules stimulate the accessory olfactory system to regulate the reproductive behavior of mice.

[Coronary artery spasm immediately after the long-standing operation for cancer of the tongue].

A 72-year-old male underwent radical operation for cancer of the tongue. Anesthesia was maintained with the combination of enflurane-N2O-vecuronium and cervical epidural block. Five minutes after the cessation of the longstanding operation, VT and circulatory collapse occurred. After administration of lidocaine and ephedrine, VPC and ST elevation were noted, followed by VT and Vf. Cardioversion successfully restored sinus rhythm with no ST change, suggesting an episode of coronary artery spasm. The possible inducing factors in this case were hypotension and acute imbalance in autonomic nervous systems caused by hypovolemia, hypothermia, insufficient anesthetic depth, loss of surgical stress, neostigmine and epidural block. The authors reviewed case reports on coronary spasm, especially looking for possible inducing factors of coronary artery spasm during anesthesia.

Undecalcified temporal bone morphology: a methodology useful for gross to fine observation and three-dimensional reconstruction.

Using a novel method of cutting undecalcified temporal bone specimens, quantitative structural analysis in the human and the Japanese monkey was undertaken. One millimeter thick serial slices made from unembedded temporal bones retained fine structure. Therefore, gross to fine observation could be performed systematically at the macroscopic, light, scanning, and transmission electron microscopic levels. The entire temporal bone three-dimensional reconstruction was completed from embedded sections; consequently, the volume of the tubotympanum and air cell system could be calculated. Available methods by embedding, tungsten carbide sectioning, grinding, and microwave irradiation for decalcification were also examined. These morphologic studies suggest that these novel methods offer timesaving advantages over any presently available techniques, and allow for elucidation of temporal bone morphology with only a few specimens.

[Analysis of the characteristics of responders to chemotherapy of advanced hepatocellular carcinoma].

To evaluate characteristics of the responders to chemotherapy for advanced hepatocellular carcinoma, clinicopathological findings of the responders (9 cases) and those of the non-responders (58 cases) have been compared. As for clinical characteristics, no difference was found in age and sex between the two groups. The responders were, however, restricted to the good cases in respect to performance status (PS) and hepatic reserve based on Child's classification. Concerning characteristics of tumors, no significant difference between the two groups was observed in size and number of tumors, or in the frequency of portal vein thrombosis and that of remote metastasis. There was no difference between them in chemotherapy regimen, either. It was concluded that PS and hepatic reserve might be the most important factors determining the efficacy of chemotherapy for advanced hepatocellular carcinoma.

Chromosomal assignment of woodchuck hepatitis virus (WHV) DNA integration sites in a woodchuck hepatocellular carcinoma-derived cell line (WH257GE10).

The chromosomal sites of woodchuck hepatitis virus (WHV) DNA integration were identified in a woodchuck hepatocellular carcinoma-derived cell line (WH257GE10) by the in situ hybridization technique using 3H-labelled WHV whole genome (WHV 2) as a probe. The G-banded chromosome spreads from WH257GE10 were identified and diagrammed schematically according to their band patterns. WHV DNA was integrated into 2 sites: 33 region of the long arm of chromosome 6 (6q33) and 31 region of the long arm of chromosome 8 (8q31). Chromosomal sites of WHV DNA integration were stable during successive passage periods analyzed in vitro.

Analysis of the chromosomal abnormalities of a woodchuck hepatocellular carcinoma-derived cell line (WH257GE10).

The normal woodchuck karyotype was determined by the G-banding technique, and the chromosomal abnormalities of a woodchuck hepatocellular carcinoma-derived cell line (WH257GE10) were analyzed and compared with it. The normal woodchuck karyotype was 38,XY in a male, and 38,XX in a female. Autosomes were classified into 2 groups (A and B). Group A consisted of 13 pairs (1-13) of metacentric or sub-metacentric chromosomes, and group B consisted of 5 pairs (14-18) of telocentric or sub-telocentric ones. The X chromosome was sub-metacentric, and the Y chromosome was telocentric. In WH257GE10, the modal number was 36. Deletion of one of the chromosome 5 pair, of the chromosome 13 pair, and of the Y chromosome was observed. Two marker chromosomes (M1 and M2) were found. These markers showed the same G-banded patterns except for a small chromosome fragment at the distal part of the long arm of M1. The chromosome aberrations were very stable through successive passages in vitro. These findings suggest that the chromosomal abnormalities found in WH257GE10 might be associated with the pathogenesis of woodchuck hepatocellular carcinoma.

Hepatocellular carcinoma with chondrosarcomatous variation. A case report and review of the literature.

An autopsy case of hepatocellular carcinoma with a sarcomatous component in a 63-year-old woman was described. A massive tumor measuring 15 X 15 X 10 cm almost replaced the left lobe of the liver and extended to the right atrium of the heart. Histologically the tumor was a typical hepatocellular carcinoma of adult type, except in the central portion where there was gradual replacement of the carcinoma by spindle-shaped atypical cells with areas of chondrosarcomatous tissue. Some of the spindle cells were immunohistochemically positive for keratin, and the sarcomatous component was not found in the metastatic foci of liver and lungs. Reported cases of mixed hepatic tumor of adults in the literature were reviewed, and discussion was made concerning the nosology of the present tumor.