Renal and hormonal effects of alpha1-adrenoceptor blockade by bunazosin in essential hypertension

Summary The renal and hormonal effects of the ₁-adrenoceptor blocker bunazosin were examined in 6 patients with essential hypertension. Oral bunazosin for 4 to 12 weeks significantly decreased mean blood pressure by 10%, increased effective renal blood flow and creatinine clearance by 34% and 37%, respectively, the plasma norepinephrine concentration was elevated by 60%, and the plasma atrial natriuretic peptide level was lowered by 22%. The plasma renin activity and aldosterone concentration were unchanged. Thus, a moderate reduction in blood pressure was produced by bunazosin treatment while maintaining renal perfusion.     

Anti-cholinergic effect of verapamil on the muscarinic acetylcholine receptor-gated K+ channel in isolated guinea-pig atrial myocytes

Summary Effects of verapamil on the acetylcholine (ACh)-induced K⁺ current were examined in single atrial cells, using the tight-seal whole-cell clamp technique. The pipette solution contained guanosine-5-triphosphate (GTP) or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolysable GTP analogue). In GTP-loaded cells, ACh induced a specific K⁺ current, which is known to be mediated by pertussis toxin-sensitive GTP-binding (G) proteins. Verapamil (0.1–100 M) depressed the ACh-induced K⁺ current in a concentration-dependent fashion. In GTP-S-loaded cells, the K⁺ current remained persistently after wash-out of ACh, probably due to irreversible activation of G proteins by GTP-S. Verapamil (0.1–100 M) also depressed the intracellular GTP-S-induced K⁺ current. However, the magnitude of verapamil-depression of the K⁺ current in GTP-S-loaded cells was significantly smaller than that in GTP-loaded cells at concentrations between 1 and 10 M of the drug. From these results, it is suggested that verapamil may block not only the function of muscarinic ACh receptors but also of G proteins and/or the K⁺ channel itself and thereby depress the ACh-induced K⁺ current in isolated atrial myocytes.Supported by grants from the Ministry of Education, Science and Culture of Japan and the Research Program on Ca Signal Control Send offprint requests to Y. Kurachi at the above address     

FARP2 triggers signals for Sema3A-mediated axonal repulsion

Sema3A, a prototypical semaphorin, acts as a chemorepellent or a chemoattractant for axons by activating a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-A1 as the signal-transducing subunit. How the signals downstream of plexin-A1 are triggered upon Sema3A stimulation, however, is unknown. Here we show that, in the presence of neuropilin-1, the FERM domain-containing guanine nucleotide exchange factor (GEF) FARP2 associates directly with plexin-A1. Sema3A binding to neuropilin-1 induces the dissociation of FARP2 from plexin-A1, resulting in activation of FARP2's Rac GEF activity, Rnd1 recruitment to plexin-A1, and downregulation of R-Ras. Simultaneously, the FERM domain of FARP2 sequesters phosphatidylinositol phosphate kinase type I isoform PIPKIgamma661 from talin, thereby inhibiting its kinase activity. These activities are required for Sema3A-mediated repulsion of outgrowing axons and suppression of neuronal adhesion. We therefore conclude that FARP2 is a key molecule involved in the response of neuronal growth cones to class-3 semaphorins.     

Population study of expression of thymidylate synthase and dihydropyrimidine dehydrogenase in patients with solid tumors

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) has been suggested to be sensitivity-limiting factors of 5-fluorouracil therapy in cancer patients. We conducted a large-scale population study on the activity of TS and DPD in patients with various solid tumors. A total of 2590 clinically removed tumors, consisting of 1112 colon, 724 gastric, 520 breast, and 236 non-small cell lung cancers, were provided to measure TS and DPD activity. TS activity in the gastric, colon, and non-small cell lung cancers was significantly higher than in matched non-cancerous tissue (P<0.0002), but there was no difference in TS expression between tumor and non-cancerous tissue from breast cancer patients. Gastric, breast, and non-small cell lung cancers showed significantly higher DPD activity than their corresponding non-cancerous tissues, but colon cancers did not. There was no correlation between TS activity and DPD activity, and thus each enzyme was considered to be an independent sensitivity-limiting factor for 5-fluorouracil therapy. The median TS activity and median DPD activity in all specimens including gastric, colorectal, breast, and non-small cell lung cancers tested were 0.041 and 110.1 pmol/mg protein, respectively. We classified each of the type of carcinoma into 4 groups by using the median activity of TS and DPD as the cutoff values: a low TS/low DPD group, high TS/low DPD group, low TS/high DPD group, and high TS/high DPD group. About 50% of the gastric, 47% of the colon, 70% of the breast and 30% of the non-small cell lung cancers had high TS activity, and 60% of the gastric, 40% of the colon, 48% of the breast, and 87% of the lung cancers had high DPD activity. Moreover, breast cancer was characterized by high TS activity and lung cancer by high DPD activity as compared with gastric and colon cancers, and their high activity levels may influence to the effectiveness of 5-fluorouracil against cancers of these organs. The results for expression of TS and DPD in clinically dissected tumors would be useful to estimate the efficacy of 5-fluorouracil in the treatment of cancer patients.     

Glyceraldehyde 3-phosphate dehydrogenase is a novel autoantigen leading autoimmune responses to proliferating cell nuclear antigen multiprotein complexes in lupus patients

Using 2-dimensional electrophoresis and ion-pair chromatography, we have identified elements of proliferating cell nuclear antigen (PCNA) multiprotein complexes that are reactive to antibodies in sera from patients with systemic lupus erythematosus. Among the various elements of the complexes, a 37 kDa protein (PI 8.5) that specifically reacted with SLE sera, but not with sera from patients with other connective tissue diseases, was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Immunoblot analysis showed that SLE sera reactive with the 37 kDa protein specifically reacted with GAPDH, as did anti-GAPDH mAbs. The purified autoantibodies to GAPDH from lupus serum showed both nuclear speckled and cytoplasmic staining patterns in immunofluorescence on Hep-2 cells. In addition, enzyme-linked immunosorbent assay (ELISA) revealed the presence of anti-GAPDH autoantibodies in 47% of lupus patients. Longitudinal analysis of the reactivity of lupus sera to PCNA complexes showed the autoimmune response to spread from GAPDH to other elements of PCNA complexes, and the presence of anti-GAPDH antibodies was significantly correlated with increased levels of serum PCNA. Taken together, these findings suggest that GAPDH interacting with PCNA in association with its cellular function is a novel autoantigen recognized by lupus sera, and that GAPDH thus plays an important role in the induction of autoimmune responses against the PCNA complex.     

Expression of interferon-gamma-inducible protein-10 in human endometrial stromal cells

Human endometrial stromal cells (ESC) can produce a variety of chemokines, especially after inflammatory stimulation. Interferon-gamma-inducible protein-10 (IP-10) is a potent chemoattractant for lymphocytes, and belongs to the family of non-ELR CXC chemokines. The expression of IP-10 in ESC after stimulation with interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), or lipopolysaccharide (LPS) was evaluated using an enzyme-linked immunosorbent assay and Northern blot analysis. A small amount of IP-10 protein was detected in the culture media of unstimulated ESC. The expression of IP-10 mRNA was detected in ESC. IFN-gamma, IL-1 beta, TNF-alpha and LPS significantly stimulated the expression of IP-10 mRNA and protein in ESC. These results suggest that the production of IP-10 by ESC is regulated by inflammatory mediators. The modulation of IP-10 concentrations in the local environment may contribute to the normal and pathological processes of human reproduction by regulating leukocyte trafficking in the endometrium.     

Translocation (1;22)(p36;q11.2) with concurrent del(22)(q11.2) resulted in homozygous deletion of <Emphasis Type="Italic">SNF5/INI1</Emphasis> in a newly established cell line derived from extrarenal rhabdoid tumor

Malignant rhabdoid tumor (MRT) is a highly malignant pediatric cancer, which arises in various sites such as the kidney, brain, and soft tissues. Cytogenetic studies have revealed alterations of 22q11 in MRT. Recently, deletions and mutations of the SNF5/INI1 locus in 22q11.2 have been reported in MRT, suggesting that SNF5/INI1 is a tumor suppressor gene for MRT. Here we report our molecular cytogenetic study for a newly established cell line from extrarenal MRT with t(1;22)(p36;q11.2). Consequently, the reciprocal translocation was associated with the interstitial deletion of a small segment including SNF5/INI1, and another, chromosome 22, showed terminal deletion, the breakpoint of which was located 70–80 kb centromeric to SNF5/INI1, resulting in homozygous deletion of SNF5/INI1 in this cell line.     

Histopathological study on the PTHrP-induced incisor lesions in rats

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). The present study elucidates the histopathological characters of incisor lesions in the HHM rat model. Nude rats were implanted with PTHrP-expressing tumor (LC-6) cells, maintained for 12 weeks, after which the mandibular incisors were collected. Incisor fractures were observed grossly. Microscopically, hypercalcified dentin, dentin niche with osteodentin, and thinning of dentin were observed. Hypercalcified dentin was observed as a basophilic line of calcified dentin without associated odontoblastic changes, whereas dentin niche and thinning of dentin occurred with osteodentin and loss of cell height, respectively. In contrast with hypercalcified dentin, which was distributed throughout the dentin, dentin niche and thinning of dentin were localized to the labial area of the apical and middle region, and to the labial and lingual areas of the middle and incisal region, respectively. These results suggest that hypercalcemia affected the entire calcification process resulting in hypercalcified dentin, and that high PTHrP concentrations affected selective populations of odontoblasts resulting in formation of dentin niche and thinning of dentin. The localization of dentin niche and thinning of dentin also suggest that PTHrP may also be involved odontoblastic development in the rat.     

Roles of the Maltese cross form in the development of parasitemia and protection against Babesia microti infection in mice

Babesia microti, a hemoprotozoan parasite of rodents, is also important as a zoonotic agent of human babesiosis. The Maltese cross form, which consists of four masses in an erythrocyte, is characteristic of the developmental stage of B. microti. Monoclonal antibody (MAb) 2-1E, which specifically recognizes the Maltese cross form of B. microti, has been described previously. In the present study, we examined the roles of the Maltese cross form during the infectious course of B. microti in mice. The number of the Maltese cross form increased in the peripheral blood of infected mice prior to the peak of parasitemia. With confocal laser scanning microscopy, MAb 2-1E was found to be reactive with the ring form, with the parasites undergoing transformation to the Maltese cross form and subsequent division, and also with extracellular merozoites. Furthermore, the Maltese cross form-related antigen (MRA) gene was isolated from a B. microti cDNA library by immunoscreening with MAb 2-1E, and the nucleotide sequence was determined. Genomic analyses indicated that the MRA gene exists as a single-copy gene in B. microti. Immunization of mice with recombinant MRA induced significant protective immunity against B. microti infection. These findings indicate that the Maltese cross form plays important roles in both the development of parasitemia and the protective response against the infection.