Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line

We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p < 0.01; one-way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.     

A case with multiple myeloma in which serum forms gel precipitation upon exposure to air

We present the case of a 69-years-old man who was admitted to hospital with multiple myeloma. IgG-kappa type monoclonal protein was detected in the serum. When we separated the serum obtained from blood sample of the patient and the lid of the collecting tube was opened, the patient's serum became gelled immediately. When the lid of the collecting tube remained closed, the patient's serum did not become gelled even at 4 degrees C. Moreover, the gelled serum of the patient did not resolve at 56 degrees C. Taken together, these results indicated that gel formation of the patient's serum may not be due to cryoglobulin. It was found that the pH of the patient's serum elevated to pH 8.0 quickly after exposed to air. It was also found that the patient's serum, but not the sera of other IgG-kappa multiple myeloma patients, became gelled as soon as PBS of pH 8.0 was added. These results highly suggest that the patient's serum becomes gelled at pH 8.0. However, the isoelectric focusing of isolated precipitation in the patient showed fractions around the pH 8.5-8.7 zone, which was different from the pH at which the precipitation began to form. We think that this may be the first report of a multiple myeloma patient whose serum becomes gelled after exposed to air.     

Effect of a spider toxin (JSTX) on excitatory postsynaptic current at neuromuscular synapse of spiny lobster

1. We studied the blocking properties of a spider (Nephila clavata) toxin (JSTX) purified from venom on the spiny lobster neuromuscular junction. 2. When a small amount of JSTX was applied to the neuromuscular junction, the excitatory postsynaptic potential (EPSP) was partially suppressed. The amplitude of EPSPs remained at a steady level for several hours during the washing of the preparation, showing that the action of JSTX is irreversible. 3. We recorded the excitatory postsynaptic current (EPSC) from synaptic site using a macro-patch electrode. The amplitude of EPSC increased linearly with hyperpolarization of the membrane potential in the presence and absence of JSTX. 4. The decay phase time constant of EPSC and spontaneous EPSC was decreased by hyperpolarizing the membrane potential both in the absence and in the presence of JSTX. The relationship between the decay time constant and the membrane potential was not modified by JSTX. 5. It is suggested that JSTX irreversibly blocks EPSC by acting on the site that is apart from the ionic channel of the glutamate receptor molecule.     

Analysis of proteins in urinary tract stones and urine of urolithic patients

In order to investigate the mechanism of urinary tract stone formation, we analyzed protein components in urine and the stone. Urinary proteins of healthy subjects and urolithic patients as well as protein components urinary tract stone of the urolithic patients were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic patterns of urinary proteins of the patients differed from those of healthy subjects after separating protein patterns into those larger than 66kDa or smaller than 30kDa. Protein constituents of urinary tract stone were mainly separated into 18 bands ranging from 26.8 to 143 kDa. Major bands among these 18 bands differed among stones from different patients. On western blotting, the developed intensities of Tamm-Horsfall protein (THP) were fainter than those of healthy subjects. Whereas intensities of albumin (ALB) were stronger than those of healthy subjects. Moreover, blotting patterns of THP of the patients on non-reducing SDS-PAGE were obviously broad. Thus, we suggest that analysis of fractionated urinary proteins or protein components of urinary tract stone may provide a tool for monitoring the prognosis or relapse in the patients.     

The expression of neuronal nitric oxide synthase and dystrophin in rat regenerating muscles

We investigated the expression of neuronal nitric oxide synthase (nNOS) and dystrophin in the regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis by immunohistochemical studies and western blot analysis. In normal muscles, nNOS was moderately immunostained on type 2B fibers, but was faintly immunostained on type 2A or type 1 fibers. In immunohistochemical studies of regenerating muscles, nNOS was first observed at the sarcolemma of type 2B fibers on day 10, when the type discrimination between types 2A and 2B was first detected by ATP reactions. Subsequently, the immunostaining of nNOS grew progressively stronger in type 2B fibers, with faint staining in type 2A and type 1 fibers until day 28. Meanwhile, the immunostaining of dystrophin grew stronger equally in all three fibers until day 21. In western blot analysis of regenerating muscles, nNOS regenerated more slowly than dystrophin. The present data suggest that the expression of nNOS is related to the muscle fiber type differentiation, and that the role of nNOS is related to the function of the type 2B fibers of the muscle. This revised version was published online in July 2006 with corrections to the Cover Date.