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A novel blocking enzyme-linked immunosorbent assay (BL-ELISA) was developed for detection of antibodies to human group C rotavirus (CHRV). The specificity of the BL-ELISA was confirmed by using animal sera hyperimmunized to group A and group C rotaviruses and paired sera from five patients with acute CHRV gastroenteritis. Furthermore, there was concordance between the BL-ELISA and a neutralization assay for CHRV in 226 (95%) of 238 samples. By using the BL-ELISA, we determined the seroprevalence of CHRV in 704 serum samples obtained from nine different age groups of inhabitants of Okayama Prefecture, Japan, in 1992, 1994, and 1996. As a result, 211 sera (30%) were found to be positive for CHRV antibodies. The seroprevalence gradually increased with age and reached 52.7% in the oldest individuals. A further analysis of the youngest age group suggested that CHRVs predominantly prevail in persons older than 3 years of age in Japan. When comparing the three sampling years, a larger percentage of antibody-positive sera was detected in 1994 than in either 1992 or 1996 in individuals between 6 and 15 years of age, reflecting the occurrence of a CHRV outbreak among children during the winter of 1992 to 1993 that was previously documented. These results indicate that CHRV infections may occur more frequently in spite of the relatively low detection rate of the virus.

1The cloned α1‐subunits of the smooth muscle Ca2+channel (α1C‐b) from rabbit lung were expressed in Chinese hamster ovary cells. The effect of large depolarizations was examined using cell‐attached patch clamp techniques.2After large, long‐duration depolarizations (to +80 mV, 4 s), the cloned smooth muscle Ca2+channels were still open, and also showed slow channel closure upon repolarization. The sum of unitary channel currents revealed that the tail current seen after large conditioning depolarizations had a slower deactivation time constant compared to that seen when the cell membrane was depolarized briefly with a test step (to +40 mV), suggesting that large depolarizations transform the conformation of the Ca2+channels to a second open state.3The decay time course of the tail current induced by large conditioning depolarizations was prolonged by reducing the negativity of the repolarization step, and vice versa.4Using the slow deactivating characteristic, the current‐voltage relationship was directly measured by applying a ramp pulse after a large depolarization. Its slope conductance was approximately 26 pS.5Since the patch pipettes contained Ca2+agonists, the transition of the Ca2+channel conformation to the second, long open state during a large depolarization was distinct from that caused by Ca2+agonists, suggesting that the cloned α1‐subunits of smooth muscle Ca2+channels preserve the characteristic features seen in native smooth muscle Ca2+channels.6In addition, when skeletal muscle β‐subunits were coexpressed with the α1‐subunits, the long channel openings after large, long‐duration depolarizations were frequently suppressed. This phenomenon could be explained if the skeletal muscle β‐subunits increased the inactivation rate during the preconditioning depolarization.

We report the detailed study of mechanically induced free radical (mechanoradical) formation of glucose-based polysaccharides such as cellulose and amylose based on electron spin resonance (ESR) on its comparison with plasma-induced radicals of polysaccharides. The observed ESR spectra of mechanically fractured samples by ball milling at room temperature have shown the multicomponent spectra, which differ in pattern from those of plasma-irradiated cellulose but are similar to those of plasma-irradiated amylose. The systematic computer simulations disclosed that the observed spectra of cellulose consist of three kinds of spectral components, an isotropic doublet (I) assigned to a hydroxylalkyl-type radical at C1, an anisotropic doublet of doublets (II) assigned to an acylalkyl-type radical at C2 and/or C3 as discrete components, and a singlet spectrum (III) assigned to dangling-bond sites (DBS), while those of amylose consist of two kinds of spectral components, I and III. One of the most intriguing facts is that the component radicals are all glucose-derived mid-chain alkyl-type radicals as in the case of plasma irradiation, although it is known that mechanoradicals are produced by the polymer main-chain scission. It can be reasonably assumed, therefore, that the mechanoradicals primarily formed by 1,4-glucosidic bond cleavage of polysaccharides at room temperature underwent a hydrogen abstraction from the glucose units to give rise to the glucose-derived mid-chain alkyl-type radicals. Furthermore, spectrum III was a major component in the simulated spectra of both cellulose and amylose, unlike those in the case of plasma irradiation, suggesting that cross-linking reactions simultaneously occur accompanied by a decrease in the molecular weight in the course of vibratory milling.

We evaluated the effect of the antioxidant, probucol, on the cytotoxic effects of oxidized low density lipoprotein (OX-LDL) or of cumene hydroperoxide (CumOOH) on cultured bovine endothelial cells (EC). The addition of CumOOH to EC caused the release of lactate dehydrogenase and the accumulation of thiobarbituric acid-reacting substances (TBARS), effects that were protected against by preincubation with either probucol or tocopherol. Similarly, preincubation of EC with those antioxidants protected against OX-LDL toxicity and the accumulation of TBARS. The content of probucol in EC measured by high performance liquid chromatography was directly correlated with the extent of protection against OX-LDL toxicity. We also found that treatment of EC with serum from patients receiving treatment with probucol resulted in the detection of probucol in the cells. We conclude that probucol is transported and incorporated into EC membranes to act as a radical-trapping antioxidant, protecting the EC against oxidative stress. Our results also indicate that lipid peroxidation in cellular membranes involves cell injury inflicted by OX-LDL.

A quadrupole-based laser ionization mass spectrometry system was developed by combining a commercial quadrupole mass filter with a laser microprobe instrument, which employs a pulse generator that synchronizes the laser pulse with the quadrupole mass scan to detect the pulsed ion signals generated by laser induced ionization. Mass spectra were measured for several solid samples of pure metals (Al,Cu), metal alloys (Inconel 601, brass), and ceramics (BN). Reproducible spectra, with relative standard deviations of the ion signals less than 1%, were obtained with this system. Moreover, isotope abundance ratios were measured and compared with the natural abundance ratios.

Advanced glycation endproduct (AGE) accumulation in extracellular matrix proteins has been demonstrated in diabetic patients with a significant correlation with the severity of diabetic complications. AGE accumulation induces matrix protein cross-link formation, resulting in an increased stiffness of matrix fibres and the reduction of the susceptibility of matrix proteins to proteolytic degradation. We examined whether glycation-induced collagen cross-linking may affect vascular endothelial cell behaviours such as invasion, proliferation and differentiation, using the in vitro angiogenesis model of capillary-like structure formation in three-dimensional matrices of collagen type I. Endothelial cells cultured on collagen gel with angiogenic factors (the combination of fibroblast growth factor-2 and vascular endothelial growth factor) invaded the underlying collagen matrix, and organized capillary-like cord structures in the gel. We found that endothelial cell invasion into glycated collagen gel was significantly attenuated without any effect on proteinase activity including cell-associated plasminogen activator and matrix metalloproteinase in the conditioned medium. In addition, subsequent capillary-like cord formation was also inhibited in glycated collagen gel. In contrast, endothelial cell proliferation was enhanced on glycated collagen gel with or without angiogenic factors compared with control collagen gel. These results suggest that the structural alterations of extracellular matrix proteins through the glycation-induced cross-link formation affect the interaction between endothelial cell and extracellular matrix, resulting in the impairment of an adequate neovascularization in diabetic patients. [Diabetologia (1998) 41: 491–499]Advanced glycation endproduct (AGE) accumulation in extracellular matrix proteins has been demonstrated in diabetic patients with a significant correlation with the severity of diabetic complications. AGE accumulation induces matrix protein cross-link formation, resulting in an increased stiffness of matrix fibres and the reduction of the susceptibility of matrix proteins to proteolytic degradation. We examined whether glycation-induced collagen cross-linking may affect vascular endothelial cell behaviours such as invasion, proliferation and differentiation, using the in vitro angiogenesis model of capillary-like structure formation in three-dimensional matrices of collagen type I. Endothelial cells cultured on collagen gel with angiogenic factors (the combination of fibroblast growth factor-2 and vascular endothelial growth factor) invaded the underlying collagen matrix, and organized capillary-like cord structures in the gel. We found that endothelial cell invasion into glycated collagen gel was significantly attenuated without any effect on proteinase activity including cell-associated plasminogen activator and matrix metalloproteinase in the conditioned medium. In addition, subsequent capillary-like cord formation was also inhibited in glycated collagen gel. In contrast, endothelial cell proliferation was enhanced on glycated collagen gel with or without angiogenic factors compared with control collagen gel. These results suggest that the structural alterations of extracellular matrix proteins through the glycation-induced cross-link formation affect the interaction between endothelial cell and extracellular matrix, resulting in the impairment of an adequate neovascularization in diabetic patients. [Diabetologia (1998) 41: 491–499]

Plasma-induced low-density polyethylene (LDPE) radicals were studied in detail by electron spin resonance (ESR) by its comparison with ESR of high-density polyethylene (HDPE). The observed ESR spectra of plasma-irradiated LDPE are largely different in pattern from those of HDPE. The systematic computer simulation disclosed that such observed spectra consist of three kinds of radicals, midchain alkyl radical (1), allylic radical (2) as discrete radical species, and a large amount of dangling bond sites (DBS) (3) at an intra- and intersegmental cross-linked region. All these component radicals are essentially identical to those of HDPE. One of the most special features unique to plasma-irradiated LDPE, however, is the fact that thermally stable DBS (3) is a major component radical instead of a midchain alkyl radical in HDPE. This can be ascribed to the difference in polymer morphology between LDPE and HDPE:  branched structure with a large amount of amorphous region for LDPE and linear structure with a large amount of crystalline region for HDPE. Since one of the characteristics of plasma irradiation is the fact that it is surface-limited, LDPE would undergo the radical formation preferentially on the surface-branched structural moiety followed by facile cross-link reactions resulting in the formation of DBS. Thus, the nature of radical formation of PE was found to be affected by the polymer morphology in a very sensitive manner.

The nature of peroxy radical formation from plasma-induced surface radicals of polyethylene (PE), both low-density polyethylene (LDPE) and high-density polyethylene (HDPE), was studied by electron spin resonance with the aid of systematic computer simulations. It was found that peroxy radical formation varies with the structure of component radicals of plasma-irradiated PE, both LDPE and HDPE:  Among three plasma-induced radicals of PE, dangling bond sites (DBS) undergo an instant conversion into the corresponding peroxy radicals in contact with oxygen, while the midchain alkyl radical is of very low reactivity with oxygen in both LDPE and HDPE. Computer simulations disclosed that ESR spectra of peroxy radicals are similar to each other in LDPE and HDPE, both being composed of two types of spectra, a partial g-averaging anisotropic spectrum and a nearly isotropic single line spectrum due to different molecular motional freedom at the trapping sites of peroxy radicals.

Nucleotide sequences for the VP7 gene of human group C rotavirus were determined for two strains isolated in Okayama, Japan, during a 1988-1990 epidemic. These isolates, OK118 and OK450, were selected as prototypes of two different electropherotypes, patterns I and II, respectively. The genes were identical in size (1,063 bp), and both contained singled open reading frames encoding 332 amino acids. The alignment of two sequences revealed 46 nucleotide substitutions, 11 of which were predicted to give amino acid changes. The deduced amino acid sequence of VP7 from OK118 was similar to published sequences of a Japanese isolate and three foreign isolates (more than 98.4% identity), whereas the VP7 sequence of OK450 revealed around 96% identity with these isolates and had nine unique amino acid substitutions. The VP7 genes of nine Okayama isolated were than analyzed by dot blot hybridization with the VP7 probes of OK118 and OK450. Under highly stringent conditions, the OK118 probe produced strong hybridization signals with the genes of five pattern I strains and one pattern II strain, while the OK450 probe strongly reacted only with those of three pattern II strains. Our results concluded that relative sequence diversity in the VP7 gene was observed between two different electropherotypes prevalent in a limited area.

To determine the mechanism of the antiatherosclerotic effect of estrogen, we investigated the effect of estrogen on endothelial nitric oxide synthase (NOS-3). Preincubation with a physiologic concentration of 17 β-estradiol (10−12-10−8 M) over 8 hours significantly enhanced the activity of NOS-3 in endothelial cells of cultured human umblical vein (HUVEC) and of bovine aortas (BAEC). 17 β-estradiol also enhanced the release of nitric oxide (NO) as measured by an NO selective meter and NO−₂/NO−₃ metabolites of NO, from endothelial cells. Western blot showed a similar effect of 17 β-estradiol on NOS-3. The estrogen receptor antagonists, tamoxifen and ICI182780, each inhibited the effect of 17 β-estradiol by 80%. The effect of 17 β-estradiol gradually decreased in cells beyond the 10th passage and was not significant in cells beyond the 16th passage. Immunocytochemistry showed the existence of estrogen receptor in HUVEC and BAEC (less than 5 passages) and the sparseness of the existence in BAEC beyond the 16th passage. Estrogen increases NOS-3 via a receptor-mediated system, and estrogen receptor, which appeared to be altered by cell senescence, could be important in the release of NO from endothelium.Copyright 1995, 1999 Academic Press, Inc.

Reverse passive hemagglutination (RPHA) tests and a latex agglutination test with monoclonal antibodies (MAbs) were developed for the rapid detection of noncultivatable human group C rotaviruses. For RPHA tests, two MAbs, MAb 5A12 recognizing the outer capsid and MAb 13A3 recognizing the inner capsid, were separately used for the coating of sheep erythrocytes (SRBCs). Forty-six fecal samples were examined to confirm the practicality of the tests. As a result, there was concordance between the RPHA test with SRBCs coated with MAb 5A12 and polyacrylamide gel electrophoresis of viral RNA (RNA-PAGE) in 44 (95.6%) of 46 samples, while the diagnoses by the RPHA test with SRBCs coated with MAb 13A3 were in complete agreement with those by RNA-PAGE. Furthermore, a latex agglutination test with MAb 13A3 was also developed, and this test was fast enough and sensitive enough to successfully detect the viruses from most fecal samples within 2 min. The present procedures would be useful for the diagnosis of human group C rotavirus infections in clinical laboratories which are not well equipped.

Estrogen is known to retard the development of atherosclerosis and to work in the brain, but the mechanism of hormonal action is completely unknown. We investigated the effect of estrogen on the activity of neuronal constitutive nitric oxide synthase (NNOS). A low concentration of estrogen (10−10-7M) enhanced the activity of homogenates of the cytosol fraction of rabbit cerebellums and also that of partially purified NNOS, and high dose (10−6-5M) attenuated them. The study using estrogen receptor antagonists, tamoxifen, clomiphene, and ICI182780 suggested that estrogen receptor did not relate significantly to those effects of 17β-estradiol. 17α-estradiol or progesterone did not change significantly it in low doses, although moderately inhibited it in high doses. Estrogen enhanced the fluorescence of dansyl-calmodulin in low doses and attenuated it in high doses, suggesting that estrogen affects Ca2+-calmodulin directly. This study demonstrated that estrogen has a biphasic effect on the activity of NNOS through a Ca2+-calmodulin.

An enzyme-linked immunosorbent assay using monoclonal antibodies was established for the detection of human group C rotaviruses. Seventeen clinical samples which were found to contain group C rotaviruses were all strongly positive, whereas 9 samples containing group A rotaviruses and 51 samples lacking rotaviruses were all negative with this test.

Cell surface proteins of phase I cultures of Bordetella bronchiseptica strains from various species of animals were compared with those of isogenic phase III cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein band with a molecular mass of 74 kilodaltons was found only in various phase I cultures. This protein disappeared in parallel with antigenic modulation and had strong antigenicity. We found it to be an appropriate phase-shift marker for two reasons: it was readily extracted and recognized, and it was specifically identified by its distinct antigenicity.