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Clostridium perfringens is known to be a major food poisoning pathogen. Classical bacteriologic methods have been used to identify food poisoning outbreaks by this bacterium: isolation from contaminated food and/or the feces of patients and detection of the most important toxin, C. perfringens enterotoxin (CPE). Several molecular assays, mainly PCR (polymerase chain reaction) assays that detect the CPE gene, have recently been developed to identify type A C. perfringens food poisoning. This article will focus on novel insights into the epidemiology and genetics of C. perfringens food poisoning strains.

It's long been possible to give users outside an actual environment that environment's visual and auditory information and thus contribute to establishing presence. However, we've yet to establish much presence when users require olfactory information - such as in environments focused on foods, flowers, perfumes, or, in some cases, more offensive smells. Recently, several VR researchers have become interested in olfaction and olfactory displays that present smells in virtual environments (VEs). In this article, we describe our interactive olfactory display. One of our development goals is to confirm the assumption that users' interactions with the system increases presence. Thus, we used our interactive olfactory display to develop a cooking game in collaboration with electronic engineers and artists.

Summary Objectives: To apply an institutional clinical data warehouse (CDW) to the assessment of adverse drug reactions (ADRs) and demonstrate its utility through a specific example. Methods: We modeled the process for assessing ADRs through retrospective cohort design by using CDW at the Osaka University Hospital as follows: 1) We defined a drug X, an adverse drug reaction (ADR) Y, and a laboratory measurement Z to assess Y during a given study period; 2) we excluded those whose Z value exceeded the defined criteria or were not available at the inception of the cohort; 3) we divided the patients into two groups based on exposure or non-exposure to X; 4) we matched the patient characteristics between the two groups through stratification and randomization; and 5) we compared the frequency of patients who presented Y during the study period between the two groups. Aminoglycoside and Cephalosporin associated nephrotoxicity in pediatric inpatients was used as an example to demonstrate the usefulness of this approach. Results: Our evaluation indicates that there is an increased risk of nephrotoxicity for pediatric inpatients who were prescribed cephalosporin either alone or in combination with aminoglycoside; further, aminoglycoside tends to increase the cephalosporin-associated nephrotoxicity. Conclusions: Our findings are consistent with those drawn from other studies, indicating thatthe method of a pplying an institutional CDW is useful for assessing ADRs.

Ansaplastic thyroid cancer (ATC) is one of the most lethal malignancies, but the carcinogenic mechanism of ATC has not been clarified. Recently, we performed a cDNA microarray analysis and identified transmembrane protein 34 (TMEM34) that down-regulated in anaplastic thyroid cancer cell lines (ACL)s as compared to normal thyroid tissues. To investigate the role of TMEM34 in ATC carcinogenesis, we examined expression levels of TMEM34 in ACLs as well as differentiated thyroid cancers (DTC)s and normal human tissues. To explore the effect of TMEM34 in ATC development, cell-growth assays with KTA2 cells were performed. Expression of TMEM34 was down-regulated in all 11 ACLs, as compared to either normal thyroid tissues or cell lines derived from papillary or follicular thyroid cancers. TMEM34 was expressed ubiquitously in normal human tissues tested. Transfection of TMEM34 into KTA2 cells led to inhibition of cell growth. Our findings suggest that TMEM34 might be a tumor suppressor gene, associated with the development of ATC from DTC.

Male siblings with intrauterine growth retardation, hydrops, mild liver dysfunction, chronic diarrhoea, failure to thrive and microcephaly are reported. In both patients, the intrauterine growth retardation was detected in the second trimester of pregnancy. Relatively severe early onset neonatal jaundice, microcytosis, anisocytosis and abnormal iron metabolism were also seen. Bone marrow examination in the second sibling showed marked ringed sideroblasts and multilobulated erythroblasts in late developmental stages. The brain was very small with enlarged cerebrospinal fluid space, a reduced number of gyri and a thin cortex. The clinical and laboratory findings in these patients appear to be unique.

Deoxycholic acid (DCA) crystal was found to be amorphised easily by mechanical grinding at room temperature. X-ray and differential scanning calorimetry (DSC) experiments showed a glass transition, crystallisation, and subsequent fusion processes characteristic of a vitreous solid. A mixture of DCA and tri-O-methyl-β-cyclodextrin (TMCD) crystals also gave a halo pattern in the x-ray experiment after grinding. The DSC curve exhibited only a single glass transition in the vitrified binary solid. This indicates that the molecules of DCA and TMCD were mixed on a microscopic level, which yields a single structural relaxation in the vitreous solid. The devitrified solid gave double fusion processes, indicating a phase separation at the devitrification temperature. Thus, the formation of a molecular alloy was possible only under nonequilibrium conditions. Glucose which could not be amorphised by itself was forced to be vitrified by coexistence with TMCD crystal. The formation of molecular alloy was discussed in relation to the disorder of the systems.

Immunohistochemical staining of human skin specimen showed the stronger localization of proopiomelanocortin peptides near the suprabasal layer of the epidermis, where keratinocytes are mostly differentiated. To test the possibilities of whether the production of proopiomelanocortin peptides or their receptor-binding activity or both is increased during differentiation of keratinocytes, we treated the cells in culture with Ca2+ to induce their differentiation. The production of proopiomelanocortin peptides and its gene expression were not induced significantly, but the binding ability of melanocortin receptor, as well as its gene expression were stimulated by Ca2+. Ultraviolet B irradiation, an inducer of differentiation, stimulated both proopiomelanocortin production and melanocortin receptor expression. These data show that normal human keratinocytes express melanocortin receptor similar to melanocytes, and that it is induced during differentiation.

Melanocyte-stimulating hormone (MSH) receptor binding activity and melanocortin-1 receptor (MC1-R) gene expression on normal human melanocytes have been studied as responses to the effects of ultraviolet B (UVB), interleukin-1 (IL-1), endothelin-1 (ET-1) and tumour necrosis factor-alpha (TNF-alpha), which are known as UV sensitive regulators of melanocytic function. MSH receptor (MSH-R) binding activity was upregulated by UVB, IL-1alpha, -1beta and ET-1, but was downregulated by TNF-alpha. Northern blot analysis showed that MC1-R mRNA expression was induced 24 h after UVB irradiation in a dose-dependent manner, and that 24-h treatment with ET-1 also induced an expression of MC1-R mRNA, whereas TNF-alpha downregulated the expression. In addition, IL-1alpha and -1beta have a small but real inductive effect on MC1-R mRNA expression. Taken together, our results suggest a model in which higher MC1-R mRNA expression is accompanied by upregulation of MSH-R binding activity, and enhanced by UVB or cytokines sensitive to UVB. Such a regulatory system would enable normal human melanocytes to respond to MSH more efficiently and induce an increase of melanization of the skin through the MSH/MSH-R system after UVB radiation.

Melanocyte-stimulating hormone (MSH) has been reported to enhance the experimental metastatic behaviour of melanoma cells in the mouse model. alpha-MSH production and MSH receptor (melanocortin 1 receptor gene) expression have been detected in cultured normal human melanocytes and metastasized melanomas. The exact role of MSH in the metastatic behaviour of human melanoma cells is, however, not yet known. To clarify a possible role of proopiomelanocortin (POMC)-derived peptides, including alpha-MSH, in melanoma development and progression, we analysed immunohistochemically the localization of alpha-MSH adrenocorticotrophic hormone (ACTH) and beta-endorphin in various kinds of benign pigmented naevocytic lesions and malignant melanomas. Three of 21 samples of common and dysplastic naevi showed detectable alpha-MSH staining in naevus cells, and five and six of 15 samples were weakly positive for ACTH and beta-endorphin staining, respectively. In melanoma samples, 24 of 45, 23 of 39 and 30 of 42 samples showed positive staining with alpha-MSH, ACTH and beta-endorphin antibodies, respectively. Furthermore, staining for all three antibodies was noted to be more intense and diffuse in samples of nodular melanoma, vertically growing acral lentiginous melanoma and superficial spreading melanoma as well as metastatic lesions compared with those of naevi. Although it is yet to be determined whether or not this strong staining for POMC-derived peptides in advanced melanoma cells indicates a role of autocrine or paracrine regulation, our results suggest a possible involvement of POMC gene products in melanoma progression.

We report the identification of a putative v-SNARE (GOS-28), localized primarily to transport vesicles at the terminal rims of Golgi stacks. In vitro, GOS-28, A Golgi SNARE of 28 kD, is efficiently packaged into Golgi-derived vesicles, which are most likely COPI coated. Antibodies directed against GOS-28 block its ability to bind alpha-SNAP, partially inhibit transport from the cis to the medial cisternae, and do not inhibit budding of COP-coated vesicles, but do accumulate docked uncoated vesicles.

Staurosporine, a potent inhibitor of protein kinases, caused the rapid outgrowth of neurites from cultured dorsal root ganglia of chick embryos and from PC12D cells, a subline of PC12 cells. Treatment of dorsal root ganglia with 1 to 20 nM staurosporine resulted in the extensive outgrowth of neurites that were indistinguishable from those induced by NGF, as assessed by phase-contrast microscopy, electron microscopy and cytochemical staining of actin and tubulin. However, neurites generated from the ganglia in response to the higher concentrations of staurosporine (40-100 nM) seemed to have different characteristics, possibly as a result of the inhibition of cell migration from ganglia. The sequential changes in morphology of PC12D cells in response to staurosporine and to NGF were revealed by staining of actin. Ruffling membranes emerged at the margins of PC12D cells within 4 min after the addition of staurosporine or of NGF. From 10 min to 24 h after the addition of either compound, the ruffles were transformed into several projections that became growing neurites. The formation of ruffles and the outgrowth of neurites were both apparent at a concentration of staurosporine of 10 nM. The neurites that emerged from PC12D cells in response to staurosporine and in response to NGF were indistinguishable under the phase-contrast microscope and after staining of actin and tubulin. However, staurosporine never promoted survival of PC12D cells in serum-free conditions as that promoted by NGF. The observations indicate that staurosporine at nanomolar concentrations may reproduce the neurogenic changes that induced by NGF in primed neuronal cells, although it can not mimic the action of NGF that supports survival of neurons.

The exploration on seafloor soil is one of the first things to be carried out when the construction is planned and performed offshore. The data on the grain size distribution obtained by the exploration are imoportant to identify the mechanical properties of seafloor soil. In this paper an accoustic method to obtain the grain size distribution of seafloor soil is shown, where the ultrasonic transducers are used to acquire the frictional sound pressure generated between soil and sledge. Then, the empirical relationship between frictional sound pressure and grain size distribution is proposed. The grain size distribution is calculated by the empirical relation based on the assumption that the grain size distribution may be the logarithmic normal distribution.

Background: After small-for-size graft (SFSG) transplantation, elevated portal venous pressure (PVP) may lead to postoperative liver damage. Herein we evaluated the impact of portocaval shunt (PCS) to control PVP on liver grafts and intestine following SFSG transplantation. Methods: Nineteen SFSG transplantations were performed with 30% of native liver in swine. Swine were divided into 3 groups: a high-flow shunt group (HS: n = 7), in which portal venous flow (PVF) was reduced with a 10-mm diameter PCS; a low-flow shunt group (LS: n = 6), in which PVF was reduced with a 5-mm diameter PCS, and a no-shunt group (NS: n = 6), in which no PCS was placed. Results: Seven-day survivals were 83.3% in NS, 100% in LS and 0% in HS (p = 0.0088). PVP was significantly higher in the NS group (p = 0.0001; mean ± SEM NS/LS/HS: 20.5 ± 0.7/14.0 ± 1.2/11.6 ± 0.5 mm Hg). The LS group exhibited the highest compliance (PVF/PVP; NS/LS/HS 42.7 ± 10.9/44.6 ± 4.9/37.7 ± 8.3 ml/min/mm Hg; p = 0.009), the lowest aspartate aminotransferase (NS/LS/HS 562 ± 18/370 ± 55/720 ± 130 IU/l; p = 0.0493), and suppressed deleterious alternations of the hepatic parenchyma and intestinal mucosa. Conclusions: Portal hypertension after SFSG transplantation impaired liver and intestinal mucosa; however, inadequate portal flow impaired not only the liver, but also survival.

Summary The localization of calmodulin in testes of patients with idiopathic male infertility was studied using the indirect immunoperoxidase method. Specimens were obtained by testicular biopsy from 55 patients. They were divided into 26 cases of hypospermatogenesis, 11 cases of maturation arrest (8 of primary spermatocyte arrest and 3 of spermatid arrest) and 18 cases of Sertoli cell-only syndrome. Regardless of the type of testicular pathology, the types of immuno-reactive cell and the intensities of staining were the same as those in the normal testis. That is, staining for calmodulin was first found to be positive in early pachytene primary spermatocytes. It became intense in late pachytene primary spermatocytes and round spermatids. By contrast, elongated spermatids and spermatozoa were not stained. Sertoli cells were stained slightly or not at all. A calmodulin-staining index (CaM-S index) was defined as the proportion of primary spermatocytes that were stained intensely for calmodulin relative to the total number of primary spermatocytes. The indices for the testes of men with complete spermatocyte maturation arrest were significantly lower than those for the testes of normal controls and of men with hypospermatogenesis. Degenerating late pachytene spermatocytes observed in the testes of men with spermatocyte arrest showed low calmodulin-specific immunoreactivity. Such a decrease in numbers of normal late pachytene spermatocytes might be responsible for the low CaM-S index in cases of complete spermatocyte arrest.

The localization of calmodulin in the normal human testis was studied by the indirect immunoperoxidase method. Three different types of fixative were used: phosphate-buffered formalin, Bouin's solution or Carnoy's solution. Immunoreactivity specific for calmodulin was not detectable in the testis fixed in Carnoy's solution. The specimens fixed in phosphate-buffered formalin and in Bouin's solution were stained. The immunostaining for calmodulin was observed in pachytene spermatocytes, secondary spermatocytes and round spermatids but not in spermatogonia, or in pre-leptotene, leptotene and zygotene spermatocytes, elongated spermatids, spermatozoa or Leydig cells. Sertoli cells were not stained or were stained slightly. Among pachytene spermatocytes, the cells at the early stage were barely stained but those at the middle and late stages were slightly and intensely stained, respectively. The present report provides the first confirmation of the localization of calmodulin in the human testis.

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.

In April 1996, a 77-year-old man initially presented with fever, rash and polyarthralgia, and was diagnosed as having low titer cold agglutinin disease with acute hemolytic anemia. The patient's condition and laboratory findings improved after administration of corticosteroid (prednisolone 60 mg). In June 1996, however, he developed acute cholecystitis and died due to sepsis, disseminated intravascular coagulation and multiple organ failure. During the course, the levels of inflammatory cytokines such as TNF-alpha and IL-6 were correlated with the pathology, and the disease was diagnosed as systemic inflammatory response syndrome (SIRS). Autopsy revealed necrotizing cholecystitis, erythrophagocytosis in the liver, and cytomegalovirus infection in the lung and gall bladder. This was considered to be a rare case of low titer cold agglutinin disease complicated by SIRS.

We investigated the levels of various cytokines and soluble factors in ITP patients, in order to determine the influence of these factors on the pathogenesis of ITP. We found increases in IL-2, IL-6, IFN-gamma, and M-CSF levels in ITP patients compared with those in healthy individuals. On lymphocyte phenotype analysis, we found no clear difference in total T cell population (CD2+ CD19- cells) or cytotoxic T cell frequency (CD8+ CD11b- cells) between these two groups. The frequency of helper/inducer T cells (CD4+ CD8- cells) was decreased in ITP patients. There was a significant increase in activated T cells (CD3+ HLA-DR+ cells) in ITP patients. Furthermore, frequencies of NK cells of potent activity (CD16+ CD56+ cells) were significantly elevated in ITP patients. Seventeen of the 54 ITP patients (31.5%) had elevated levels of sFas, and 11 of the 54 patients (20.4%) of sFasL. In addition, a significant increase of sFasL was observed in sFas-positive ITP patients, and in these patients the sFasL level was correlated with that of sFas (r = 0.687, p0.01). We found significant increases in IL-2 and sIL-2R levels in sFas-positive ITP patients. For other factors examined, however, there were no differences in level between sFas-positive and -negative ITP patients. Percentages of activated T cells (CD3+ and HLA-DR+ cells) and NK cells (CD16+ and CD56+ cells) were significantly higher in sFas-positive ITP patients than in sFas-negative ITP patients. These findings suggests that the pathogenesis of ITP includes alteration of the Fas/FasL pathway.

PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui et al. reported that the ectopic expression of alpha1-antitrypsin variant Portland (alpha1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y. et al. (1998) EMBO J. 17, 4735-4743]. TGFbeta-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to alpha1-PDX, have been reported for PACE4. In this study, we examined whether alpha1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that alpha1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An alpha1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease(s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca(2+)-dependent protease with an optimal Ca(2+) requirement of 2 mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by alpha1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by alpha1-PDX causes abnormal embryogenic development.

Lymph follicles are frequently found on histological examination of a surgically removed gallbladder. The significance of these lymph follicles is not clearly understood. The aim of this study was to examine the clinicopathological correlation between the lymph follicles in the gallbladder morphologically and the mucosa-associated lymphoid tissue (MALT) in the gut.The gallbladders were fixed and cut serially. The tissue slices were processed in the routine manner for a histological examination. The histological criteria for MALT in this study was defined as the presence of lymph follicles with germinal centers in the lamina propria mucosae in approximately equal numbers in all portions of the gallbladders from the neck to the fundus. Biliary bile obtained at surgery was cultured for a bacteriological examination in the hospital laboratory. The types of gallstones were classified according to the Classification of Gallstones by the Japanese Society of Gastroenterology.Of the 1341 patients, 158 (11.8%) patients fulfilled the histological criteria, including 64 men and 94 women with an average age of 64.2 years. Gallstones were present in 89.2% of the patients, and 74.5% of these were calcium bilirubinate gallstones. Cultures of the bile were positive in 95.4% of the patients. A variety of bacterial species were thus found, most commonly Escherichia coli and Klebsiella spp. Grossly, the gallbladders usually showed a granular appearance of the mucosa.The MALT in the gallbladder is not a rare condition and is frequently encountered in clinical practice. This lymphoid tissue may represent a mucosal and morphological immune phenomenon for infection rather than a substrate for the development of low-grade B-cell lymphoma.

Clostridium perfringens epsilon-toxin bound to the Madin Darby canine kidney (MDCK) cells and aggregated. The complex of the toxin was formed in a dose- and a time-dependent manner. The formation of the complex increased with a decrease in viable counts of MDCK cells and with increasing K+ release from the cells. The inactivated toxin heated at 100 degrees C did not aggregate under the condition. In addition, the prototoxin dose-dependently bound to the cells, but did not form the complex. Incubation of the toxin with MDCK cell membranes also showed the formation of the complex, but that with membrane preparations prepared from Vero cells or sheep erythrocytes, which are insensitive for the toxin, showed no formation of the complex. Incubation of the toxin with mouse brain homogenates resulted in formation of the complex, but that with brain homogenates heated at 80 degrees C or mouse liver homogenates showed no formation of the complex. These observations show that the complex formation of epsilon-toxin is essential for toxicity of the toxin.

Melanocyte-stimulating hormone (MSH) receptor binding activity and melanocortin-1 receptor (MC1-R) gene expression on normal human melanocytes have been studied as responses to the effects of ultraviolet B (UVB), interleukin-1 (IL-1), endothelin-1 (ET-1) and tumour necrosis factor-alpha (TNF-alpha), which are known as UV sensitive regulators of melanocytic function. MSH receptor (MSH-R) binding activity was upregulated by UVB, IL-1alpha, -1beta and ET-1, but was downregulated by TNF-alpha. Northern blot analysis showed that MC1-R mRNA expression was induced 24 h after UVB irradiation in a dose-dependent manner, and that 24-h treatment with ET-1 also induced an expression of MC1-R mRNA, whereas TNF-alpha downregulated the expression. In addition, IL-1alpha and -1beta have a small but real inductive effect on MC1-R mRNA expression. Taken together, our results suggest a model in which higher MC1-R mRNA expression is accompanied by upregulation of MSH-R binding activity, and enhanced by UVB or cytokines sensitive to UVB. Such a regulatory system would enable normal human melanocytes to respond to MSH more efficiently and induce an increase of melanization of the skin through the MSH/MSH-R system after UVB radiation.

Advanced cancer of the pancreatic head, especially in its caudal portion, has a limited surgical resectability due to its frequent invasion to the superior mesenteric vein (SMV). A patient with advanced carcinoma of the pancreatic head with vascular invasion to the major bifurcation of the SMV, underwent a pancreatico-duodenectomy under a two-step passive bypass of the portal flow. The marginal colic veins were dilated and formed a long collateral route to the inferior mesenteric vein and the splenic vein due to stenosis of the SMV. The venous flow in this vein was then easily interrupted with the surgical manipulation of the transverse colon. The first passive bypass was thus inserted into the accessory right colic vein to preserve the venous return from both the small intestine and the colon, and also to enable surgical dissection of the invaded SMV itself. After a thorough dissection of the invaded SMV, a second bypass was inserted into the main trunk of the SMV through the dissected vascular stump. Subsequently the passive bypass was maintained until the portal reconstruction with an interposition of the left common iliac vein could be performed. The patient recovered well from the surgery without any complications throughout the postoperative course. In summary, the two-step portal bypass describes, is thus considered a stable and safe procedure for the reconstruction of the superior mesenteric vein, during pancreatoduodenectomy for advanced carcinoma of the pancreatic head.

A 76-year-old female with villous adenoma of the gallbladder is herein reported. She presented complaining of upper abdominal pain for some months prior to admission. No tumor mass could be detected by transabdominal ultrasonography, but CT scan demonstrated a sessile tumor measuring some 2 cm in diameter. A simple cholecystectomy was thus performed. The resected gallbladder was filled with mucinous material, and showed a sessile tumor with finger-like processes in the fundus. Both the macro- and microscopical findings of the lesion were almost identical to a typical villous adenoma of the colon. Little information is available on villous adenoma of the gallbladder, because such cases are extremely rare. In this paper, is described e a rare villous adenoma lesion while paying special attention to the morphological findings.

We have studied the cryopreservation of thyroid pieces for the purpose of autotransplantation. In this paper, the optimal freezing condition was investigated by comparing the survival cells of the cryopreserved thyroid pieces under various freezing conditions, including cooling rate, freezing medium, cryoprotectant, size of pieces and prefreezing incubation. The recovery regarded as survival rate was also evaluated. Based on the results, the optimal freezing condition was that 1 mm 3 of the thyroid pieces in the freezing medium, consisted of culture medium supplemented with 10% of fetal bovine serum and 10% of dimethylsulfoxide, were cooled slowly until -80 degrees C with prefreezing incubation at 4 degrees C for 1 hour and kept in liquid nitrogen for preservation. The number of survival cells under the optimal condition was 1.24-2.03 (1.71 +/- 0.40) x 10(6)/0.1 g tissue, and recovery was 25.2-58.0 (45.5 +/- 14.6)%. This high survival rate suggests the possibility that the cryopreserved thyroid pieces are utilized as the autografts maintaining the intact follicles.

In an experimental study using adult cats, we analyzed the distribution of the sympathetic postganglionic neurons in the superior cervical ganglion (SCG) which innervate the pupillary dilator and tarsal muscles to determine whether there are direct fiber projections from the spinal cord to the iridic and tarsal muscles. This study consisted of three experiments. In Experiments 1 and 2, respectively, horseradish peroxidase (HRP) was injected unilaterally into the iridic stroma or superior tarsal muscles. The SCG and spinal cord were then removed for microscopic analysis. In Experiment 3, the ganglion was examined microscopically after the injection of bisbezimide into the iris and of HRP into the ipsilateral superior tarsal muscle. Experiment 1 revealed more numerous HRP-labeled neurons in the rostral part than in any other region of the ipsilateral superior cervical ganglion. In Experiment 2, HRP-labeled neurons were evenly distributed over the whole ipsilateral SCG. No HRP-labeled neurons were detected in the spinal cord in either of these experiments. In Experiment 3, no doubly-labeled neurons were discernible in the ganglion. These results clearly indicate the localization of internal carotid nerve cells in a specific region of the SCG, and provide evidence for the absence of postganglionic neurons that innervate both the dilator muscle of the pupil and the tarsal muscles.

The localization of a GTP-binding protein G(o) was examined immunohistochemically in rat ileum using antibodies against the alpha-subunit of G(o) (G(o) alpha). G(o) alpha-positive fibers clearly demonstrated the topography and structure of the enteric nervous system of the ileum. G(o) alpha was present only in the nervous system and was generally distributed both in synapse-rich and in nonsynaptic areas. The distribution of G(o) alpha in a nonsynaptic area suggests the possibility that G(o) may be involved not only in neurotransmission mechanisms but also in other functions.

It is well known that precursor cleavage at paired basic amino acids (e.g., Lys-Arg, Arg-Arg) within the regulated secretory pathway is one of the key steps to produce bioactive peptides. On the other hand, we have recently shown that precursors with an Arg residue at the fourth residue upstream of the cleavage site besides the basic pair, i.e. with the Arg-X-Lys/Arg-Arg (RXK/RR) motif, are cleaved within the constitutive secretory pathway. To discriminate between the precursor cleavage at RXK/RR sites within the constitutive pathway and that at dibasic sites within the regulated pathway, we examined the effects of drugs affecting the secretory process, intracellular Ca2+ depletion, and a protease inhibitor on these cleavages. Chloroquine (a weak base), depletion of intracellular Ca2+ by A23187 (a Ca2+ ionophore), and the Pittsburgh-type mutant of alpha 1-protease inhibitor differentially affected these two cleavages. Brefeldin A, which impedes protein transport from the endoplasmic reticulum to the Golgi complex, inhibited both cleavages. Colchicine (an anti-microtubular drug) had no discernible effect on either cleavage. These observations support the notion that the precursor cleavages at dibasic and RXK/RR sites occur in different subcellular compartments, and are catalyzed by different processing endoproteases.

An enzyme-linked immunosorbent assay (ELISA) with antibodies specific to beta, epsilon and iota ib toxins of Clostridium perfringens was developed to detect beta, epsilon and iota ib toxins, respectively. The ELISA was sensitive enough to detect as little as 1.0 ng/ml of purified beta and iota ib toxins and 0.1 ng/ml of purified epsilon toxin. By means of the ELISA method, 192 isolates of C. perfringens from food samples in Japan and Thailand, and 58 isolates from patients suffering from gas gangrene or gastroenteritis were examined. One isolate from food samples in Japan, three from food samples in Thailand and five from stools of patients with gastroenteritis were C. perfringens type D. One type B and one type C were detected from the stools of patients with gastroenteritis.

Effects of glucose and ascorbic acid on in vitro collagen and DNA synthesis were evaluated in human umbilical vein endothelial cells. Ascorbic acid significantly potentiated collagen synthesis. Incubation of cells with elevated glucose concentration for 24h significantly reduced [3H]-thymidine uptake (2h). However, the addition of ascorbic acid (0.1 mM) dramatically prevented the inhibition of thymidine uptake. Based on these data, it is suggested that ascorbic acid supplementation in diabetics may prevent or ameliorate diabetic angiopathy.

The effects of an aldose reductase (AR) inhibitor, elevated glucose and other compounds were evaluated on in vitro 2-[3H] myo-inositol (MI) uptake in cultured human endothelial cells (ECs). Significant AR activity was present in ECs (1,373 +/- 170 mumol/mg.min: incubated with 28 mM glucose for 48 hr). Since Na(+)-deprivation and the addition of Ouabain (5 mM) significantly reduced MI uptake, MI incorporation into ECs might be dependent on an active transport system via Na(+)-K+ ATPase activity. MI uptake was reduced significantly (21 +/- 6, 39 +/- 7% reduction) in the presence of excess glucose (27.5, 55 mM). However, addition of the AR inhibitor (ONO-2235 100 microM) prevented the glucose mediated inhibition of MI uptake (15 +/- 5, 21 +/- 6% reduction). These results suggest that inhibition of AR might prevent glucose-mediated toxicity via an increment of MI uptake.

Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the virus into the cerebral ventricles at the late gestation. After deliveries, offspring were fed by the mothers until 4 weeks. Cystic brain lesions, regarded as porencephaly or paraventricular cysts, were observed in about 20% of the MCMV-injected offspring 3 to 4 weeks after birth. The porencephaly involved the cerebral cortex and the white matter, and sometimes opened to the ventricles, while the paraventricular cysts involved the white matter. The inner surfaces of the cysts were covered with thin monolayer cells. Around the cystic lesions, perivascular cuffings were sometimes observed in the meninges and the basal regions. Viral antigen-positive cells were observed in the cortex and the hippocampus but were hardly observed along the cystic walls. Immunohistochemical double staining using antibodies specific for the viral antigen and specific for factor VIII-related antigen showed that the brain capillary endothelial cells had susceptibility to MCMV infection, and in addition that some neurons in the cortex and the hippocampus had the same susceptibility. These findings suggest that there are at least two ways by which MCMV induce abnormalities in the developing mouse brains; migration of MCMV-infected neurons and affinity to the endothelial cells of the brain vessels to this virus.

Lethal and pressor activities, and the contractile responses of rat isolated ileum to Clostridium perfringens epsilon toxin, were significantly prevented by the prior administration of epsilon toxin inactivated by 1-ethyl-3-(3-diethyl-aminopropyl) carbodiimide in the presence of glycine methyl ester (EDC), 2,4,6-trinitrobenzene sulfonic acid (TNBS), succinic anhydride (SA) and ethoxyformic anhydride (EFA). However, the prior administration of the toxin inactivated by N-acetylimidazole (NAI), tetranitromethane (TNM) and N-bromosuccinimide (NBS) resulted in no inhibition of these biological activities. These data suggest that the toxin interacts with specific site(s) on target organs or tissues. The relationship between amino acid residues and the actions of the toxin is described.

The localization of GTP-binding protein (G-protein) subunits, Go alpha, Gi2 alpha and beta, in subcellular fractions of rat cerebral cortex was determined by means of immunoassays specific for the respective subunits. High concentrations of all three subunits were observed in both crude mitochondrial and microsomal fractions. Muscarinic cholinergic receptors were also densely localized in these fractions. Then the crude mitochondrial and microsomal fractions were subfractionated by sucrose density gradient centrifugation. Each fraction obtained was evaluated morphologically by electron microscopy and biochemically by determination of membrane markers. The crude mitochondrial fraction was subfractionated into myelin, synaptic plasma membrane, and mitochondrial fractions. All the G-protein subunits examined and muscarinic receptors were exclusively localized in the synaptic plasma membrane fraction. Among the submicrosomal fractions, the heavy smooth-surfaced microsomal fraction showed the highest concentrations of all G-protein subunits and receptors, while the rough-surfaced microsomal fraction contained low amounts of them. The heavy smooth-surfaced microsomal fraction also contained high specific activity of (Na(+)-K+)-ATPase, a marker of the plasma membrane. These results indicated that the Go alpha, Gi2 alpha and beta subunits are mainly localized in the plasma membrane in the brain.

It is generally accepted that preganglionic neurons of the cervical sympathetic nerve arise from the ciliospinal center, which exists in the spinal cord over C8-T3. However, its distribution within the spinal cord has not been exactly demonstrated. Therefore, horseradish peroxidase (HRP) was injected into the cervical sympathetic trunk in order to demonstrate the distribution of preganglionic neurons in the spinal cord in the cat. Four adult cats were used. HRP was injected into the superior cervical sympathetic trunk. Two or three days after the injection cats were sacrificed and prepared for histochemical demonstration of HRP. The superior cervical ganglion, sympathetic trunk and spinal cord were examined. Labelled cells with HRP were found in the intermediolateral nucleus over C8-T6 on the ipsilateral side of the injection. These neurons were identified as preganglionic neurons of the cervical sympathetic nerve. In addition, a large number of labelled cells were seen at the T2 and T3 levels, but a few labelled cells were observed at C8, T5 and T6. T1 and T4 showed the moderate distribution. Preganglionic neurons in the ciliospinal center were located at the level from C8 to T6 of the spinal cord.

Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.

Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the virus into the cerebral ventricles in the late stage of gestation; the brains of the offspring were than analyzed using the histological and immunohistochemical methods. Brains of the offspring, which were injected with relatively high titers of MCMV [1 X 10(4) plaque-forming units (pfu)] on day 13 of gestation exo utero or on day 15 of gestation in utero, showed massiv necrosis of the cerebral cortex with gliomesodermal proliferation around 9 to 10 days after birth. In these brains, viral antigen-positive cells were observed in zonal arrangement in the lesion-free cortex and in the hippocampus. Immunohistochemical double staining showed that some of the viral antigen-positive cells had also reacted with antibody to neuron-specific enolase at the same time, but had hardly reacted with antibodies to brain-type creatine kinase or glial fibrillary acidic protein. Brains of the offspring, which were injected with relatively low titers of virus (1 x 10(3) pfu) on day 15 of gestation, showed zonal arrangement of viral antigen-positive cells mainly in the cerebral cortex and in the hippocampus 7 days after birth, although the numbers of the positive cells were low. Fourteen days after birth, some of these offspring showed atrophy of the cerebral cortex and the hippocampus. These results suggest that some of the neuronal cells in the cerebral cortex and the hippocampus have special susceptibility to MCMV infection.

Hemolysins purified from non-O1 Vibrio cholerae (non-O1 hemolysin) and a Vibrio cholerae O1, biotype El Tor (El Tor hemolysin) were investigated for their homology. The hemolysins were isolated from the culture supernatant fluids by ammonium sulfate precipitation and gel filtration on Sephadex G-100 columns. The purified hemolysins gave single bands with an identical mobility on conventional polyacrylamide gel disc electrophoresis. The molecular weights of the non-O1 and El Tor hemolysins were estimated to be about 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the amino acid compositions of the hemolysins were very similar. The specific activities of the hemolysins were identical, and both hemolysins were neutralized to the same extent with antisera against the homologous and heterologous hemolysins. Ouchterlony double immunodiffusion tests with both hemolysins and antihemolysin serum gave a common (fused) precipitin line. These data indicate that the non-O1 hemolysin is biologically, physicochemically, and immunologically indistinguishable from the El Tor hemolysin.