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Obesity is associated with perturbations in incretin production and whole-body glucose metabolism, but the precise underlying mechanism remains unclear. Here, we tested the hypothesis that nicotinamide phosphoribosyltransferase (NAMPT), which mediates the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a key regulator of cellular energy metabolism, plays a critical role in obesity-associated intestinal pathophysiology and systemic metabolic complications. To this end, we generated a novel mouse model, namely intestinal epithelial cell-specific Nampt knockout (INKO) mice. INKO mice displayed diminished glucagon-like peptide-1 (GLP-1) production, at least partly contributing to reduced early-phase insulin secretion and postprandial hyperglycemia. Mechanistically, loss of NAMPT attenuated the Wnt signaling pathway, resulting in insufficient GLP-1 production. We also found that diet-induced obese mice had compromised intestinal NAMPT-mediated NAD+ biosynthesis and Wnt signaling pathway, associated with impaired GLP-1 production and whole-body glucose metabolism, resembling the INKO mice. Finally, administration of a key NAD+ intermediate, nicotinamide mononucleotide (NMN), restored intestinal NAD+ levels and obesity-associated metabolic derangements, manifested by a decrease in ileal Proglucagon expression and GLP-1 production as well as postprandial hyperglycemia in INKO and diet-induced obese mice. Collectively, our study provides mechanistic and therapeutic insights into intestinal NAD+ biology related to obesity-associated dysregulation of GLP-1 production and postprandial hyperglycemia., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glucocorticoid causes hyperglycemia, which is common in patients with or without diabetes. Prolonged hyperglycemia can be experienced even after the discontinuation of glucocorticoid use. In the present study, we examined the time course of blood glucose level in hospital patients who received transient glucocorticoid treatment. In addition, the mechanism of prolonged hyperglycemia was investigated by using dexamethasone (Dexa)-treated mice and cultured cells. The blood glucose level in glucose tolerance tests, level of insulin and glucagon-like peptide 1 (GLP-1), and the activity of dipeptidyl peptidase 4 (DPP-4) were examined during and after Dexa loading in mice, with histone acetylation level of the promoter region. Mice showed prolonged hyperglycemia during and after transient Dexa loading accompanied by persistently lower blood GLP-1 level and higher activity of DPP-4. The expression level of Dpp-4 was increased in the mononuclear cells and the promoter region of Dpp-4 was hyperacetylated during and after the transient Dexa treatment. In vitro experiments also indicated development of histone hyperacetylation in the Dpp-4 promoter region during and after Dexa treatment. The upregulation of Dpp-4 in cultured cells was significantly inhibited by a histone acetyltransferase inhibitor. Moreover, the histone hyperacetylation induced by Dexa was reversible by treatment with a sirtuin histone deacetylase activator, nicotinamide mononucleotide. We identified persistent reduction in blood GLP-1 level with hyperglycemia during and after Dexa treatment in mice, associated with histone hyperacetylation of promoter region of Dpp-4. The results unveil a novel mechanism of glucocorticoid-induced hyperglycemia, and suggest therapeutic intervention through epigenetic modification of Dpp-4., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Context: T-cadherin (T-cad) is a glycosylphosphatidylinositol (GPI)-anchored cadherin that mediates adiponectin to induce exosome biogenesis and secretion, protect cardiovascular tissues, promote muscle regeneration, and stimulate therapeutic heart protection by transplanted mesenchymal stem cells. CDH13, the gene locus of T-cad, affects plasma adiponectin levels most strongly, in addition to affecting cardiovascular disease risk and glucose homeostasis. Recently, it has been suggested that T-cad exists in human serum, although the details are still unclear., Objective: To validate the existence of T-cad forms in human serum and investigate the association with clinical parameters of type 2 diabetes patients., Methods: Using newly developed monoclonal antibodies against T-cad, pooled human serum was analyzed, and novel T-cad enzyme-linked immunosorbent assays (ELISAs) were developed. The serum T-cad concentrations of 183 Japanese type 2 diabetes patients were measured in a cross-sectional observational study. The main outcome measure was the existence of soluble T-cad in human serum., Results: There were 3 forms of soluble T-cad: a 130-kDa form with a prodomain, a 100-kDa mature form, and a 30-kDa prodomain in human serum. Using newly developed ELISAs to measure them simultaneously, we found that the 130-kDa form of T-cad positively correlated with plasma adiponectin (r = 0.28, P < .001), although a physiological interaction with adiponectin was not observed in serum. The unique 30-kDa prodomain was associated with several clinical parameters in diabetes patients., Conclusion: We identified 3 novel forms of soluble T-cad. Their importance as disease markers and/or biomarkers of adiponectin function and the possible bioactivity of the respective molecules require further investigation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Objectives: The effect of anthropometric factors on the fingertip vibrotactile perception threshold (VPT) of industrial vibrating tool operators (IVTOs) is not well known. The purpose of this study was to investigate the associations between anthropometric factors and fingertip VPT., Methods: We included for analysis two groups of IVTOs: Group 1, predominantly forestry workers (n=325); and Group 2, public servants (n=68). These IVTOs regularly received medical examinations to evaluate hand-arm vibration syndrome. In the examination, measurements of their fingertip VPTs were taken before and after cold-water immersion (10 minutes at 10°C for Group 1 and 5 minutes at 12°C for Group 2). Their body height and weight were measured to calculate the body mass index (BMI). The presence of peripheral neuropathy (PN) was defined as a VPT ≥17.5 dB at 10 minutes after finishing immersion., Results: In the univariate analysis, weight and BMI were associated with a decreased risk of PN in both Groups 1 and 2. The negative association between BMI and PN remained in the multivariate analysis consistently, but weight reached marginal significance only in the multivariate analysis without BMI in both the groups. Age was positively associated with PN consistently in Group 1 but not in Group 2. Years exposed to vibration showed positive association with PN only in the univariate analysis of Group 1., Conclusions: Among IVTOs, factors reflecting body heat production, such as weight and BMI, were associated with a decreased risk of VPT-defined PN, regardless of the task engaged.

Because a physical decline correlates with an increased risk of a wide range of disease and morbidity, an improvement of physical performance is expected to bring significant clinical benefits. The primary cause of physical decline in 5/6 nephrectomized (5/6Nx) chronic kidney disease model mice has been regarded as a decrease in muscle mass; however, our recent study showed that a decrease in muscle mitochondria plays a critical role. In the present study, we examined the effects of a gastric hormone ghrelin, which has been reported to promote muscle mitochondrial oxidation, on the physical decline in the chronic kidney disease model mice, focusing on the epigenetic modulations of a mitochondrial activator gene, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Ghrelin treatment improved a decline in exercise endurance of 5/6Nx mice, associated with an increase in both of the muscle mass and mitochondrial amount. The expression level of PGC-1α was decreased in the skeletal muscle of 5/6Nx mice, which was associated with an increase in the methylation ratio of the cytosine residue at 260 base pairs upstream of the initiation point. Conversely, ghrelin treatment de-methylated the cytosine residue and increased the expression of PGC-1α. A representative muscle anabolic factor, IGF-1, did not affect the expression of PGC-1α and muscle mitochondrial amount, although it increased muscle mass. As a result, IGF-1 treatment in 5/6Nx mice did not increase the decreased exercise endurance as effectively as ghrelin treatment did. These findings indicate an advantage of ghrelin treatment for a recovery of physical decline.

Aims: Alcohol-related disorders (ARD) have been shown to be accompanied by a variety of other comorbid mental disorders. This study evaluated the associations between a variety of mental disorders and genetic alcohol sensitivity., Methods: A total of 1944 Japanese workers were interviewed regarding their mental disorders by the Mini-International Neuropsychiatric Interview (M.I.N.I.). We investigated the relationship of ADH1B rs1229984 and ALDH2 rs671 polymorphisms' combination with mental disorder risks. Logistic regression analysis was used to evaluate the associations between those polymorphisms and mental disorders, adjusting for sex, age, and job rank., Results: The degree of alcohol sensitivity was classified into five groups according to the combination of ADH1B and ALDH2 genotypes (Group I-V in order starting from the lowest alcohol sensitivity). Those with ALDH2 *1/*1 and ADH1B *1/*1 or with ALDH2 *1/*1 and ADH1B *1/*2,*2/*2 (low sensitivity) were significantly or nearly significantly associated with an increased risk of ARD compared with those with ALDH2 *1/*2 and ADH1B *1/*2,*2/*2 as a reference. Those with ALDH2 *1/*1 and ADH1B *1/*1 were also likely to be at an increased risk of any mental disorder except ARD, as well as disorders without comorbid ARD. This tendency was more apparent among women (OR 11.94, 95% CI 0.73-195.63) and non-drinkers (OR 5.43, 95% CI 1.05-28.23)., Conclusion: The genotype combination of ALDH2 *1/*1 and ADH1B *1/*1 is significantly associated with an increased risk of any mental disorder, especially ARD. Non-drinkers or women with ALDH2 *1/*1 and ADH1B *1/*1 are likely to suffer from any mental disorder except ARD., (© The Author 2014. Medical Council on Alcohol and Oxford University Press. All rights reserved.)

A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein 3 (MCP-3, known as CCL7) a total of 33 541 genes in the spinal cord, which lasted for 4 weeks. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at Lys27 at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation. Consistent with these findings, a single intrathecal injection of recombinant proteins of interleukin 6 significantly increased MCP-3 messenger RNA with a decrease in the level of Lys27 trimethylation of histone H3 at the MCP-3 promoter in the spinal cord of mice. Furthermore, deletion of the C-C chemokine receptor type 2 (CCR2) gene, which encodes a receptor for MCP-3, failed to affect the acceleration of MCP-3 expression in the spinal cord after partial sciatic nerve ligation. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. On the other hand, the increases in both microglia and astrocytes in the spinal cord by partial sciatic nerve ligation were mostly abolished in interleukin 6 knockout mice. Moreover, this increase in microglia was almost abolished by CCR2 gene deletion, whereas the increase in astrocytes was not affected in nerve-ligated mice that lacked the CCR2 gene. We also found that either in vivo or in vitro treatment with MCP-3 caused robust microglia activation. Under these conditions, intrathecal administration of MCP-3 antibody suppressed the increase in microglia within the mouse spinal cord and neuropathic pain-like behaviours after nerve injury. With the use of a functional magnetic resonance imaging analysis, we demonstrated that a single intrathecal injection of MCP-3 induced dramatic increases in signal intensity in pain-related brain regions. These findings suggest that increased MCP-3 expression associated with interleukin 6 dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord and could play a critical role in the neuropathic pain-like state.

The dasD gene is located just downstream of the dasABC gene cluster, encoding components of an ABC transporter for uptake of a chitin-degradation product N,N'-diacetylchitobiose [(GlcNAc)(2) ] in Streptomyces coelicolor A3(2). To clarify the roles of the DasD protein in the degradation and assimilation of chitin, we obtained and characterized a recombinant DasD protein and a dasD-null mutant of S. coelicolor A3(2). The recombinant DasD protein produced in Escherichia coli showed N-acetyl-β-d-glucosaminidase (GlcNAcase) activity and its optimum temperature and pH were 40 °C and 7, respectively. dasD transcription was strongly induced in the presence of chitin, weakly by chitosan, but not by cellulose or xylan in S. coelicolor A3(2). Immuno-blot analysis demonstrated that DasD is a cytoplasmic protein. The dasD-null mutant exhibited cellular GlcNAcase activity which was comparable with that of the parent strain M145. DasD, thus, did not seem to be a major GlcNAcase. Induced extracellular chitinase activity in the dasD-null mutant was, interestingly, higher than M145, in the presence of colloidal chitin or (GlcNAc)(2) . In contrast to M145, (GlcNAc)(2) temporally accumulated in the culture supernatant of the dasD-null mutant in the presence of colloidal chitin., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Objectives: The aim of this study was to investigate the clinical characteristics of HAVS in a tropical environment in comparison with a temperate environment., Methods: We conducted a series medical examinations among the forestry, construction and automobile industry workers in Malaysia adopting the compulsory medical examination procedure used by Wakayama Medical University for Japanese vibratory tools workers. We matched the duration of vibration exposure and compared our results against the Japanese workers. We also compared the results of the Malaysian tree fellers against a group of symptomatic Japanese tree fellers diagnosed with HAVS., Results: Malaysian subjects reported a similar prevalence of finger tingling, numbness and dullness (Malaysian=25.0%, Japanese=21.5%, p=0.444) but had a lower finger skin temperature (FST) and higher vibrotactile perception threshold (VPT) values as compared with the Japanese workers. No white finger was reported in Malaysian subjects. The FST and VPT of the Malaysian tree fellers were at least as bad as the Japanese tree fellers despite a shorter duration (mean difference=20.12 years, 95%CI=14.50, 25.40) of vibration exposure., Conclusions: Although the vascular disorder does not manifest clinically in the tropical environment, the severity of HAVS can be as bad as in the temperate environment with predominantly neurological disorder. Hence, it is essential to formulate national legislation for the control of the occupational vibration exposure.

Lactoferrin (LF) is a multifunctional glycoprotein found in mammalian milk. We have shown in a previous clinical study that enteric-coated bovine LF tablets decreased visceral fat accumulation. To address the underlying mechanism, we conducted in vitro studies and revealed the anti-adipogenic action of LF in pre-adipocytes. The aim of this study was to assess whether LF could increase the lipolytic activity in mature adipocytes. Pre-adipocytes were prepared from rat mesenteric fat and differentiated into mature adipocytes for assays of lipolysis. The addition of LF significantly increased the glycerol concentration in the medium in a dose-dependent manner, whereas pepsin-degraded LF did not. A DNA microarray analysis demonstrated that LF decreased the expression of perilipin and affected the cAMP pathway. These findings are supported by the results of quantitative RT-PCR of perilipin and assays of cAMP. These data collectively indicate that visceral fat reduction by LF may result from the promotion of lipolysis and the additional anti-adipogenic activity of LF.

Background: Endothelial lipase (EL) regulates the metabolism of HDL cholesterol (HDL-C). However, the role of EL in regulating plasma HDL-C concentrations and EL's potential involvement in atherosclerosis in humans has not been fully investigated due to the lack of reliable assays for EL mass. We developed an ELISA system for serum EL mass., Methods: Human recombinant EL proteins, purified from cultured media of human EL-transfected Chinese hamster ovary cells, were used as antigen and calibrator. Two specific monoclonal antibodies were generated in mice against recombinant EL protein for a sandwich ELISA. We measured EL mass in human serum using EL recombinant protein as a calibration standard., Results: The EL antibodies did not cross-react with lipoprotein lipase and hepatic triglyceride lipase. The detection limit of the ELISA was 20 pg/mL, which is approximately 10 times lower than that of previous ELISA systems. Recovery of spiked EL in serum was 90%-105%. Assay linearity was intact with a >4-fold dilution of serum. Intra- and interassay CVs were <5%. The serum EL mass in 645 human subjects was [mean (SE)] 344.4 (7.7) pg/mL (range 55.2-1387.7 pg/mL). Interestingly, serum EL mass was increased in patients with diagnosed cardiovascular disease and inversely correlated with serum HDL-C concentrations. There was no difference in EL mass between pre- and postheparin plasma samples., Conclusions: This ELISA should be useful for clarifying the impact of EL on HDL metabolism and EL's potential role in atherosclerosis., (© 2012 American Association for Clinical Chemistry)

Objectives: The purpose of this study was to clarify the occurrence of Raynaud's phenomenon among workers using an impact wrench for a long time., Methods: The subjects were 704 workers regularly using an impact wrench and taking special medical examinations for vibration syndrome from 1981 to 2008. Raynaud's phenomenon was observed in 39 subjects during the observation period., Results: The mean operating years at the occurrence of Raynaud's phenomenon was 25.5 ± 8.3 (standard deviation) yr. The mean total operating time (geometric average) at the occurrence of Raynaud's phenomenon was 11,689 h. By the person-year method, the incidence rate of Raynaud's phenomenon was 6.27 persons per 1,000 person-years. The estimated risk of developing Raynaud's phenomenon did not increase until 12 years after starting to operate an impact wrench but increased exponentially after that. The vibration level of an impact wrench (from 4.9 m/s(2) to 22.6 m/s(2)) exceeded the occupational exposure limit value (4.9 m/s(2)). Various countermeasures, such as introducing automatically apparatus and keeping the working environment warm to protect from cold exposure, were taken at the factory., Conclusions: These findings showed that the rate of occurrence of Raynaud's phenomenon was not high, although the vibration level of the impact wrench was high. This may result from various countermeasures to prevent the occurrence of Raynaud's phenomenon. A long period of exposure to vibration had the potential to lead to the occurrence of Raynaud's phenomenon even under various countermeasures.

Unlabelled: The internationally accepted limit values and the health effects of hand-transmitted vibration exposure have been described extensively in the literature from temperate climate countries but not from a tropical climate environment., Objectives: We conducted a systematic review of the health effects of hand-transmitted vibration exposure in tropical countries to determine the characteristics of hand-arm vibration syndrome in a warm environment and compared the findings with the results of the systematic reviews published by the US NIOSH., Methods: We searched major medical databases including MEDLINE, PubMed, Embase, CINAHL, Ovid and Cochrane based on the terms "hand arm vibration syndrome," "hand transmitted vibration," "vibration white finger" and "Raynaud" up to January 2011. Only studies conducted in a tropical or subtropical environment were selected for the review. The quality of the selected papers was assessed independently by two investigators using predefined criteria. A standard set of information was abstracted from the papers for review., Results: Only six papers from tropical countries and three papers from subtropical countries were available in the literature. No vibration white finger was reported in the tropical countries. Neurological symptoms were prevalent in the vibration-exposed workers. Finger coldness seems to be an important surrogate for vascular disorder in a tropical environment. Meta-analysis could not be performed due to inadequacy of the information reported in these papers., Conclusions: The current dose-response relationship in ISO5349-1 for hand-transmitted vibration exposure is not applicable to a tropical environment. Further studies on hand-arm vibration syndromes in tropical countries are needed.

Context: Adrenal venous sampling is the "gold standard" test in the diagnosis of an aldosterone-producing adenoma (APA) among patients with primary aldosteronism (PA) but is available only in specialized medical centers. Meanwhile, an APA is reported to be generally more sensitive to ACTH than idiopathic hyperaldosteronism., Objective: The aim was to evaluate the diagnostic accuracy of the ACTH stimulation test in the diagnosis of an APA among those with suspicion of PA., Patients and Setting: Fifty-nine patients admitted to Kyoto University Hospital on suspicion of PA were included in the study., Interventions: ACTH stimulation tests with 1-mg dexamethasone suppression were performed., Main Outcome Measure: Plasma aldosterone concentrations (PAC) were examined every 30 min after ACTH stimulation. Receiver-operated characteristics curve analysis was used to evaluate the diagnostic accuracy., Results: PAC after ACTH stimulations were significantly higher in patients with an APA than in patients with idiopathic hyperaldosteronism or non-PA. Receiver-operated characteristics curve analyses showed that the PAC after ACTH stimulation was effective for the diagnosis of an APA among patients suspected of PA. The diagnostic accuracy was highest at 90 min after ACTH injection, with the optimal cutoff value greater than 37.9 ng/dl corresponding with sensitivity and specificity of 91.3 and 80.6% for the diagnosis of an APA., Conclusions: Our study indicates that the ACTH stimulation test is useful in the diagnosis of an APA among patients suspected of PA. This test can be used to select patients who are highly suspected of an APA and definitely require adrenal venous sampling.

Although Streptomyces species are major chitin-degraders in soil ecosystems, the expression of the diverse chitinase genes within Streptomyces coelicolor grown in soil has not been assessed. As a first step, the induction pattern of nine chitinase genes in S. coelicolor growing in autoclaved soil was compared with those in liquid cultures. The relative expression levels of nine chitinase genes were measured using real-time reverse transcription PCR. The expression of all chitinase genes was induced by chitin in both autoclaved soil and liquid cultures, but to different levels. The expression levels of five chitinase genes in autoclaved soil were significantly higher than those in the liquid cultures. In particular, a putative chitinase gene, chitinase H, showed the highest induction in autoclaved soil. The same induction pattern was confirmed in nonautoclaved soil, indicating that soil contains some factors affecting the expression of chitinase genes. The chiH gene product, ChiH, cloned in Streptomycetes lividans was secreted and exhibited chitin degradation activity that was stable within a wide range of acidic pHs. The disruption of dasR, a transcriptional regulator for the uptake of N-acetylglucosamine, abolished the expression of chiH, demonstrating that DasR is required for the regulation of ChiH expression., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 under the control of a modified cauliflower mosaic virus 35S promoter was introduced into a hybrid poplar (Populus tremula x P. tremuloides). Integration of the cbnA gene in transgenic poplar was confirmed by PCR and genomic Southern blot analysis. Expression of the cbnA gene was analyzed by Western blot analysis. Transgenic poplar calli efficiently converted 3-chlorocatechol to 2-chloro-cis,cis-muconate.

Mineralocorticoid receptors (MRs) are classically known to be expressed in the distal collecting duct of the kidney. Recently it was reported that MR is identified in the heart and vasculature. Although MR expression is also found in the brain, it is restricted to the hippocampus and cerebral cortex under normal condition, and the role played by MRs in brain remodeling after cerebral ischemia remains unclear. In the present study, we used the mouse 20-min middle cerebral artery occlusion model to examine the time course of MR expression and activity in the ischemic brain. We found that MR-positive cells remarkably increased in the ischemic striatum, in which MR expression is not observed under normal conditions, during the acute and, especially, subacute phases after stroke and that the majority of MR-expressing cells were astrocytes that migrated to the ischemic core. Treatment with the MR antagonist spironolactone markedly suppressed superoxide production within the infarct area during this period. Quantitative real-time RT-PCR revealed that spironolactone stimulated the expression of neuroprotective or angiogenic factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), whereas immunohistochemical analysis showed astrocytes to be cells expressing bFGF and VEGF. Thereby the incidence of apoptosis was reduced. The up-regulated bFGF and VEGF expression also appeared to promote endogenous angiogenesis and blood flow within the infarct area and to increase the number of neuroblasts migrating toward the ischemic striatum. By these beneficial effects, the infarct volume was significantly reduced in spironolactone-treated mice. Spironolactone may thus provide therapeutic neuroprotective effects in the ischemic brain after stroke.

Peripheral arterial diseases are caused by arterial sclerosis and impaired collateral vessel formation, which are exacerbated by diabetes, often leading to leg amputation. We have reported that an activation of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway accelerated vascular regeneration and blood flow recovery in murine legs, for which ischemia had been induced by a femoral arterial ligation as a model for peripheral arterial diseases. In this study, ip injection of carperitide, a human recombinant atrial natriuretic peptide, accelerated blood flow recovery with increasing capillary density in ischemic legs not only in nondiabetic mice but also in mice kept upon streptozotocin-induced hyperglycemia for 16 wk, which significantly impaired the blood flow recovery compared with nondiabetic mice. Based on these findings, we tried to apply the administration of carperitide to the treatment of peripheral arterial diseases. The study group comprised a continuous series of 13 patients with peripheral arterial diseases (Fontaine's classification I, one; II, five; III, two; and IV, five), for whom conventional therapies had not accomplished appreciable results. Carperitide was administrated continuously and intravenously for 2 wk to Fontaine's class I-III patients and for 4 weeks to class IV patients. The dose was gradually increased to the maximum, with the patient's systolic blood pressure being kept above 100 mm Hg. Carperitide administration improved the ankle-brachial pressure index, intermittent claudication, rest pain, and ulcers. In conclusion, this study showed a therapeutic potential of carperitide to treat peripheral arterial diseases refractory to conventional therapies.

Our work involves the design and synthesis of various types of bridged nucleic acid (BNA). Here, we describe a novel 2'-O, 5'-N-bridged nucleic acid, 2',5'-BNA(ON), whose sugar puckering is fixed to the S-type conformation by N-O linkage. A 2',5'-BNA(ON)-containing dimer unit was synthesized via a coupling reaction between a protected 2',5'-BNA(ON)-U monomer and a thymidine derivative. Introduction of 2',5'-BNA(ON)-U into DNA was carried out using conventional phosphoramidite chemistry with a DNA synthesizer. The hybridization abilities of 2',5'-BNA(ON)-U-modified oligonucleotides with their DNA or RNA complements were evaluated.

We found that soybean beta-conglycinin peptone (BconP) suppresses food intake through cholecystokinin (CCK) release from enteroendocrine cells in association with binding of the peptone to rat small intestinal brush border membrane (BBM). The aim of the present study was to find new appetite suppressing peptides. Peptones from chicken, pork, beef, beef liver, and egg white were examined for activities to bind with rat BBM, CCK-release from enteroendocrine cell line STC-1, and induce satiety in rats. Chicken and pork peptone (ChickP and PorkP) bound to BBM with highest ability as evaluated with a surface plasmon biosensor. PorkP and ChickP released CCK in higher amounts than BconP from STC-1 cells dose-dependently, with highest stimulation by PorkP. An orogastric preload of PorkP, but not ChickP, suppressed food intake similarly to BconP, dose-dependently. These results suggest that PorkP interacts directly with the small intestinal CCK cells to release CCK, and that it suppresses appetite in rats.

Background: Regular physical exercise may improve a variety of physiological and psychological factors in depressive persons. However, there is little experimental evidence to support this assumption for adolescent populations. We conducted a randomized controlled trial to investigate the effect of physical exercise on depressive state, the excretions of stress hormones and physiological fitness variables in adolescent females with depressive symptoms., Methods: Forty-nine female volunteers (aged 18-20 years; mean 18.8 +/- 0.7 years) with mild-to-moderate depressive symptoms, as measured by the Centre for Epidemiologic Studies Depression (CES-D) scale, were randomly assigned to either an exercise regimen or usual daily activities for 8 weeks. The subjects were then crossed over to the alternate regimen for an additional 8-week period. The exercise program consisted of five 50-min sessions per week of a group jogging training at a mild intensity. The variables measured were CES-D rating scale, urinary cortisol and epinephrine levels, and cardiorespiratory factors at rest and during exercise endurance test., Results: After the sessions of exercise the CES-D total depressive score showed a significant decrease, whereas no effect was observed after the period of usual daily activities (ANOVA). Twenty-four hour excretions of cortisol and epinephrine in urine were reduced due to the exercise regimen. The training group had a significantly reduced resting heart rate and increased peak oxygen uptake and lung capacity., Conclusions: The findings of this study suggest that a group jogging exercise may be effective in improving depressive state, hormonal response to stress and physiological fitness of adolescent females with depressive symptoms.

Streptomyces coelicolor A3(2) has 13 chitinase genes encoding 11 family 18 and two family 19 chitinases. To compare enzymatic properties of family 19 chitinase and family 18 chitinases produced by the same organism, the four chitinases (Chi18bA, Chi18aC, Chi18aD, and Chi19F), whose genes are expressed at high levels in the presence of chitin, were produced in Escherichia coli and purified. The effect of pH on the hydrolytic activity was very different not only among the four chitinases but also among the substrates. The hydrolytic activity of Chi19F, family 19 chitinase, against soluble substrates was remarkably high as compared with three family 18 chitinases, but was the lowest against crystalline substrates among the four chitinases. On the contrary, Chi18aC, a family 18-subfamily A chitinase, showed highest activity against crystalline substrates. Only Chi19F exhibited significant antifungal activity. Based on these observations, the roles of family 19 chitinases are discussed.

Adrenomedullin (AM) is a vasodilating hormone secreted mainly from vascular wall, and its expression is markedly enhanced after stroke. We have revealed that AM promotes not only vasodilation but also vascular regeneration. In this study, we focused on the roles of AM in the ischemic brain and examined its therapeutic potential. We developed novel AM-transgenic (AM-Tg) mice that overproduce AM in the liver and performed middle cerebral artery occlusion for 20 min (20m-MCAO) to examine the effects of AM on degenerative or regenerative processes in ischemic brain. The infarct area and gliosis after 20m-MCAO was reduced in AM-Tg mice in association with suppression of leukocyte infiltration, oxidative stress, and apoptosis in the ischemic core. In addition, vascular regeneration and subsequent neurogenesis were enhanced in AM-Tg mice, preceded by increase in mobilization of CD34(+) mononuclear cells, which can differentiate into endothelial cells. The vasculo-neuro-regenerative actions observed in AM-Tg mice in combination with neuroprotection resulted in improved recovery of motor function. Brain edema was also significantly reduced in AM-Tg mice via suppression of vascular permeability. In vitro, AM exerted direct antiapoptotic and neurogenic actions on neuronal cells. Exogenous administration of AM in mice after 20m-MCAO also reduced the infarct area, and promoted vascular regeneration and functional recovery. In summary, this study suggests the neuroprotective and vasculo-neuro-regenerative roles of AM and provides basis for a new strategy to rescue ischemic brain through its multiple hormonal actions.

Background: The onset of intracerebral hemorrhage (ICH) has a circadian variation, with a lower risk during the asleep period. It is unclear, however, whether ICH during the asleep period differs from that during the awake period in pathophysiologic nature. The purpose of this study is to elucidate the incidences and clinical features of ICH during the asleep period., Methods: We studied 129 consecutive patients with primary ICH and classified them into two groups according to the circumstance of their stroke onset, either during the awake period (awake ICH group) or the asleep period (asleep ICH group). Demographic and clinical characteristics were then compared between the two groups., Results: Of the patients, 19 (14.7%) had ICH during the asleep period. The mortality rate at 1 month after the stroke was significantly higher in the asleep ICH group than in the awake ICH group (21.1% v 4.9%, P = .0325). The hemorrhage volume in the asleep ICH group was also significantly larger than that in the awake ICH group (mean volume, 32.6 mL v 16.7 mL, P = .0122)., Conclusions: Our findings indicate that ICH during the asleep period may be more detrimental compared with ICH during the awake period, causing larger hematoma and higher mortality rates.

The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.

2',4'-BNA(NC), a bridged nucleic acid analogue, which was designed and synthesized in our laboratory, showed very high binding affinity towards complementary RNA and DNA strands. Its duplex-forming ability towards a single-stranded RNA was similar to or slightly higher than that of 2',4'-BNA and the overall triplex-forming ability against a double-stranded DNA was also better than that of 2',4'-BNA. 2',4'-BNA(NC) exhibited higher RNA selectivity than 2',4'-BNA.

Microbial modification of vegetable fatty acids can often lead to special changes in their structure and in biological function. A bacterial strain, Pseudomonas aeruginosa PR3, is known to carry out multiple hydroxylations on polyunsaturated fatty acids containing 1,4-cis, cis diene structural units, resulting in antibacterial activity. In this paper, in an effort to understand the overall mechanism involved in the varied biological functions of the complicated metabolites of bio-converted polyunsaturated fatty acids, we performed bioconversion of several polyunsaturated fatty acids using PR3, and determined their oxidative activities against fish oil. Bio-converted linoleic acid, eicosapentanoic acid, and docosahexanoic acid promoted effectively oxidation of fish oil. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of these bio-converted fatty acids.

A simple and highly sensitive method for determining the fatty acid composition of food lipids containing conjugated linoleic acid (CLA) is described. The method is based on the separation of the 9-anthrylmethyl ester derivatives of saturated and unsaturated (conjugated and non-conjugated) fatty acids by reversed-phase high-performance liquid chromatography with fluorescence detection. Just like the other fatty acids, CLA reacts readily with 9-anthryldiazomethane at room temperature to produce 9-anthrylmethyl esters without isomerization and decomposition of the conjugated double bonds. Clear resolution of the individual fatty acids as their 9-anthrylmethyl esters is achieved on a highly efficient octadecylsilylated silica column (150- x 3-mm i.d., 3-microm particle size) using a stepwise gradient elution with methanol-water. The method is standardized with commercially available CLA isomers (cis-9, trans-11 and trans-10, cis-12-octadecadienoic acids, and their cis,cis and trans,trans isomers) and applied for determination of the fatty acid compositions of milk and sdairy products.

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharides, plays an important role in the innate immune response. In this study, we investigated the role of TLR4 in the development of experimental colitis with regard to the biological actions of macrophage migration inhibitory factor (MIF) using TLR4 null ((-/-)) mice. TLR4(-/-) mice were given 2% dextran sulphate sodium (DSS) in drinking water to induce colitis, which was clinically and histologically as severe as that seen in wild-type (WT) mice. The level of tumour necrosis factor (TNF)-alpha in colon tissues was increased in WT mice but unchanged in TLR4(-/-) mice. The level of myeloperoxidase (MPO) activity in colon tissues was increased by DSS administration in both TLR4(-/-) and WT mice. The expression of MIF was up-regulated in the colons of TLR4(-/-) mice with acute DSS-induced colitis. An anti-MIF antibody significantly suppressed colitis and elevation of matrix metalloproteinase-13 in TLR4(-/-) mice. The current results obtained from TLR4(-/-) mice provide evidence that MIF plays a critical role in the development of acute DSS-induced colitis.

Enhanced production of macrophage migration inhibitory factor (MIF) is recognized in patients with inflammatory bowel disease (IBD) and mice with experimental colitis; however, the precise molecular function of MIF in colitis is not fully understood. To further investigate this matter, we examined the pathological features of MIF transgenic mice with dextran sulphate sodium (DSS)-induced colitis. We generated transgenic mice carrying a murine MIF cDNA driven by a cytomegalovirus enhancer and a beta-actin/beta-globin promoter. Mice were orally administered 1-4% DSS in drinking water for 7 days. Clinical disease activity, survival and histological features were evaluated. The level of myeloperoxidase (MPO) activity in the colon tissue was measured to assess neutrophil infiltration. The level of corticosterone in the serum was measured by enzyme linked-immunosorbent assay (ELISA). MIF mRNA and protein were markedly up-regulated in the colon and serum obtained from MIF transgenic mice. The severity of the colitis induced by 1% DSS treatment was markedly higher in MIF transgenic mice than in wild-type mice. We also found that MPO activity was significantly higher in MIF transgenic mice than wild-type mice in response to DSS stimulation. Interestingly, the corticosterone level remained unchanged in MIF transgenic mice. MIF enhances DSS-induced colitis, in part via neutrophil accumulation and inhibition of glucocorticoid bioactivity.

A protein that binds specifically to the promoter region of chiA was purified from the cell lysate of Streptomyces lividans by using an affinity purification method. Determining the amino-terminal amino acid sequence of the purified protein led to cloning of a gene (cpb1) encoding the chitinase promoter-binding protein, Cpb1. The deduced amino acid sequence of Cpb1 showed significant similarity to the sequences of a group of hypothetical proteins of S. coelicolor that have been revealed by the genome project, and the amino-terminal region of Cpb1 showed similarity to the DNA-binding domains of several transcription factors. The Cpb1 proteins expressed in S. lividans or Escherichia coli showed specific binding activity to the chiA promoter. The disruption of cpb1 resulted in partial relief of the glucose repression of chitinase production, indicating that cpb1 took part in the regulation of chitinase expression in S. lividans.

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of < or =6 bp. Type B changes are more drastic and involve modifications of > or =8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Lipid peroxidation of human heptoma cell line, HepG2, after incorporation of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was measured with a fluorescent probe and gas chromatography-mass spectrometry (GC-MS) analysis. The analysis with a fluorescent probe showed that incorporation of each polyunsaturated fatty acid (PUFA) enhanced the cellular lipid peroxidation level, but there was little difference in the effect of LA, AA, or DHA on the enhancement of cellular lipid peroxidation. The fluorescent analysis also showed that the addition of H(2)O(2) (0.5 mM) enhanced the cellular lipid peroxidation levels in LA and AA supplemented cells as compared with those without H(2)O(2). However, the enhancement of lipid peroxidation by H(2)O(2) was not observed in DHA-supplemented cells. The same result was obtained in the GC-MS analysis of total amounts of monohydroperoxides (MHP) formed in the cellular phospholipid oxidation. In this case, the main source for MHP was LA in LA-, AA-, and DHA-supplemented cells. A significant amount of AA-MHP and a small amount of DHA-MHP were observed in AA- and DHA-supplemented cells respectively. GC-MS analysis also indicated the specific positional distribution of DHA-MHP isomers. The isomers were formed only by hydrogen abstraction at the C-18 (16-MHP + 20-MHP; 46.5%), C-6 (4-MHP + 8-MHP; 38.5%), and C-12 (10-MHP + 14-MHP; 15.1%) positions, but not at the C-9 or C-15 positions.

We have recently designed and synthesized a novel bridged nucleic acid analogue 2',4'-BNA(NC), bearing an N-O bridged structure, which furnished both higher duplex and triplex-forming abilities and sequence selectivity towards complementary RNA and/or DNA, respectively, and showed excellent resistance against nuclease degradation. Duplex and triplex-forming abilities were slightly higher or similar to those of 2',4'-BNA and nuclease resistance was as high as that of S-oligo.

Aims: To evaluate the efficacy of electrolysed NaCl solutions (EW) for disinfecting bacterial isolates from carp, and the potential application of EW to reducing the bacterial load in whole carp and carp fillets., Methods and Results: EW was produced by using a two-compartment batch-type electrolysed apparatus. Pure cultures (in vitro), whole carp (skin surface) and carp fillets were treated with EW to detect its antimicrobial effects. The anodic solution [EW (+)] completely inhibited growth of the isolates. Furthermore, dipping the fish samples in EW (+) reduced the mean total count of aerobic bacteria on the skin of whole carp and in fillets by 2.8 and 2.0 log(10), respectively. The cathodic solution [EW (-)] also reduced growth of the isolates from carp by ca 1.0 log(10). Moreover, the total counts of aerobic bacteria in whole carp (on the skin) and fillets were reduced by 1.28 and 0.82 log(10), respectively., Conclusions: EW (+) has a strong bactericidal effect on bacteria isolated from carp., Significance and Impact of the Study: Treatment with EW (+) could extend the shelf life of these fish.

We report here that 6.9% (68/987) of randomly selected cDNA clones from an S. pombe cDNA library lack apparently long open reading frames which we denote prl. One of them, prl1, was examined further because multiple bands were observed when it was used as a probe in northern blot analysis. These multiple bands appear to be derived from overlapping transcripts from both DNA strands, including non-coding RNAs and antisense RNAs in addition to mRNA. Such mechanisms may increase the transcriptional variation in S. pombe cells.

Synthesized PCs containing docosahexaenoic acid (DHA), arachidonic acid (AA), linoleic acid (LA), and palmitic acid (PA) at known positions in the glycerol moiety were oxidized in liposomes, bulk, and organic solvent. In bulk and organic solvent, the oxidative stability of PC decreased with increasing degrees of unsaturation. However, the degree of unsaturation had little effect on the stability of PC in liposomes. The oxidative stability of PC in liposomes would be affected by the chemical reactivity based on the degree of unsaturation and by the conformation of fatty acyl component in PC bilayers. When the oxidative stability of 1-PA-2-LA-PC or 1-PA-2-AA-PC was compared with that of a 1:1 (mol ratio) mixture of 1,2-diPA-PC + 1,2-diLA-PC, or 1,2-diPA-PC + 1,2-diAA-PC, respectively, the former PC was more oxidatively stable than that of the latter PC mixture in all oxidation systems, although the degree of unsaturation of 1-PA-2-PUFA-PC was the same as that of the corresponding mixture of diPA-PC + diPUFA-PC. The higher oxidative stability of 1-PA-2-PUFA-PC than that of a corresponding mixture of diPA-PC + diPUFA-PC in liposomes was suggested to be due to the different conformation of PC bilayers and the different rate of hydrogen abstraction by free radicals from intermolecular and intramolecular acyl groups.

In order to isolate meiosis-specific genes in Schizosaccharomyces pombe, we have constructed a subtracted cDNA library enriched in clones whose expression is enhanced during meiosis induced by nitrogen starvation. Using northern blot analysis, we isolated 31 kinds of clones whose expression was induced in a meiosis/sporulation-specific manner. We comprehensively named them meu after meiotic expression upregulated. The transcription of 20 meu genes was found to be dependent on the mei4(+) gene, which encodes a transcription factor required for the progression of meiosis. DNA sequencing indicated that most of the meu genes encode novel proteins. Notably, five of the meu genes harbor no apparent protein coding sequences, and the transcripts form stable hairpin structures, suggesting that they may generate non-coding RNAs or antisense RNAS: The results presented here imply that RNAs are also important for the comprehensive characterization of genomic expression.

Induction and repression of a gene for chitinase (chiA) in Streptomyces lividans was investigated using a catechol 2,3-dioxygenase gene (xylE) as the reporter gene. Of various substrates examined, expression of the promoter (PchiA) was observed after a delay when colloidal chitin or small chitin-oligosaccharides were added to the medium. N-acetylglucosamine completely repressed the chiA promoter. The duration of the delay in expression of PchiA differed with the inducer used, with chitobiose inducing the activity most rapidly. The minimum concentration of chitobiose needed for induction was 1 microM. It appears, therefore, that an efficient inducer of the gene for chitinase in S. lividans is chitobiose.

A monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 microm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINE), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.

Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants.

The effectiveness was evaluated of a cathodic solution prepared by the electrolysis of an NaCl solution in inhibiting the aqueous oxidation of ethyl linoleate and ethyl docosahexaenoate. The decrease in unoxidized substrate and the formation of total peroxides during oxidation indicate that the cathodic solution completely inhibited the oxidation of both ethyl esters, while these lipids were easily oxidized in an NaCl solution and in distilled water. The antioxidative activity of the cathodic solution was confirmed after open incubation for 3 days and 7 days at 37°C, although the scavenging ability of the cathodic solution toward DPPH radicals disappeared during this incubation.

The oxidative stability of phosphatidylcholines (PCs) from soybean, chicken egg, and salmon egg in liposomes was compared with that in aqueous micelles. When each PC was oxidized in aqueous micelles, salmon egg PC was the most oxidatively stable, followed by chicken egg PC and soybean PC, however, no significant difference in the oxidative stability was apparent between chicken egg PC and salmon egg PC in liposomes. The main molecular species of soybean PC was 1,2-dilinoleoyl-PC, while most of the PUFAs in chicken egg PC and salmon egg PC were not esterified at the sn-1 position but at the sn-2 position. Therefore, it is suggested that the oxidative stability of PC liposomes would be strongly influenced by the positional distribution of PUFAs in the PC molecule. Further studies on the oxidation of PC liposomes showed that chicken egg albumin and soybean protein protected PC bilayers against attack by free radicals generated in the aqueous phase.

Coxsackievirus 3C proteinase (3Cpro) cleaves between Gln and Gly, but additional amino acids are required to constitute a cleavage site. To investigate the additional sequence requirements, cleavages of the peptide substrate, and its derivatives were examined. Substitutions of each residue from the P2 to P5 positions showed the importance of the P2 Phe and P4 Ala for recognition by 3Cpro.

In order to identify the specific regions in the human TSH receptor for TSAb and thyroid stimulation-blocking antibody (TSBAb), we produced rabbit antibodies raised against several peptides of the extracellular domain of the human TSH receptor, where sequences are not conserved in the LH/CG receptor, and measured the TSAb activity and TSBAb activity of those antibodies using Chinese hamster ovary cells expressing human TSH receptors. Only antisera from rabbits that were immunized with a peptide of amino acid 32-56, including the small insertion near the N-terminal end of the extracellular domain, showed apparent TSAb activities and have been shown to be significantly precipitated by IgG of patients with Graves' disease. TSAb activity positively correlated with the antibody titers against the peptide in those rabbits. In contrast, antisera from rabbits immunized with a peptide of amino acid 352-378, including a part of the large insertion near the C-terminal end of the extracellular domain, showed the obvious TSBAb activities. TSBAb activity also positively correlated with the degree of antibody titers against the peptide in those rabbits. Moreover, this peptide was significantly immunoprecipitated by the IgG from hypothyroid patients who had TSBAb, and the immunoprecipitation of this peptide positively correlated with TSBAb activities. These results suggest that the epitope responsible for TSAb is quite different from that for TSBAb in the extracellular domain of the human TSH receptor.

The substrate requirements of coxsackievirus 3C proteinase (3Cpro) were investigated on the C-terminal side of the scissile bond using C-terminal truncated peptides of the substrate peptide Ac-EALFQGPPV. Not only the Gln-Gly bond of Ac-EALFQG-NH2 but also the C-terminal amide group of Ac-EALFQ-NH2 was hydrolyzed by 3Cpro, suggesting that the essential residues for cleavage by coxsackievirus 3Cpro would exist within the N-terminal 5 residues.

Human thyrotropin (TSH) receptors were expressed in Chinese hamster ovary (CHO) cells using eukaryotic expression plasmid pCXN2, which contains beta-actin promoter. We measured cAMP stimulation in CHO cells expressing human TSH receptors (CHO-hTSH-R cells) by immunoglobulin G (IgG) of patients with Graves' disease and Hashimoto's thyroiditis, and compared the results with a conventional thyroid-stimulating antibody (TS-Ab) assay using porcine thyroid cells and a TSH-binding inhibiting immunoglobulin (TBII) assay. Nineteen untreated patients with Graves' disease, including a case who developed hyperthyroidism after interferon -alpha therapy for chronic hepatitis C, and 13 treated patients with Graves' disease, 10 patients with Hashimoto's thyroiditis and 8 control subjects were studied. In 19 untreated patients with Graves' disease, 17 patients showed positive CHO-hTSH-R cell stimulation, 11 patients showed positive porcine thyroid cell stimulation and 15 patients showed positive TBII. All the untreated patients showed positive results in at least one assay. Although significantly positive correlations were observed among CHO-hTSH-R cell stimulation, porcine thyroid cell stimulation and TBII activities, the IgG of several patients showed significant discrepancy in the assay results. In a patient with interferon-induced hyperthyroidism only CHO-hTSH-R cell stimulation was positive, while porcine thyroid cell stimulation and TBII were negative. After the treatment with propylthiouracil for 6 months, CHO-hTSH-R cell stimulation became negative. The IgG of patients with Hashimoto's thyroiditis did not show significant stimulation of CHO-hTSH-R cells. These results suggest that the CHO-hTSH-R cell stimulation assay is clinically useful for the diagnosis and follow-up of patients with Graves' disease.

Single amino acid substitutions were introduced into the putative substrate-binding site of 3C proteinase (3Cpro) of coxsackievirus B3, a member of the picornavirus family. Mutations at either Thr142, His161, Gly164, Gly169, or Ala172 severely impaired or abolished the proteolytic activity except that a conservative Thr142 to Ser mutant had detectable activity. These results, which have shown the participation of the 5 residues in 3Cpro activity, are consistent with the earlier predictions that these residues might be involved in substrate binding.

A chitinase gene (chiA) from Streptomyces lividans was characterized and its nucleotides sequenced. Although the deduced amino acid sequence of chitinase A1 did not show any similarity to those of other Streptomyces chitinases that has been sequenced, the C-terminal part, containing both a putative catalytic domain and type-III-like repeating units, showed a similarity (36%) to that of chitinase D from Bacillus circulans. A site of initiation of transcription was found approximately 51 bp upstream from the GTG initiation codon. The promoter region of the chiA gene was subcloned on a 178-bp fragment into the promoter-probe vector pIJ486, resulting in the chitin stimulated expression of the neomycin resistance gene. One of the deleted subclones, which contained a 114-bp sequence upstream from the translation start codon, retained both chitin stimulated production and glucose repression. Chitin stimulated production was lost in an other deleted mutant containing the 104-bp upstream sequence.