Mycoplasma hyopneumoniae increases intracellular calcium release in porcine ciliated tracheal cells

We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]i of 103 +/- 3 nM (n = 217 cells). The [Ca2+]i increased by 250 +/- 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 microg/ml), and this increase lasted approximately 60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 microg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenic M. hyopneumoniae still increased [Ca2+]i in tracheal cells. Pretreatment with thapsigargin (1 microM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 microM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins G(i) and G(o), increased [Ca2+]i in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to G(i) or G(o), which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.     

Mechanisms of AVP-induced glucagon release in clonal alpha-cells in-R1-G9: involvement of Ca(2+)-dependent and -independent pathways

1. The mechanisms underlying AVP-induced increase in [Ca(2+)](i) and glucagon release in clonal alpha-cells In-R1-G9 were investigated. 2. AVP increased [Ca(2+)](i) and glucagon release in a concentration-dependent manner. After the administration of AVP, glucagon was released within 30 s, quickly reached the maximum within 2 min, and maintained a steady-state concentration for at least 15 min. 3. In Ca(2+)-containing medium, AVP increased [Ca(2+)](i) in a biphasic pattern; a peak followed by a sustained plateau. In Ca(2+)-free medium, the Ca(2+) response to AVP became monophasic with lower amplitude and no plateau. Both the basal and AVP-induced glucagon releases were lower in the absence than in the presence of extracellular Ca(2+). When [Ca(2+)](i) was stringently deprived by BAPTA, a Ca(2+) chelator, AVP still significantly increased glucagon release. 4. Pretreatment with thapsigargin, a microsomal Ca(2+) ATPase inhibitor, abolished both the Ca(2+) peak and sustained plateau. 5.AVP increased intracellular concentration of IP(3). 6. U-73122 (8 microM), a phospholipase C inhibitor, abolished AVP-induced increases in [Ca(2+)](i), but only reduced AVP-induced glucagon release by 39%. 7. Pretreatment with nimodipine, an L-type Ca(2+) channel blocker failed to alter AVP-induced glucagon release or increase in [Ca(2+)](i). 8. The results suggest that AVP causes glucagon release through both Ca(2+)-dependent and -independent pathways. For the Ca(2+)-dependent pathway, the G(q) protein activates phospholipase C, which catalyzes the formation of IP(3). IP(3) induces Ca(2+) release from the endoplasmic reticulum, which, in turn, triggers Ca(2+) influx. Both Ca(2+) release and Ca(2+) influx may contribute to AVP-induced glucagon release.     

Glucose dependency of arginine vasopressin-induced insulin and glucagon release from the perfused rat pancreas

The purpose of this study was to investigate the glucose dependency of arginine vasopressin (AVP)-induced insulin, glucagon, and somatostatin release from the perfused rat pancreas. AVP (30 or 300 pmol/L) was tested in the presence of a glucose concentration of 0, 1.4, 5.5 (basal level), or 20 mmol/L. The rates of insulin release at 0 and 1.4 mmol/L glucose were approximately 70% to 80% and 60% to 70% less, respectively, than that at the baseline level. AVP (30 or 300 pmol/L) failed to change insulin release at 0 and 1.4 mmol/L glucose. At the basal glucose level, AVP (300 pmol/L) induced a biphasic insulin release, a peak followed by a sustained phase. In addition, the combination of glucose (20 mmol/L) and AVP (300 pmol/L) induced a higher insulin peak and sustained phase than 20 mmol/L glucose alone. The rates of glucagon release at 0 and 1.4 mmol/L glucose were about 3- and 2-fold more, respectively, than that at the baseline level. At 0 and 1.4 mmol/L glucose, both 30 and 300 pmol/L AVP caused a higher glucagon peak and sustained phase than 0 and 1.4 mmol/L glucose alone. At the basal glucose level, AVP (30 or 300 pmol/L) induced a biphasic glucagon release, a peak followed by a sustained phase. The rate of glucagon release at 20 mmol/L glucose was approximately 60% to 70% less than that at the baseline level. When AVP (300 pmol/L) was administered in 20 mmol/L glucose, it induced a transient glucagon peak, which was 2.4-fold of the baseline level. At all glucose concentrations tested, AVP (30 or 300 pmol/L) failed to change somatostatin release. These results suggested that (1) hypoglycemia directly increases glucagon and decreases insulin release; (2) AVP induces insulin and glucagon release by a direct action on beta and alpha cells, respectively; (3) AVP induces insulin and glucagon release in a glucose-dependent manner-the higher the glucose concentration, the greater the enhancement of AVP-induced insulin release, whereas the lower the glucose concentration, the higher the enhancement of AVP-induced glucagon release; and (4) alpha cells are more sensitive to AVP than beta cells in hormone release.     

Grape seed extract supplementation prevents high-fructose diet-induced insulin resistance in rats by improving insulin and adiponectin signalling pathways

Recent evidence strongly supports the contention that grape seed extract (GSE) improves hyperglycaemia and hyperinsulinaemia in high-fructose-fed rats. To explore the underlying molecular mechanisms of action, we examined the effects of GSE on the expression of muscle proteins related to the insulin signalling pathway and of mRNA for genes involved in the adiponectin signalling pathway. Compared with rats fed on a normal diet, high-fructose-fed rats developed pathological changes, including insulin resistance, hyperinsulinaemia, hypertriacylglycerolaemia, a low level of plasma adiponectin and a high level of plasma fructosamine. These disorders were effectively attenuated in high-fructose-fed rats supplemented with GSE. A high-fructose diet causes insulin resistance by significantly reducing the protein expression of insulin receptor, insulin receptor substrate-1, Akt and GLUT4, and the mRNA expression of adiponectin, adiponectin receptor R1 (AdipoR1) and AMP-activated protein kinase (AMPK)-α in the skeletal muscle. Supplementation of GSE enhanced the expression of insulin signalling pathway-related proteins, including Akt and GLUT4. GSE also increased the mRNA expression of adiponectin, AdipoR1 and AMPK-α. In addition, GSE increased the mRNA levels of glycogen synthase and suppressed the mRNA expression of glycogen synthase kinase-3-α, causing an increase in glycogen accumulation in the skeletal muscle. These results suggest that GSE ameliorates the defective insulin and adiponectin signalling pathways in the skeletal muscle, resulting in improved insulin resistance in fructose-fed rats.     

Cytotoxicity, acute oral toxicity, and skin irritation of 2-ethylhexyl-2,4,5-trimethoxycinnamate and di(2-ethylhexyl)-2,4,5-trimethoxybenzalmalonate

Safety of two new ultraviolet (UV) filters, 2-ethylhexyl-2,4,5-trimethoxycinnamate (E8) and 2-ethylhexyl-2,4,5-trimethoxybenzalmalonate (B8), has been evaluated through the human melanoma cytotoxicity test and seven-day acute oral toxicity studies in rats. At 2.5 mg/mL, both compounds gave similar cell viability to the control. LD50 values for E8 and B8 are more than 5000 and 1000 mg/kg body weight, respectively. No significant difference in body weight and hematological parameters among the 0, 5, 50, 500, and 5000 mg/Kg E8-treated animals could be detected. Pathological examination of rat tissues collected at the end of the study period revealed no significant difference between the control and all E8-administered rats. There was no significant difference in all clinical blood chemistry parameters (aspartate aminotransferase, creatinine, blood urea nitrogen, and cholesterol), except alanine aminotransferase (ALT), between the control and the E8-treated animals. All ALT values were, however, in the normal range of SD rats. E8 showed negative results for the skin irritation study on human volunteers, using patch and photopatch tests. Excitation of respiratory signs of dypsnea in 10, 100, and 1000 mg/Kg B8-treated rats could be observed during 1-24 h. All groups were, however, normal during the second to the seventh day. Hematological parameters of the 0, 10, 100, and 1000 mg/Kg B8-treated animals showed no significant difference. Pathological examination revealed no significant difference between the control and all B8-administered rats. However, significant differences in some clinical blood chemistry parameters and body weights between the control and some B8-treated animals could be detected. All values, however, were in the normal ranges of the SD rats.     

Eubacterium bacteremia and colon cancer

Eubacterium bacteremia is rare. We report a senile patient who presented with 3 episodes of bacteremia, caused by Clostridium perfringens, Eubacterium limosum and Escherichia coli, respectively, which led to the diagnosis of adenocarcinoma of the colon. A differential diagnosis of colon cancer should be considered in patients with eubacterium bacteremia.     

Rapid detection of apolipoprotein E genotypes in Alzheimer's disease using polymerase chain reaction-single strand conformation polymorphism

Apolipoprotein E (APOE) gene on chromosome 19q13.2 is encoded by three common alleles designated as epsilon2, epsilon3 and epsilon4. In Alzheimer's disease (AD) the epsilon4 allele is over-represented and is considered to be a major genetic risk factor. Several methods have been developed to determine APOE genotypes. Among them, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) appears to be highly reliable. In this study, we improved the nonisotopic PCR-SSCP method for determining APOE genotypes in 42 cases of AD patients, 40 cases of non-AD dementia patients, and 49 cases of age-matched controls. DNA from the target sequence on APOE was amplified by PCR from peripheral blood genomic DNA. PCR products were electrophoresed in a non-denaturing polyacrylamide gel and visualized by silver staining. We found that the epsilon4 allele had a significantly high frequency of occurrence in AD patients (33.3%) compared with age-matched controls (13.3%) (chi(2) = 10.43, p = 0.001) and non-AD dementia (10%) (chi(2) = 13.02, p<0.001) whereas the epsilon3 allele was of high frequency in non-AD dementia (90%) compared with age-matched controls (85.7%) and AD patients (66.7%). APOE epsilon4 homozygotes were found only in AD groups. On the other hand, the epsilon2 allele was found only in an age-matched control. This study confirmed that the APOE psilon4 allele is a risk factor in Thai AD subjects and that the PCR-SSCP method is a rapid and useful means of detecting the APOE genotype in AD.     

Effects of Orthosiphon stamineus aqueous extract on plasma glucose concentration and lipid profile in normal and streptozotocin-induced diabetic rats

The objective of this study was to investigate the effects of Orthosiphon stamineus Benth. aqueous extract on plasma glucose concentration and lipid profile in normal and streptozotocin-induced diabetic rats. The chemical screening of the extract showed phenolic compound and flavonoid content were 13.24+/-0.33 mg/g and 1.73+/-0.14 microg/g, respectively. In oral glucose tolerance test, the extract (0.2-1.0 g/kg) significantly decreased plasma glucose concentration in a dose-dependent manner in both normal and diabetic rats. The extract at 1.0 g/kg was most effective in decreasing plasma glucose concentrations and the response was closed to the result of glibenclamide (5 mg/kg). After repeated daily oral administrations of the extract (0.5 g/kg) for 14 days, the extract significantly reduced plasma glucose concentration in diabetic rats at days 7 and 14. By the end of the study, plasma triglyceride concentration was lower in the extract-treated diabetic rats than untreated ones. Furthermore, plasma HDL-cholesterol concentration was significantly increased in diabetic rats treated with the extract. In perfused rat pancreas, the extract did not increase insulin secretion in the presence of 5.5 mM glucose, but 100 microg/ml extract potentiated glucose-induced insulin secretion. Our findings suggested that Orthosiphon stamineus aqueous extract is effective for alleviating hyperglycemia and improving lipid profile in diabetic rats.     

Modified Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE) as a screening test for dementia for Thai elderly

A potential test for early detection of dementia in the elderly is the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE), which is based on information from the informant for the elderly about the changes of the elderly in everyday cognitive functioning associated with dementia. The present study aimed to modify and assess the reliability and validity of the modified IQCODE consisting of 32 items. The study consisted of two methods of assessing dementia: DSMIV diagnosis carried out by clinicians, and informants responding to the IQCODE. The subjects were 200 pairs of elderly subjects and their informants who visited the Geriatric Clinic, Ramathibodi Hospital. The optimal cutoff score on the modified IQCODE was 3.42, with 90% sensitivity and 95% specificity. The positive predictive values, negative predictive values, and accuracy were 0.94, 0.90, and 0.92, respectively. The IQCODE items had high internal consistency. The IQCODE associated with the elderly person's age, but not with their gender and educational level; nor were they associated with the demographic characteristics of the informant. Therefore, the IQCODE could be used as an alternative screening test for dementia in Thailand with acceptable sensitivity and specificity. This tool may be useful for dementia screening in the community and the geriatric clinic for early detection of disease.     

Undiagnosed dementia and value of serial cognitive impairment screening in developing countries: A population-based study

Background:   We conducted a population-based prospective study in 420 older persons to examine the prevalence of undiagnosed dementia and validity of the Chula Mental Test (CMT) as well as value of serial administration of the CMT and its score evolution over 2 years.
Methods:   The CMT score was obtained in 1997 and 1999 surveys. In 1999, all participants were evaluated by a geriatrician to make diagnoses of dementia according to the 4th edn of the Diagnostic and Statistical Manual criteria. Information on previous diagnosis of dementia by physicians was collected. Validity of the CMT was determined by the receiver–operator curve. The pattern of cognitive evolution over 2 years was analyzed.
Results:   Of 420 subjects, 23 had dementia, of which 22 (95.6%) were undiagnosed. The prevalence (95% confidence interval) of dementia and undiagnosed dementia were 5.5% (3.3–7.7%) and 5.3% (4.1–6.3%), respectively. With original cut-off (15/14) of the CMT, the sensitivity and specificity were 0.74 and 0.86, respectively. The best cut-off found in this study was 16/15 which provided better sensitivity (0.91) but worse specificity (0.76) than those of the original cut-off. Pattern of cognitive evolution was heterogeneous. The heterogeneous change was substantial in subjects with mild low CMT score. Cognitive evolution pattern showed that serial administration of the CMT could reduce workload of primary care physicians and might be useful in a screening protocol.
Conclusion:   The prevalence of undiagnosed dementia in community-dwelling Thai older persons was high. The CMT was valid for use in a community. Heterogeneous evolution of cognitive function and value of serial cognitive impairment screening was found.