Probiotic Escherichia coli Nissle inhibits IL-6 and MAPK-mediated cardiac hypertrophy during STZ-induced diabetes in rats.

Escherichia coli Nissle (EcN), a probiotic bacterium protects against several disorders. Multiple reports have studied the pathways involved in cardiac hypertrophy. However, the effects of probiotic EcN against diabetes-induced cardiac hypertrophy remain to be understood. We administered five weeks old Wistar male (271±19.4 g body weight) streptozotocin-induced diabetic rats with 109 cfu of EcN via oral gavage every day for 24 days followed by subjecting the rats to echocardiography to analyse the cardiac parameters. Overexpressed interleukin (IL)-6 induced the MEK5/ERK5, JAK2/STAT3, and MAPK signalling cascades in streptozotocin-induced diabetic rats. Further, the upregulation of calcineurin, NFATc3, and p-GATA4 led to the elevation of hypertrophy markers, such as atrial and B-type natriuretic peptides. In contrast, diabetic rats supplemented with probiotic EcN exhibited significant downregulated IL-6. Moreover, the MEK5/ERK5 and JAK2/STAT3 cascades involved during eccentric hypertrophy and MAPK signalling, including phosphorylated MEK, ERK, JNK, and p-38, were significantly attenuated in diabetic rats after supplementation of EcN. Western blotting and immunofluorescence revealed the significant downregulation of NFATc3 and downstream mediators, thereby resulting in the impairment of cardiac hypertrophy. Taken together, the findings demonstrate that supplementing probiotic EcN has the potential to show cardioprotective effects by inhibiting diabetes-induced cardiomyopathies.

KHC-4 inhibits ß-catenin expression in prostate cancer cells.

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of ß-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting ß-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit ß-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total ß-catenin expression and concomitantly decreased ß-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited ß-catenin expression and that of its target proteins, PI3K, AKT, GSK3ß and TBX3. We monitored the stability of ß-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal ß-catenin degradation. We verified CDK1/ß-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and ß-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits ß-catenin signaling in DU145 prostate cancer cells.

Phosphorylation of cofilin-1 by ERK confers HDAC inhibitor resistance in hepatocellular carcinoma cells via decreased ROS-mediated mitochondria injury.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Despite the availability of several treatment strategies, resistance to chemotherapeutic agents, which limits the effectiveness of anticancer drugs, is a major problem in cancer therapy. In this study, we used a histone deacetylases inhibitor (HDACi) to establish drug-resistant HCC cells and further analyzed the molecular mechanisms underlying the development of resistance in HCC cells. Compared with the parental cells, HDACi-resistant cells showed high metastatic and pro-survival abilities. Two-dimensional electrophoresis data showed that the cofilin-1 (CFL-1) protein was altered in HDACi-resistant cells and was highly expressed in resistant cells compared with parental cells. The molecular function of CFL-1 is actin depolymerization, and it is involved in tumor metastasis. In this study, we showed that CFL-1 inhibition decreased cell migration and increased cell apoptosis in HDACi-resistant cells. We observed that HDACi induced ROS accumulation in cells and apoptosis via promotion of the CFL-1 interaction with Bax and CFL-1 translocation to the mitochondria, resulting in cytochrome C release. Importantly, phosphorylation of CFL-1 by activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) confers strong protection against HDAC inhibitor-induced cell injury. p-CFL-1 shows a loss of affinity with Bax and will not translocate to mitochondria, stably remaining in the cytoplasm. These results indicate that phosphorylation to inactivate CFL-1 decreased the chemosensitivity to HDAC inhibitors and resulting in drug resistance of HCC cells.

ß-Catenin overexpression causes an increase in inflammatory cytokines and NF-κB activation in cardiomyocytes.

ß-Catenin has been implicated in various developmental and physiological processes. Defective Wnt signaling can result in different cardiac and vascular abnormalities and is activated under pathological conditions such as inflammation and obesity. In this study, roles of ß-catenin in inflammation in cardiomyocytes were investigated. 10 samples from hearts of patients with acute infarction and 10 from normal ones were collected in order to access roles of ß-catenin in cardiomyocytes. H9c2 cardiomyoblasts and primary neonatal rat cardiomyocytes were transfected with porcine cytomegalovirus (pCMV)-ß-catenin plasmid in order to overexpress ß-catenin. Protein level of ß-catenin protein was increased in human acute infarction tissues compared to ones from normal patients. The transcription factor had increased nuclear localization in cardiomyocytes of the Wistar rats with cardiac hypertension. Furthermore, expression of fibrosis protein markers increased. Protein expression of ß-catenin was increased in human acute infarction inflammatory heart tissues and in hearts of inflammatory obesity rats. After pCMV-ß-catenin plasmid was transfected in a dose-dependent manner, inflammation protein markers, TNF-α and IL-8, were upregulated in hypertensive neonatal rat cardiomyocytes and H9c2 cardiomyoblasts. In addition, overexpression of ß-catenin induced activation and nuclear localization of NF-κB. Therefore, ß-catenin is a potential molecular target for treatment of inflammation and fibrosis in cardiomyocytes.

Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen.

Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.

Regulation of thyroid hormones in the secretion of insulin and gastric inhibitory polypeptide in male rats.

The effect of thyroid hormones on glucose-induced secretion of gastric inhibitory polypeptide (GIP) and insulin was studied. Male rats were thyroidectomized (Tx) or sham Tx. Sham Tx rats were injected with either propylthiouracil ([PTU] 20 mg/kg intraperitoneally) or saline for 2 weeks. In addition, thyroid-intact rats were injected intravenously with triiodothyronine ([T3]5 microg/kg) or saline 10 minutes before an oral glucose load (3.2 g/kg). Blood samples were collected from each animal via a jugular catheter at 0, 10, 20, 30,45, 60, and 90 minutes following glucose ingestion. Plasma levels of GIP and insulin were measured by specific radioimmunoassays (RIAs). Thyroidectomy-induced hypothyroidism increased the basal level of plasma GIP, but decreased that of insulin. Insulin levels at 10, 20, and 30 minutes following oral glucose were lower in hypothyroid rats than in euthyroid rats. Conversely, GIP levels at 60 and 90 minutes following glucose ingestion in PTU-induced hypothyroid rats were higher than those in euthyroid rats. Furthermore, glucose-stimulated insulin secretion was unaltered by pretreatment with T3, whereas the glucose-induced increase in plasma GIP was completely abolished by preinjection of T3 in thyroid-intact rats. These results suggest that thyroid functions are involved in the regulation of insulin and GIP secretion in rats.

Age-related differences in gastric acid secretion and response of gastric inhibitory polypeptide after oral glucose in male rats.

Effects of aging on the secretion of gastric acid and the response of gastric inhibitory polypeptide (GIP) and insulin after oral glucose were studied. Male rats with the age of 3 and 20 months (mo) were fasted overnight before anesthetization with pentobarbital (25 mg/kg BW, ip). The right jugular vein was catheterized for pentagastrin infusion. A PE-320 cannula was introduced into the stomach through an incision in the duodenum and was ligated about 0.5 cm from the pylorus. The stomach was flushed through the esophagus cannula via a peristaltic pump with 10 ml saline at room temperature and then irrigated with saline. Acid output was determined by titration of the flushed perfusate with 0.01 N NaOH to pH 7.0. Basal secretions were collected for 45 min before intravenous infusion of pentagastrin (0.5 microgram/kg/min) for 90 min, then for an addition 45 min. In another experiment, rats were fasted for 4 hours before an oral glucose load (3.2 g/kg). Blood samples were collected from each animal via a jugular catheter at -10, 0, 10, 20, 30, 45, 60, and 90 min following glucose ingestion. The concentrations of plasma GIP, insulin and triiodothyronine (T3) were measured by specific radioimmunoassays. The plasma T3 concentration was decreased in male rats during aging. Pentagastrin infusion stimulated gastric acid secretion in both old and young rats. The spontaneous gastric acid secretion in 20 mo rats was not significantly different from that in 3 mo rats. However, the secretion of gastric acid in response to pentagastrin was greater in 20 mo rats than in 3 mo rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related differences in the spontaneous and thyrotropin-releasing hormone-stimulated release of prolactin and thyrotropin in ovariectomized rats.

Effect of estradiol on the spontaneous and thyrotropin-releasing hormone (TRH)-stimulated release of prolactin (PRL) and thyrotropin (TSH) in young and aged ovariectomized (Ovx) rats was investigated. Old (22-26 months) and young (3 months) female rats were Ovx 3 weeks before use. They were injected subcutaneously with estradiol benzoate (EB, 25 micrograms/kg) or sesame oil for 3 days and were catheterized via the right jugular vein. Twenty hours after the last administration of EB, rats were injected with TRH (10 micrograms/kg) through the catheter. Blood samples were collected before and 5, 10, 20, 40 and 60 min after TRH injection. On the day following blood sampling, all rats were decapitated. The anterior pituitary glands (APs) were excised, and incubated with or without TRH (10 ng/ml) at 37 degrees C for 30 min. The basal level of PRL concentration in plasma samples was 5-fold higher in old Ovx rats than in young Ovx rats. Five min after TRH injection, the increase in plasma PRL was greater in old animals than in young animals. Plasma PRL remained higher in old animals than in young animals at 10, 20, 40 and 60 min following TRH challenge. Administration of EB to old and to young Ovx rats produced increases in both basal and TRH-stimulated secretions of PRL, but did not affect the difference in plasma PRL patterns between old and young animals. The release of PRL from APs was increased significantly in all rats after a 30-min incubation with TRH. In Ovx rats injected with oil, the basal release of PRL in vitro was increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro studies on the release of thyrotropin in aging rats.

Effects of aging on the concentration of serum thyrotropin (TSH) and the release of TSH in vitro from anterior pituitary glands (APs) of male and female rats were investigated. Rats with 3-5 (young) and 24-26 (old) months of age were used. Some female rats were ovariectomized (Ovx) 10 weeks before use. After decapitation, trunk blood was collected. APs were bisected and preincubated with Krebs-Ringer phosphate buffer (KRB) at 37 degrees C for 60 min. They were then incubated with or without 10 nM thyrotropin-releasing hormone (TRH) for 30 min. The media were collected and replaced by fresh KRB. Incubations were continued and KRB media were changed three times, once every 30 min. One hemi-AP per flask and 7 or 8 flasks per group were used. The concentration of TSH in the medium and serum samples was measured by radioimmunoassay. The level of serum TSH in the diestrous, estrous and Ovx rats was reduced by aging. Thirty min after incubation with TRH, the release of TSH from APs of old intact female or Ovx rats was significantly (P less than 0.01) less than that of corresponding young rats. The concentration of serum TSH and TRH-stimulated release of TSH from APs of male rats was not altered by aging. There was no difference in the basal release of TSH from APs between old and young animals. These data suggest that the release of TSH from APs in response to TRH is decreased by aging in female but not in male rats.