Maintenance of cellular annexin A1 level is essential for PI3K/AKT/mTOR-mediated proliferation of pancreatic beta cells.

Annexin A1 (AnxA1, also known as lipocortin-1), is a calcium-dependent phospholipid binding protein with diverse functions. Previous studies have indicated that AnxA1 is associated with age-related ß-cell dysfunction and aging, which lead to decreased ß-cell proliferation capacity. However, it has been uncertain whether AnxA1 affects the proliferation of pancreatic beta (ß) cells. In the present study, we reduced AnxA1 expression in the MIN6 islet ß-cell line using small interfering RNA (AnxA1-siRNA), then measured cell cycle distribution and cellular proliferation. We also measured the expression levels of cell cycle-related proteins such as cyclin D1, cyclin E, and cyclin-dependent kinase 2 (CDK2) by Western blot analysis. We investigated the phosphatidylinositol 3-kinase (PI3K)/ serine/threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway to explore the potential mechanism underlying the observed effects. Knockdown of AnxA1 expression using siRNA reduced the rates of MIN6 cell proliferation. The proportions of cells in S and G2/M phases also decreased upon inhibition of AnxA1. Moreover, AnxA1 protein expression in MIN6 cells was positively related to the protein levels of cyclin D1, cyclin E, and CDK2. Activation of the PI3K/Akt/mTOR signaling pathway by AnxA1 may be involved in the signaling cascade to regulate cell proliferation. This study identified a positive correlation between AnxA1 protein and pancreatic ß-cell proliferation. AnxA1 protein expression might affect the proliferation of MIN6 cells via regulation of cyclin D1, cyclin E, and CDK2 proteins, as well as the PI3K/Akt/mTOR signaling pathway.

Influence of MiR-154 on myocardial apoptosis in rats with acute myocardial infarction through Wnt/ß-catenin signaling pathway.

OBJECTIVE: To explore the influence of micro ribonucleic acid (miR)-154 on myocardial apoptosis in rats with acute myocardial infarction (AMI), and to analyze whether Wnt/ß-catenin signaling pathway was involved in the regulation. MATERIALS AND METHODS: The Sprague-Dawley (SD) rat model of AMI was established via ligation of left anterior descending artery. Rats were randomly divided into model group (M group, n=12) and ICG-001 intervention group (I group, n=12). At the same time, sham operation group (S group, n=12) was established. In I group, ICG-001 (5 mg/kg) was intraperitoneally injected every day after operation. Meanwhile, an equal amount of normal saline was injected in rats of S group and M group. 21 d after operation, the cardiac function of rats in each group was detected via echocardiography. After that, the rats were immediately executed. MI area in each group was detected via 2,3,5-triphenyltetrazolium chloride (TTC) staining. Myocardial apoptosis level in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Moreover, the changes of apoptotic proteins in rat myocardial cells were detected via Western blotting. Moreover, the expression level of miR-154 in myocardial cells of rats was detected via quantitative polymerase chain reaction (qPCR). Furthermore, the influence of miR-154 on Wnt/ß-catenin signaling pathway was detected via Western blotting. RESULTS: Compared with S group, left ventricular ejection fraction (LVEF, %) and left ventricular fractional shortening (LVFS, %) were significantly decreased in M group (p<0.01). However, left ventricular internal diameter at end-diastole (LVIDd) and left ventricular internal diameter at end-systole (LVIDs) were significantly increased (p<0.01). In I group, LVEF (%) and LVFS (%) were significantly higher than those of M group (p<0.05), whereas LVIDs and LVIDd were significantly lower (p<0.05). MI area in M group was remarkably larger than that of S group (p<0.01). Meanwhile, MI area in I group was significantly smaller than that of M group (p<0.01). Compared with S group, the number of apoptotic myocardial cells and the protein expression level of cleaved caspase-3 were significantly increased in M group (p<0.01). However, the expression level of B-cell lymphoma-2/Bcl-2 associated X protein (Bcl-2/Bax) was significantly decreased (p<0.01). The number of apoptotic myocardial cells and the protein expression level of cleaved caspase-3 were significantly declined in I group when compared with those of M group (p<0.01). However, the expression level of Bcl-2/Bax was significantly increased in I group (p<0.01). The expression level of miR-154 in myocardial cells of M group and I group was remarkably increased when compared with that of S group (p<0.01). Furthermore, the expression levels of ß-catenin and Cyclin D1 in myocardial cells of M group were remarkably higher than those of S group and I group (p<0.01). CONCLUSIONS: AMI significantly increases the expression level of miR-154. Moreover, miR-154 can activate Wnt/ß-catenin signaling pathway, eventually promoting myocardial apoptosis.

Is bag urine culture useful in monitoring urinary tract infection in infants?

OBJECTIVES: We studied the false-positive rate of bag urine cultures in diagnosing bacteriuria in infants and examined the factors responsible for contamination. METHODS: One hundred asymptomatic patients with previous urinary tract infections (UTI; age range 5-23 months; sex ratio M : F 73:27) were screened by bag urine cultures and confirmed by suprapubic aspiration or catheterization. Those producing contaminated and clean samples were interviewed with a standard questionnaire. RESULTS: Sixty patients had insignificant growth and 40 had doubtful or mixed growth. After further interview and instructions, the latter group produced a second bag urine sample with negative results in 23 and positive results in 17. Among the latter, UTI was confirmed in five and excluded in 12 cases. The false-positive rate of first urine collection was 36.8%. Comparing patients with contaminated urine to those with negative urine samples, we found the former were associated with waiting for more than one void to complete urine collection, and with uncircumcised boys. Other factors did not reach statistical significance. Proper counselling and repeating a second culture reduced the overall false-positive rate to 12.6%. CONCLUSION: In our population, the contamination rate of one bag urine culture was 36.8%, which is unacceptable and alternative methods need to be sought. Contamination was associated with improper collection procedures and with uncircumcised boys. Proper instructions and doing a second urine culture reduced the overall rate to 12.6%, but at the expense of delaying diagnosis and treatment.

Correlation between disulfide reduction and conformational unfolding in bovine pancreatic trypsin inhibitor.

The native-like two-disulfide intermediate of bovine pancreatic trypsin inhibitor (BPTI), with the disulfide between Cys14 and Cys38 reduced, plays a particularly important role in the disulfide-coupled folding pathway of BPTI because of its participation in the rate-determining step of the reaction [Creighton & Goldenberg (1984) J. Mol. Biol. 179, 497-526; Weissman & Kim (1991) Science 253, 1386-1393]. In order to study directly the relationship between conformational stability and reductive unfolding kinetics, and to gain insight concerning the rate-limiting transition state in the thiol/disulfide-mediated folding/unfolding reaction of BPTI, BPTI variants based on a native-like two-disulfide analog of this intermediate, BPTI(Ala14)Ala38, were examined. The amino acid replacements introduced into BPTI(Ala14)Ala38 rendered it thermodynamically less stable. The kinetic stability, with respect to reduction by dithiothreitol, of the disulfides in these BPTI(Ala14)Ala38 variants was also decreased by the substitutions. The stabilization free energy (deltaG), obtained from chemical denaturation measurements, and the activation energy of the conformational transition (deltaG(++)conf), from the reductive unfolding reaction for this series of variants, were highly correlated. The observed correlation implies a direct coupling of disulfide reduction to conformational stability in this set of protein variants. It also strongly suggests that the transition state in the rate-limiting step of the reductive unfolding reaction involves a highly unfolded conformation of the protein. These data are consistent with a conformation-coupled redox folding pathway for BPTI(Ala14)Ala38 involving two parallel paths with unfolded (30-51) and unfolded (5-55) as the reactive species. Furthermore, the results provide a theoretical explanation for the observed 1000-fold diminution in the rate of 5-55 disulfide bond formation, relative to that of 14-38 bond formation, from the one-disulfide (30-51) intermediate in the wild-type BPTI refolding reaction. The data fit a general paradigm for protein disulfide formation during protein folding whereby native-like structure in folding intermediates accelerates formation of solvent-exposed disulfides but inhibits formation of core disulfides. This model predicts that a "rearrangement" mechanism (i.e., with non-native disulfides involved in the rate-limiting step) to form buried disulfides at a late stage in the folding reaction may be a common feature of redox folding pathways for surface disulfide-containing proteins of high stability.

Psychosocial stresses and adjustment of nasopharyngeal carcinoma patients in Hong Kong: a panel study.

PURPOSE: This article reports the results of a study conducted in Hong Kong from November 1992 to May 1994 on patients with nasopharyngeal carcinoma. The objective was to understand stresses and the short-term psychosocial adjustment outcome throughout the course of the illness. DESCRIPTION OF STUDY: A cognitive appraisal model of stress was used, and short-term psychosocial adjustment outcome was measured by psychological distress, social maladjustment to illness, and health status as perceived by the patients and as viewed by the oncologist. A panel study was adopted as the research design. A total of 125 patients were interviewed in the diagnostic phase, dropping to 119 in the treatment phase and 111 in the post-treatment phase. Data were gathered through face-to-face interviews by a structured instrument, the reliability of which had been evaluated immediately before the start of the study. RESULTS: The patients' level of anxiety was highest in the diagnostic phase, and gradually declined in the treatment and the post-treatment phases. Level of depression remained unchanged in the diagnostic and treatment phases, and decreased in the post-treatment phase; social maladjustment to illness and health status were poorest in the treatment phase. CLINICAL IMPLICATIONS: Health professionals can sharpen the focus of their work by helping nasopharyngeal carcinoma patients to lower their psychological distress in the diagnostic phase, enhance their social functioning, and improve their health status in the treatment and the post-treatment phases.