Characterization of SNARE protein expression in beta cell lines and pancreatic islets

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.     

Association of electrocardiographic left ventricular hypertrophy with the incidence of new congestive heart failure

OBJECTIVE: To investigate the association of electrocardiographic (ECG) left ventricular hypertrophy (LVH) with the incidence of new congestive heart failure (CHF) in older people. DESIGN: In a prospective study of 2638 older people, ECGs were obtained at study entry, at 1 month after study entry, when clinically indicated, and at least yearly after study entry. ECG LVH was diagnosed if the point score of Romhilt and Estes was > or = 5. Persistent LVH was diagnosed if all of the ECGs showed LVH. New LVH was diagnosed if the baseline ECG showed no LVH but LVH was present on the last ECG. Regression of LVH was diagnosed if the baseline ECG showed LVH but no LVH was present on the last ECG. No LVH was diagnosed if all of the ECGs showed no LVH. Persistent LVH, new LVH, regression of LVH, and no LVH were correlated with the incidence of new CHF at follow-up. SETTING: A large long-term health care facility. PATIENTS: The patients included 1805 women and 833 men, mean age 81 +/- 9 years (range 60 to 103). MEASUREMENTS AND MAIN RESULTS: Of the 2,638 older persons studied, 281 (11%) had persistent ECG LVH, 31 (1%) had new ECG LVH, 12 (0.5%) had regression of ECG LVH, and 2314 (88%) had no ECG LVH. At 42 +/- 24 months (range 1 to 154 months) follow-up, new CHF developed in 168 of 281 persons (60%) with persistent LVH, in 16 of 31 persons (52%) with new LVH, in 4 of 12 persons (33%) with regression of LVH, and in 507 of 2314 persons (22%) with no LVH. Kaplan-Meier survival curves showed that the development of new CHF was higher in persons with persistent LVH versus regression of LVH (P = .013), in persons with persistent LVH versus no LVH (P = .001), in persons with new LVH versus regression of LVH (P = .039), and in persons with new LVH versus no LVH (P = .001). CONCLUSION: Older persons with persistent or new ECG LVH have a higher incidence of new CHF and an earlier time to the development of new CHF than older persons without ECG LVH.     

Direct action of nitric oxide on osteoblastic differentiation

The effect of nitric oxide (NO) on osteoblastic differentiation was examined in cultured mouse osteoblasts. Interleukin-1beta and tumor necrosis factor-alpha expressed inducible NO synthase gene with little effect on constitutive NO synthase gene. These cytokines increased NO production, which was inhibited by L-NMMA pretreatment, and decreased alkaline phosphatase (AIPase) activity, which was not restored by L-NMMA. Furthermore, NO donors, sodium nitroprusside and NONOate dose-dependently elevated AIPase activity and expression of osteocalcin gene. These results suggest that NO directly facilitates osteoblastic differentiation and the cytokine-induced inhibition of AIPase activity is mediated via mechanism other than NO.     

Functional characterization of the Tn5 transposase by limited proteolysis

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.     

Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation

This study investigates the significance of the glucose-6-phosphate dehydrogenase (G6PD) catalyzed oxidative and the transketolase (TK) catalyzed nonoxidative pentose cycle (PC) reactions in the tumor proliferation process by characterizing tumor growth patterns and synthesis of the RNA ribose moiety in the presence of respective inhibitors of G6PD and TK. Mass spectra analysis of 13C-labeled carbons revealed that these PC reactions contribute to over 85% of de novo ribose synthesis in RNA from [1,2-(13)C]glucose in cultured Mia pancreatic adenocarcinoma cells, with the fraction synthesized through the TK pathway predominating (85%). Five days of treatment with the TK inhibitor oxythiamine (OT) and the G6PD inhibitor dehydroepiandrosterone-sulfate (0.5 microM each) exerted a 39 and a 23% maximum inhibitory effect on cell proliferation in culture, which was increased to 60% when the two drugs were administered in combination. In vivo testing of 400 mg/kg OT or dehydroepiandrosterone-sulfate in C57BL/6 mice hosting Ehrlich's ascitic tumor cells revealed a 90.4 and a 46% decrease in the final tumor mass after 3 days of treatment. RNA ribose fractional synthesis through the TK reaction using metabolites directly from glycolysis declined by 9.1 and 23.9% after OT or the combined treatment, respectively. Nonoxidative PC reactions play a central regulating role in the carbon-recruiting process toward de novo nucleic acid ribose synthesis and cell proliferation in vitro and in vivo. Therefore, enzymes or substrates regulating the nonoxidative synthesis of ribose could also be the sites to preferentially target tumor cell proliferation by new anticancer drugs.