Involvement of RhoA/ROCK in insulin secretion of pancreatic β-cells in 3D culture

Cell-cell contacts and interactions between pancreatic β-cells and/or other cell populations within islets are essential for cell survival, insulin secretion, and functional synchronization. Three-dimensional (3D) culture systems supply the ideal microenvironment for islet-like cluster formation and functional maintenance. However, the underlying mechanisms remain unclear. In this study, mouse insulinoma 6 (MIN6) cells were cultured in a rotating 3D culture system to form islet-like aggregates. Glucose-stimulated insulin secretion (GSIS) and the RhoA/ROCK pathway were investigated. In the 3D-cultured MIN6 cells, more endocrine-specific genes were up-regulated, and GSIS was increased to a greater extent than in cells grown in monolayers. RhoA/ROCK inactivation led to F-actin remodeling in the MIN6 cell aggregates and greater insulin exocytosis. The gap junction protein, connexin 36 (Cx36), was up-regulated in MIN6 cell aggregates and RhoA/ROCK-inactivated monolayer cells. GSIS dramatically decreased when Cx36 was knocked down by short interfering RNA and could not be reversed by RhoA/ROCK inactivation. Thus, the RhoA/ROCK signaling pathway is involved in insulin release through the up-regulation of Cx36 expression in 3D-cultured MIN6 cells.     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.     

Biosynthesis of meteloidine from 3α-tigloyloxytropane in Datura innoxia

The administration of 3α-tigloyl-[1-¹⁴C]-oxytropane-[3β-³H] (³H/¹⁴C = 11·0 to Datura innoxia plants for 7 days led to the formation of radioactive meteloidine (³H/¹⁴C = 11·6). Degradation of the meteloidine indicated that the alkaloid was labeled specifically with ³H at C-3 of its teloidine moiety, and on the carbonyl group of its tigloyl residue with ¹⁴C. These results strongly favor the hypothesis that hydroxylation of tropine occurs after formation of its tigloyl ester.     

Two Novel SNPs in ATXN3 3’ UTR May Decrease Age at Onset of SCA3/MJD in Chinese Patients

Spinocerebellar ataxia type 3 (SCA3), or Machado—Joseph disease (MJD), is an autosomal dominantly-inherited disease that produces progressive problems with movement. It is caused by the expansion of an area of CAG repeats in a coding region of ATXN3. The number of repeats is inversely associated with age at disease onset (AO) and is significantly associated with disease severity; however, the degree of CAG expansion only explains 50 to 70% of variance in AO. We tested two SNPs, rs709930 and rs910369, in the 3’ UTR of ATXN3 gene for association with SCA3/MJD risk and with SCA3/MJD AO in an independent cohort of 170 patients with SCA3/MJD and 200 healthy controls from mainland China. rs709930 genotype frequencies were statistically significantly different between patients and controls (p = 0.001, α = 0.05). SCA3/MJD patients carrying the rs709930 A allele and rs910369 T allele experienced an earlier onset, with a decrease in AO of approximately 2 to 4 years. The two novel SNPs found in this study might be genetic modifiers for AO in SCA3/MJD.     

Overexpression of osteopontin induces angiogenesis of endothelial progenitor cells via the avβ3/PI3K/AKT/eNOS/NO signaling pathway in glioma cells

Angiogenesis, a hallmark of tumor growth, is regulated by various angiogenic factors. Recent studies have shown that osteopontin (OPN) is a secreted, integrin-binding protein that contributes to glioma progression. However, its effect on the angiogenesis of gliomas is not fully understood. To elucidate the role of OPN in the process of glioma angiogenesis, endothelial progenitor cells (EPCs) were treated with conditioned media of human glioma SHG44 cells overexpressing OPN. Here, we identified that OPN secreted by glioma cells accelerated EPCs angiogenesis in vitro, including proliferation, migration, and tube formation. OPN also induced the activation of AKT and endothelial nitric oxide synthase (eNOS) and increased NO production without affecting the expression of VEGF, VEGFR-1, or VEGFR-2. Moreover, the avβ3 antibody, the PI3-K inhibitor LY294002 and the eNOS inhibitor NMA suppressed the OPN-mediated increase in NO production and angiogenesis in EPCs. Taken together, these results demonstrate that OPN directly stimulates angiogenesis via the avβ3/PI3-K/AKT/eNOS/NO signaling pathway and may play an important role in tumorigenesis by enhancing angiogenesis in gliomas.     

Identification of guanosine 3′:5′-monophosphate in the fruit of Zizyphus jujuba

Evidence is presented here confirming the identification of guanosine 3′: 5′-monophosphate (c GMP) in the tissue of higher plants. The c GMP activity detected in fruits of Zizyphus jujuba was separated from the c AMP activity also present. The separated sample was extensively purified by Bio-Rad AG 1 × 4 and aluminium oxide CC, and by TLC. The purified sample showed the same physicochemical properties as authentic c GMP by TLC using different solvents and by UV spectroscopy, and was decomposable by cyclic nucleotide-specific phosphodiesterase. The identification was further supported by HPLC. The amount of c GMP present increases 90-fold during fruit ripening.     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

Synthesis of 4-hydroxy-β3-homoprolines and their insertion in α/β/α-tripeptides

The stereoselective syntheses of 2-cyclopropyl- and (2S)-2-hydroxymethyl-(3R,4S)-4-hydroxy-β³-homoproline are described. The reported amino acids were constructed through 1,3-dipolar cycloaddition of strained alkylidenecyclopropanes with enantiopure pyrroline N-oxides derived from malic acid followed by thermal rearrangement of the adducts in the presence of trifluoroacetic acid. The two-step sequence afforded the homoprolines suitably protected to be directly used as building blocks in peptidomimetic synthesis as proved by the synthesis of the two model mixed α/β/α tripeptides Phe-β³-HPro-Val.     

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.     

Existence of independent C/EBPβ 3＇-UTR RNA in human tissues

Two years ago, it was found that the RNA expressed from the promoter in the 3＇-untranslated region （UTR） of a gene, i.e. independent 3＇-UTR RNA, exists widely in human, mouse, and fly [1].     

Effect of concentration of iodide on the bound species of I2/I−3 in the amylose-iodine complex

A liquid-liquid distribution method, with heptane as the organic solvent, involving evaluation of the concentration of free 1⁻ by magnetic circular dichroism, has been developed for determining the bound amounts of I₂/I⁻₃ in the amylose-iodine complex in unbuffered aqueous solutions. The effect of I₂ and I⁻ concentrations on the bound species of iodine in the complex was investigated by using this method. We found that the stoichiometric bound species of I₂/I⁻₃ is independent of the concentration of I₂ at a given I⁻ concentration. However, the bound species strongly depends on I⁻ concentration, and varies from I⁻₃ at 10 mM KI to I⁻₁₅ at 0M KI. Moreover, the number of d-glucosyl residues required for including one iodine atom is within the range of 2.7 to 3.0, regardless of I⁻ concentration. It was concluded that the bound species are governed by the distribution of the actual species I₂·I₂ (I₄), (I₄), I₂·I⁻₃ (I⁻₅), and I⁻₃·I⁻₃ (I²⁻₆), which are responsible for the blue color of the complex.     

Does estradiol stimulate in vivo production of 1,25-dihydroxyvitamin D3 in the rat?

Female rats were injected with estradiol benzoate (3 mg/kg daily) or corn oil for 8 days before receiving 325 picomoles of 25-[26,27³H]-hydroxyvitamin D₃ intravenously. Eighteen hours later lipids were extracted from plasma, gut mucosa and kidneys and vitamin D₃ metabolites were separated using Sephadex LH-20 and chloroform: hexanes (65:35) column chromatography. Estradiol treatment increased 1,25-[26,27-³H]-dihydroxyvitamin D₃ content in all three isssues.     

Genome organization and variation in the 3′-partial sequence of garlic latent virus in China

Ten different isolates of a carlavirus were detected by degenerate PCR from 12 garlic samples collected from 6 provinces in China, and the complete genome sequence of the Zhejiang isolate ZJ1 and 3'-terminal sequences of 9 other isolates were determined. The RNA genome of isolate ZJ1 consisted of 8363nts excluding the 3'-poly (A) tail, and the genome organization was similar to other carlaviruses with 6 open reading frames encoding a replicase, TGB1, TGB2, TGB3, CP and NABP respectively. Sequence comparisons showed that all 10 isolates were Garlic latent virus (GarLV). The variations in the TGB2, TGB3 and NABP were more significant than those in the CP. High homology was also detected between those isolates and Shallot latent virus (ShLV). Phylogenetic analysis suggested that GarLV isolates from garlic can be divided into 4 main groups and Chinese isolates belonged to each group. This is the first reported molecular analysis of members of the genus Carlavirus in China.     

Anti‐adipogenic Effects of αAL14 Mediated by Modulation of PI3K/Akt Pathways in 3T3-L1 Cells

International Journal of Peptide Research and Therapeutics - A model peptide, αAL14, was designed from the primary structure of a novel antimicrobial peptide purified form the abalone....     

The Role of dinB in the Appearing of Antibiotics Resistance in E.coli

The role of dinB gene in the appearing of antibiotics resistance was studied. Plasmid containing multi-copy dinB gene was transfected into E. coli to create an overexpression. The strains carrying multi-copies of dinB gene demonstrate a significant survival advantage over the wild strain. In vitro experiment, the dinB-overexpressed strain evolved resistance within 8 hours, while wild strain could not.In vivo experiment with mice model infected with dinB-overexpressed strain, resistant clones emerged significantly earlier and demonstrated significant higher level of resistance than those infected with the wild control strain. The results showed that dinB gene made a contribution in the appearing of the antibiotics resistance and has a potential as a target for prevention from the appearing of antibiotic resistance.     

A high proportion of mutations in the BRCA1 gene in German breast/ovarian cancer families with clustering of mutations in the 3′ third of the gene

We have analyzed 61 German breast and breast/ovarian cancer families for BRCA1 mutations using single-strand conformation polymorphism analysis (SSCP) followed by sequencing. Forty-seven of the families had at least three cases (at least two under 60 years) and 14 families had only two cases of breast/ovarian cancer (at least one under 50 years). Twenty-eight families were breast/ovarian and 33 were breast cancer-only families. Eighteen mutations in BRCA1 were detected in 11/28 breast/ovarian cancer families and 7/33 breast cancer families and none in the families with only two cases. We identified 17 truncation mutations (8 frameshift, 7 nonsense and 2 splice variants) and one missense mutation. Seven of these are novel and two, the 5382insC and 5622C→T mutations, occurred in two apparently unrelated families. The genotype of the two families with the 5382insC mutation is compatible with the rare haplotype segregating with the 5382insC mutation in different populations, further supporting its European origin. One unclassified missense alteration, R841W, was found in one family but did not segregate with the disease, suggesting that it is more likely a polymorphism. We also report and discuss the sequence of several new unclassified single-nucleotide changes first identified by SSCP. Of the 18 mutations, 13 occurred in the 3′ third of the gene (end of exon 11–24) and ovarian cancers were found in eight of these families. Received: 5 February 1998 / Accepted: 7 April 1998