Isoform-specific induction of PKC-epsilon by high glucose protects heart-derived H9c2 cells against hypoxic injury

We investigated which PKC isoforms are involved in high glucose-induced protection against hypoxic injury. Treatment for 48 h with high glucose (22 mM) markedly increased the expression of PKC- epsilon in the particulate fraction (213+/-22.1% of the control) but had no effect on other types of PKC isoforms, suggesting that the high glucose-induced increase in PKC expression is isoform-specific. The mRNA level for PKC- epsilon was also substantially increased, reaching its peak after 4h of high glucose treatment. The high glucose increased PKC-epsilon activity in the particulate fraction up to 183+/-32.2% of the control. During hypoxia, the amount of PKC-epsilon in the particulate fraction was remarkably diminished in the low glucose-treated cells, but remained at a higher level in high glucose-treated cells. The treatment with epsilon V1-2 (10 microM), a specific inhibitor of PKC epsilon, abolished the protective effect of high glucose against hypoxia. These results suggest that isoform-specific induction of PKC-epsilon is involved in high glucose-induced protection against hypoxic injury in heart-derived H9c2 cells.     

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca(2+)](c) in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca(2+) entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca(2+)](c) by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn(2+) quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca(2+) influx pathway of WEHI-231 was hardly permeable to Mn(2+). The intractable increase of [Ca(2+)](c) may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.     

Helicobacter pylori in familial clusters based on antibody profile

Studies have shown a high prevalence of Helicobacter pylori infection in close communities and that intrafamilial spread during early childhood may be a route of transmission. A total of 72 household members from 21 families were enrolled in this study. Sera from individuals showed 50/72 (69.4%) seropositive for IgG against H. pylori by ELISA. Western blots showed diversity in the protein profiles with molecular masses ranging from approximately 8 to 130 kDa. Cohen's kappa statistical analysis of the blot patterns showed that nine families demonstrated similar profiles (100%), while 4 other families showed varying similarities (17-50%). The results support the hypothesis of intrafamilial transmission of H. pylori. Furthermore, serological studies can be used as an effective approach to determine the familial status in relation to H. pylori infection.     

ATP-sensitive K(+) channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes

The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K(+) (K(ATP)) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated K(ATP) channel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 microM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 microM) increased K(ATP) channel activity in cell-attached patches. PKG (5 U/microl) added together with ATP and cGMP (100 microM each) directly to the intracellular surface increased the channel activity. Activation of K(ATP) channels was abolished by the replacement of ATP with ATPgammaS. Rp-pCPT-cGMP (100 microM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the K(ATP) channels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated K(ATP) channel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of K(ATP) channels in rabbit ventricular myocytes.     

The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.     

Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase

Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.     

A cell-free protein synthesis system as an investigational tool for the translation stop processes

Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.     

Paragonimus westermani: molecular cloning,expression, and characterization of a recombinant yolk ferritin

Ferritin is an intracellular protein involved in iron metabolism. A cDNA PwYF-1 cloned from the adult Paragonimus westermani cDNA library encoded a putative polypeptide of 216 amino acids homologous with ferritins of vertebrates and invertebrates. Febinding motifs identified in PwYF-1 polypeptide were conserved and predicted to form a ferroxidase center. PwYF-1 polypeptide contained an extended peptide of 45 amino acids at its C-terminus. Recombinant PwYF-1 protein, expressed and purified from Escherichia coli, showed iron-uptake ability and ferroxidase activity. Ferroxidase activity of recombinant PwYF-1 protein was reactivated by secondary addition of apotransferrin to assay mixture. Mouse immune serum raised against the recombinant PwYF-1 protein recognized specifically 24 kDa protein from adult P. westermani lysate. PwYF-1 protein was localized to vitelline follicles and the eggs of P. westermani. Collectively, PwYF-1 protein was identified as a P. westermani yolk ferritin.