Effect of insulin on the glucose utilization in isolated cardiac myocytes from adult rat

The acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 microM to 5.5 mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 microM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (greater than 100 microM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5 mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressive effects of 4-phenylbutyrate on the aggregation of Pael receptors and endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.     

A Putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren Syndrome Chromosome Region 17 (WBSCR17) Regulates Lamellipodium Formation and Macropinocytosis

We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294-3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.     

8-Hydroxyguanine levels and repair capacity during mouse embryonic stem cell differentiation

To evaluate the defence capacities of embryonic stem (ES) cells against gene impairment, this study measured the levels of 8-hydroxyguanine (8-OH-Gua), a well-known marker of oxidative stress in DNA, and its repair capacity during differentiation. Undifferentiated ES cells (EB3) were cultured without leukaemia inhibitory factor (LIF) for 0, 4 and 7 days and are referred to as ES-D0, ES-D4 and ES-D7, respectively. These three cell lines were treated with 300 μM hydrogen peroxide (H(2)O(2)) for 48 and 72 h. After treatment, the amounts of 8-OH-Gua in the cells were determined by the high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) method. The levels of 8-OH-Gua in ES-D7 treated with H(2)O(2) were higher than those in ES-D0 and ES-D4, suggesting that the DNA in the undifferentiated cells was protected against gene impairment, as compared to that in the differentiated cells. To examine the repair capacity for 8-OH-Gua, this study analysed the expression of 8-OH-Gua repair-associated genes, 8-oxoguanine DNA glycosylase 1 (OGG1), MutY homolog (MUTYH) and Mut T homolog 1 (MTH1), in ES-D0, ES-D4 and ES-D7. The mRNA levels of MUTYH and MTH1 showed no significant change, whereas OGG1 mRNA was significantly decreased in ES-D7 treated with H(2)O(2). Moreover, it was observed that ES-D7 treated with H(2)O(2) readily underwent apoptosis, in comparison to its undifferentiated counterparts, ES-D0 and ES-D4. Taken together, ES cells are more resistant to DNA oxidative stresses than differentiated cells.     

Schizosaccharomyces pombe RanGAP homolog, SpRna1, is required for centromeric silencing and chromosome segregation

We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S. pombe Ran homolog), rescued a lethality of Sprna1ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR+ rescued a centromeric silencing defect and Deltaclr4+ was synthetic lethal with Sprna1ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.     

Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400

SEA0400 is a potent and selective Na(+)/Ca(2+) exchanger (NCX) inhibitor. We evaluated the inhibitory effects of SEA0400 on Na(+)(i)-dependent (45)Ca(2+) uptake and whole-cell Na(+)/Ca(2+) exchange currents in NCX-transfected fibroblasts. SEA0400 preferentially inhibited (45)Ca(2+) uptake by NCX1 compared with inhibitions by NCX2, NCX3, and NCKX2. SEA0400 also selectively blocked outward exchange currents from NCX1 transfectants. We searched for regions that may form the SEA0400 receptor in the NCX1 molecule by NCX1/NCX3 chimeric analysis. The results suggest that the first intracellular loop and the fifth transmembrane segment are mostly responsible for the differential drug responses between NCX1 and NCX3. Further site-directed mutagenesis revealed that multiple mutations at Phe-213 markedly reduced sensitivity to SEA0400 without affecting that to KB-R7943. We also found that Gly-833-to-Cys mutation (within the alpha-2 repeat) greatly reduced the inhibition by SEA0400, but unexpectedly the NCX1 chimera with an alpha-2 repeat from NCKX2 possessed normal drug sensitivity. In addition, exchangers with mutated exchanger inhibitory peptide regions, which display either undetectable or accelerated Na(+)-dependent inactivation, had a markedly reduced sensitivity or hypersensitivity to SEA0400, respectively. To verify the efficacy of the NCX inhibitor, we examined the renoprotective effect of SEA0400 in a hypoxic injury model using porcine renal tubular cells. SEA0400 protected against hypoxia/reoxygenation-induced cell damage in tubular cells expressing wild-type NCX1 but not in cells expressing SEA0400-insensitive mutants. These results suggest that Phe-213, Gly-833, and residues that eliminate Na(+)-dependent inactivation are critical determinants for the inhibition by SEA0400, and their mutants are very useful for checking the pharmacological importance of NCX inhibition by SEA0400.     

Fine needle aspiration cytology of adenocarcinoma of the rectovaginal septum. A case report

BACKGROUND: Adenocarcinoma arising in the rectovaginal septum is exceedingly rare and is difficult to diagnose by pathologic examination prior to surgery because of the anatomic position of the tumor. CASE: A 42-year-old woman presumed to have adenocarcinoma of the rectovaginal septum underwent fine needle aspiration for diagnosis. Although a previously performed biopsy from the posterior vaginal fornix was unsuccessful, fine needle aspiration cytology via the posterior vaginal wall detected adenocarcinoma cells. The cell clusters were composed of cells with enlarged and hyperchromatic nuclei. The nuclei themselves demonstrated round and/or irregular morphologic patterns, with high nuclear/cytoplasmic ratios, and often contained an enlarged, round nucleolus and sometimes multiple ones in a single nucleus. Aniso-nucleosis was severe, and the chromatin patterns ranged from coarse to finely granular. The cytoplasm was narrow and lightly stained. Following fine needle aspiration, the patient underwent posterior exenteration on the basis of the cytologic diagnosis. CONCLUSION: Fine needle aspiration cytology was useful in establishing the preoperative diagnosis of adenocarcinoma of the rectovaginal septum, and curative exenterative surgery could be then performed. To our knowledge, this is the first report of fine needle aspiration cytology of adenocarcinoma at this location.     

Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8 Å Reveals Rearrangement between Two GroEL Rings

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES₂ complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES₂-ATP₁₄ complex from Escherichia coli at 3.8 Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~ 7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.     

Leu309 plays a critical role in the encapsulation of substrate protein into the internal cavity of GroEL

In the crystal structure of the native GroEL.GroES.substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL.GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K).substrate complex failed to bind GroES to produce a stable GroEL(L309K).GroES.substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K).GroES.substrate complex was stabilized with BeF(x), but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu(309)), appears to play a critical role in encapsulation of the substrate.