Up-regulation of sodium-dependent glucose transporter by interaction with heat shock protein 70

Heat shock stress induces some heat shock proteins, including Hsp70, and activates sodium-dependent glucose transport in porcine renal LLC-PK(1) cells, but its mechanisms have not been described in detail. We investigated whether sodium-dependent glucose transporter (SGLT1) interacts with Hsp70 to increase SGLT1 activity. Heat shock stress increased SGLT1 activity without changing SGLT1 expression. The increase of SGLT1 activity was completely inhibited by an anti-transforming growth factor-beta1 (TGF-beta1) antibody. Instead of heat shock stress, TGF-beta1 increased SGLT1 activity dose- and time-dependently without changing SGLT1 expression. We found that the amount of Hsp70 immunoprecipitated from TGF-beta1-treated cells with an anti-SGLT1 antibody was higher than that of the control cells. Transfection of an anti-Hsp70 antibody into the cells inhibited the increase of SGLT1 activity. With confocal laser microscopy, both SGLT1 and Hsp70 was localized near the apical membrane in the TGF-beta1-treated cells, and an anti-Hsp70 antibody disturbed this localization. Furthermore, we clarified that an anti-Hsp70 antibody inhibited interaction of SGLT1 with Hsp70 in vitro. These results suggest that Hsp70 forms a complex with SGLT1 and increases the expression level of SGLT1 in the apical membrane, resulting in up-regulation of glucose uptake.     

The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.     

Characterization of Ca(2+) signaling pathways in human mesenchymal stem cells

Human mesenchymal stem cells (HMSC) have the potential to differentiate into many cell types. The physiological properties of HMSCs including their Ca(2+) signaling pathways, however, are not well understood. We investigated Ca(2+) influx and release functions in HMSCs. In Ca(2+) imaging experiments, spontaneous Ca(2+) oscillations were observed in 36 of 50 HMSCs. The Ca(2+) oscillations were completely blocked by the application of 10 micro M cyclopiazonic acid (CPA) or 1 micro M thapsigargin (TG). A brief application of 1 micro M acetylcholine (ACh) induced a transient increase of [Ca(2+)](i) but the application of caffeine (10 mM) did not induce any Ca(2+) transient. When the stores were depleted with Ca(2+)-ATPase blockers (CPA or TG) or muscarinic agonists (ACh), store-operated Ca(2+) (SOC) entry was observed. Using the patch-clamp technique, store-operated Ca(2+) currents (I(SOC)) could be recorded in cells treated with ACh or CPA, but voltage-operated Ca(2+) currents (VOCCs) were not elicited in most of the cells (17/20), but in 15% of cells examined, small dihydropyridine (DHP)-sensitive Ca(2+) currents were recorded. Using RT-PCR, mRNAs were detected for inositol 1,4,5-trisphosphate receptor (InsP(3)R) type I, II, and III and DHP receptors alpha1A and alpha1H were detected, but mRNA was not detected for ryanodine receptor (RyR) or N-type Ca(2+) channels. These results suggest that in undifferentiated HMSCs, Ca(2+) release is mediated by InsP(3)Rs and Ca(2+) entry through plasma membrane is mainly mediated by the SOCs channels with a little contribution of VOCCs.     

Diabetes is not a potent inducer of neuronal cell death in mouse sensory ganglia,but it enhances neurite regeneration in vitro

We examined the effects of diabetes on the morphological features and regenerative capabilities of adult mouse nodose ganglia (NG) and dorsal root ganglia (DRG). By light and electron microscopy, no apoptotic cell death was detected in the ganglia obtained from either streptozotocin (STZ)-induced diabetic or normal C57BL/6J mice in vivo. Neurite regeneration from transected nerve terminals of NG and DRG explants in culture at normal (10 mM) and high (30 mM) glucose concentrations was significantly enhanced in the diabetic mice. Chromatolytic changes (i.e. swelling and migration of the nucleus to an eccentric position in the neurons, and a loss of Nissl substance in the neuronal perikarya) and apoptotic cell death (less than one-fifth of the neurons) in the cultured ganglia were present, but neither hyperglycemia in vivo nor high glucose conditions in vitro altered the morphological features of the ganglia or the ratios of apoptotic cells at 3 days in culture. By semiquantitative RT-PCR analysis, the mRNA expressions of ciliary neurotrophic factor (CNTF) in DRG from both mice were down-regulated at 1 day in culture. The expression in diabetic DRG, but not in control DRG, was significantly up-regulated at later stages (3 and 7 days) in culture. In summary, hyperglycemia is unlikely to induce cell death in the sensory ganglia, but enhances the regenerative capability of vagal and spinal sensory nerves in vitro. The up-regulation of CNTF mRNA expression during the culture of diabetic DRG may play a role in the enhanced neurite regeneration.     

Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn2+ and Mn2+

Salts at high concentrations may cause oxidative damage to plant cells since many studies indicated the involvement of reactive oxygen species in salt-stress response. Recently, we have demonstrated that treatment of tobacco ( Nicotiana tabacum ) cell suspension culture with various salts result in an immediate burst of superoxide production via activation of NADPH oxidase by ions of alkali metals (Li⁺, Na⁺, K⁺), alkali earth metals (Mg²⁺, Ca²⁺) or lanthanides (La³⁺, Gd³⁺). In this study, we tested the effect of extracellular supplementation of Zn²⁺ and Mn²⁺ on the cation-induced oxidative burst in tobacco cell suspension culture, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent. Extracellular supplementation of Zn²⁺ and Mn²⁺ inhibited the generation of superoxide in response to addition of salts. Although both Zn²⁺ and Mn²⁺ inhibited the salt-induced generation of superoxide, the modes of inhibition by those ions seemed to be different since Mn²⁺ simply inhibited total production of superoxide while Zn²⁺ inhibited the early phase of superoxide production and induced the slow release of superoxide. Roles of Mn²⁺ and Zn²⁺ in protection of plant cells from salt stress, as an effective superoxide scavenger and an effective inhibitor of plasma membrane-bound NADPH oxidase, respectively, are discussed.     

Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase,in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.     

Arginine residue at position 573 in Enterococcus hirae vacuolar-type ATPase NtpI subunit plays a crucial role in Na+ translocation

The 76-kDa NtpI subunit constitutes the membrane-embedded V(0) moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, I. (2001) J. Biol. Chem. 276, 48337-48340). The negative cooperativity for the Na+ dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 +/- 0.03 and 0.40 +/- 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase.     

Structure of the antimicrobial peptide tachystatin A

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded beta-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist omega-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that omega-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and omega-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and omega-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe(9) of tachystatin A might be an essential residue for binding to chitin.     

Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography

Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid. We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography. The crystal structure of ryRNase NW bound to 5'-GMP was determined at 1.5 A resolution by molecular replacement with tomato RNase LE as a search model. The RNase NW structurally belongs to the (alpha + beta) class of proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a uridylic acid preferential RNase MC1 from bitter gourd seeds. The guanine ring of 5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89. In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79. Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the guanine base by RNase NW, findings which provide insight into the manner in which RNase NW preferentially cleaves guanylic acid.     

Dual roles of photosynthetic electron transport in photosystem I biogenesis: light induction of mRNAs and chromatic regulation at post-mRNA level

Light regulation of photosystem I (PSI) biogenesis was studied in a unicellular green alga, Chlamydomonas reinhardtii. When Chlamydomonas cells were transferred from darkness to the light, mRNAs for both nuclear- and chloroplast-encoded PSI subunits were induced in concert. This light induction was inhibited by photosynthetic electron transport (PET) inhibitors, 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone, but not by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone. This indicated that PET plays a pivotal role in the light induction of PSI subunit mRNAs, but that photophosphorylation is not necessary. When we irradiated the Chlamydomonas cells with PSI-light (695 nm) or PSII-light (644 nm), which makes the plastoquinone pool oxidative and reductive, respectively, PSII-light caused the accumulation of PSI proteins more abundantly than did PSI-light. However, there was no difference for the PSI subunit mRNA levels between these light sources. From these results, we conclude that PET plays dual roles in the regulation of PSI biogenesis in Chlamydomonas: when cells are illuminated, PET first induces the PSI subunit mRNAs irrespective of the redox state of the intersystem electron carriers, and then their redox state fine-tunes PSI biogenesis at translational and/or post-translational steps to fulfil the chromatic adaptation.