Overexpression of a 60-kDa heat shock protein enhances cytoprotective function of small intestinal epithelial cells

AimsWith the advancement of small intestinal (double balloon and capsule) endoscopy technology, incidence of small intestinal lesion caused by nonsteroidal anti-inflammatory drugs (NSAIDs) has been known to be high. However, therapy for small intestinal mucosal lesion has not yet been developed. Previous studies have shown that heat shock proteins (HSPs) are involved in cytoprotection mediated by their function as a molecular chaperone. In this study, we examined the effect of HSP60 or HSP70 overexpression on hydrogen peroxide-induced (H₂O₂) or indomethacin-induced cell damage in the small intestinal epithelial cells.Main methodscDNA of human HSP60 or HSP70 was transfected to rat small intestinal (IEC-6) cells, and HSP60- or HSP70-overexpressing cells were cloned. IEC-6 cells transfected with vector only were used as control cells. These cells were treated with H₂O₂ (0–0.14 mM) or indomethacin (0–2.5 mM). The cell viability was determined by MTT-assay. Cell necrosis was evaluated by LDH-release assay. Further, apoptosis was evaluated by caspases-3/7 activity and TUNEL assay.Key findingsCell viability after H₂O₂ or indomethacin treatment was significantly higher in HSP60-overexpressing cells compared with that in control cells and HSP60-overexpressing cells. Apoptotic cells were also reduced in HSP60-overexpressing. Conclusion: These results indicate that HSP60 plays an important role in protecting small intestinal mucosal cells from H₂O₂-induced or indomethacin-induced cell injury. HSP70-overexpressing cells did not show anti-apoptotic ability.SignificanceThese findings possibly suggest that function of each HSP is different in the small intestine. Therefore, for the therapy of small intestinal mucosal lesion, HSP60-induction therapy could be a new therapeutic strategy.     

Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.     

The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures

Various cytopathological structures, known as inclusion bodies, are formed upon infection of cultured leafhopper cells by Rice dwarf virus, a member of the family Reoviridae. These structures include tubules of approximately 85 nm in diameter which are composed of the nonstructural viral protein Pns10 and contain viral particles. Such tubular structures were produced in heterologous non-host insect cells that expressed Pns10 of the virus. These tubules, when associated with actin-based filopodia, were able to protrude from the surface of cells and to penetrate neighboring cells. A binding assay in vitro revealed the specific binding of Pns10 to actin. Infection of clusters of cells was readily apparent 5 days after inoculation at a low multiplicity of infection with the virus, even in the presence of neutralizing antibodies. However, treatment of host cells with drugs that inhibited the elongation of actin filaments abolished the extension of Pns10 tubules from the surface of cells, with a significant simultaneous decrease in the extent of infection of neighboring cells. These results together revealed a previously undescribed aspect of the intercellular spread of Rice dwarf virus, wherein the virus exploits tubules composed of a nonstructural viral protein and actin-based filopodia to move into neighboring cells.     

Unusual hydrophobic linker region of β-glucosidase (BGLII) from Thermoascus aurantiacus is required for hyper-activation by organic solvents

A gene encoding a putative β-glucosidase was isolated from Thermoascus aurantiacus IFO9748 and designated as bgl2. The recombinant enzyme showed β-glucosidase activity when p-nitrophenyl-β-glucose (pNP-Glc) was used as substrate. We also found that the enzyme activity was increased in the presence of organic solvents. An addition of 20 % (v/v) 1-octanol resulted in 54-fold higher activity of pNP-Glc hydrolysis, and transglycosylation activity was also found to be activated. The results of tryptophan fluorescence spectral analysis revealed the changes in the tertiary structure of the enzyme in the presence of 1-hexanol that may cause increased enzyme activity. BGLII has a distinctive hydrophobic linker region between N- and C-terminal domains. A chimeric enzyme in which the linker region was substituted by the corresponding region of another β-glucosidase failed to be activated by organic solvents, suggesting that the hydrophobic linker region may act as a molecular switch in BGLII.     

The effect of dimethyl sulfoxide on the function of cytochrome P450 2D6 in HepG2 cells upon the co-expression with NADPH-cytochrome P450 reductase

HepG2 cells, a human hepatoma cell line, stably expressing NADPH-cytochrome P450 reductase (OR) and/or cytochrome P450 2D6 wild-type (CYP2D6-WT) or its variants (Pro34Ser, Gly42Arg, Arg296Cys and Ser486Thr) were established in the present study. The cultivation of HepG2 cells expressing CYP2D6-WT in the culture medium containing dimethyl sulfoxide (DMSO, 0.1% of final concentration) markedly increased the bufuralol (BF) 1'-hydroxylase activity compared with that of control cells when cultivated without DMSO. A similar effect was also observed in HepG2 cells stably expressing CYP2D6 and OR. The addition of hemin in place of DMSO to the culture medium resulted in no increase in the enzyme activity. Western blot analysis revealed that the levels of CYP2D6 protein were similar between DMSO-treated and non-treated HepG2 cells regardless of OR expression. Spectrophotometric analysis of reduced carbon monoxide-difference spectra of HepG2 cells expressing CYP2D6-WT and/or OR demonstrated that the addition of DMSO increased the peak height of functional CYP2D6 at 450 nm. These results suggest that the increase in CYP2D6 activity is attributable to the radical-scavenging effect of DMSO. The HepG2 cell lines stably expressing OR and CYP2D6 or its variants in combination with DMSO treatment may be useful for screening the cytotoxicity of chemical compounds which undergo oxidation by CYP2D6.     

Symbolic-numeric estimation of parameters in biochemical models by quantifier elimination

The sequencing of complete genomes allows analyses of the interactions between various biological molecules on a genomic scale, which prompted us to simulate the global behaviors of biological phenomena on the molecular level. One of the basic mathematical problems in the simulation is the parameter optimization in the kinetic model for complex dynamics, and many estimation methods have been designed. We introduce a new approach to estimate the parameters in biological kinetic models by quantifier elimination (QE), in combination with numerical simulation methods. The estimation method was applied to a model for the inhibition kinetics of HIV proteinase with ten parameters and nine variables, and attained the goodness of fit to 300 points of observed data with the same magnitude as that obtained by the previous estimation methods, remarkably by using only one or two points of data. Furthermore, the utilization of QE demonstrated the feasibility of the present method for elucidating the behavior of the parameters and the variables in the analyzed model. Therefore, the present symbolic-numeric method is a powerful approach to reveal the fundamental mechanisms of kinetic models, in addition to being a computational engine.     

Effect of nutrient availability on the C, N, and P elemental ratios in the cyanobacterium Microcystis aeruginosa

To clarify whether nutrients limit the growth of Microcystis aeruginosa (Kütz.) Kütz during the growing season in Lake Yogo, we examined the cellular ratios of carbon (C), nitrogen (N), and phosphorus (P) in the populations of M. aeruginosa from August to December 2001. We also measured cellular C, N, and P ratios of M. aeruginosa under batch culture conditions. The cellular levels of N and P of M. aeruginosa in natural population changed more than twofold. The atomic N: C ratio of natural populations of Microcystis fluctuated from 0.11 to 0.26. The atomic P: C ratio fluctuated from 0.0080 to 0.024. The N: C, P: C, and N: P ratios of exponentially growing M. aeruginosa in N-and P-rich medium were 0.19, 0.013, and 15 on average. The growth of M. aeruginosa was suppressed below the N: C ratio of 0.13 under the N-free condition and below the P: C ratio of 0.0026 in the P-free condition. In the natural population, the N: C ratio was low on August 1-2 (0.11) and the P: C ratio was low (less than 0.011) until September. The Microcystis population on August 1-2 was N limited, judging from the results of the culture experiment. In other periods, the population seemed to be supplied with a sufficient amount of N. Although the P: C ratio was low (approximately 0.01) during August and September, it was several times larger than the value of the reduction of growth rate that occurred in culture. P limitation did not occur during the study period. N became more of a limiting factor than P for the formation of blooms of Microcystis. No blooms were observed in August and September, in spite of the increase of cellular levels of N. The formation of Microcystis blooms in Lake Yogo seems to be affected by artificial manipulations such as pumping from Lake Biwa and outflow.     

Addition of an extra substrate binding site and partial destabilization of stem structures in HDV ribozyme give rise to high sequence-specificity for its target RNA

Because the substrate binding site (P1) of HDV ribozyme consists of only seven nucleotides, cleavage of undesired RNA is likely to occur when applied for a specific long RNA target such as mRNA. To overcome this problem, we designed modified trans-acting HDV ribozymes with an extra substrate-binding site (P5) in addition to the original binding site (P1). By inserting an additional seven base-pair stem (P5 stem) into the J1/2 single-stranded region of the ribozyme core system and partial destabilization of the P2 or P4 stem, we succeeded in preparation of new HDV ribozymes that can cleave the target RNA depending on the formation of P5 stem. Moreover, the ribozyme with a six-nucleotide P1 site was able to distinguish the substrate RNA with a complete match from that with a single mismatch in the P1 region. These results suggest that the HDV ribozyme system is useful for the application in vivo.