Targeting Superoxide Dismutase to Renal Proximal Tubule Cells Attenuates Vancomycin-induced Nephrotoxicity in Rats

Vancomycin hydrochloride (VCM), a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). As it is known to induce renal dysfunction, the dose and the duration of its administration are limited. Moreover, the mechanism of VCM-induced renal dysfunction remains to be unclear. To evaluate the involvement of free radical on VCM-induced renal dysfunction, we carried out analysis with a hexamethylenediamine-conjugated superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Male Wistar rats (weighing 200-210 g) were intraperitonealy administered with 200 mg/kg of VCM twice a day for 7 days. AH-SOD 5 mg/kg/day was subcutaneously injected 5 min before every VCM injection. VCM induced renal injury dose-dependently. Biochemical analyses revealed that plasma levels of blood urea nitrogen and creatinine significantly increased in the VCM-treated group by an AH-SOD-inhibitable mechanism. VCM simultaneously elicited an increase of 8-OHdG levels and chemiluminescence intensity of free radical generation in the kidney. Histological examination revealed that VCM also elicited a marked destruction of glomeruli and necrosis of proximal tubules. AH-SOD inhibited these phenomena in the kidney. These results suggested that oxidative stress might underlie the pathogenesis of VCM-induced nephrotoxicity and targeting SOD and/or related antioxidants to renal proximal tubules might permit the administration of higher doses of VCM sufficient for eradication of MRSA without causing renal injury.     

N- and C-terminal Upf1 phosphorylations create binding platforms for SMG-6 and SMG-5:SMG-7 during NMD

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors.     

Conformations of helical Aib peptides containing a pair of l‐amino acid and d‐amino acid

A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, ¹H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 3₁₀‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 3₁₀‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis

Chicken B lymphocyte precursors and DT40 cells diversify their immunoglobulin-variable (IgV) genes through homologous recombination (HR)-mediated Ig gene conversion. To identify DNA polymerases that are involved in Ig gene conversion, we created DT40 clones deficient in DNA polymerase eta (poleta), which, in humans, is defective in the variant form of xeroderma pigmentosum (XP-V). Poleta is an error-prone translesion DNA synthesis polymerase that can bypass UV damage-induced lesions and is involved in IgV hypermutation. Like XP-V cells, poleta-disrupted (poleta) clones exhibited hypersensitivity to UV. Remarkably, poleta cells showed a significant decrease in the frequency of both Ig gene conversion and double-strand break-induced HR when compared to wild-type cells, and these defects were reversed by complementation with human poleta. Our findings identify a DNA polymerase that carries out DNA synthesis for physiological HR and provides evidence that a single DNA polymerase can play multiple cellular roles.     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

Ionic mechanisms and Ca2+ dynamics underlying the glucose response of pancreatic β cells: a simulation study

To clarify the mechanisms underlying the pancreatic β-cell response to varying glucose concentrations ([G]), electrophysiological findings were integrated into a mathematical cell model. The Ca(2+) dynamics of the endoplasmic reticulum (ER) were also improved. The model was validated by demonstrating quiescent potential, burst-interburst electrical events accompanied by Ca(2+) transients, and continuous firing of action potentials over [G] ranges of 0-6, 7-18, and >19 mM, respectively. These responses to glucose were completely reversible. The action potential, input impedance, and Ca(2+) transients were in good agreement with experimental measurements. The ionic mechanisms underlying the burst-interburst rhythm were investigated by lead potential analysis, which quantified the contributions of individual current components. This analysis demonstrated that slow potential changes during the interburst period were attributable to modifications of ion channels or transporters by intracellular ions and/or metabolites to different degrees depending on [G]. The predominant role of adenosine triphosphate-sensitive K(+) current in switching on and off the repetitive firing of action potentials at 8 mM [G] was taken over at a higher [G] by Ca(2+)- or Na(+)-dependent currents, which were generated by the plasma membrane Ca(2+) pump, Na(+)/K(+) pump, Na(+)/Ca(2+) exchanger, and TRPM channel. Accumulation and release of Ca(2+) by the ER also had a strong influence on the slow electrical rhythm. We conclude that the present mathematical model is useful for quantifying the role of individual functional components in the whole cell responses based on experimental findings.     

Living wood fibers act as large-capacity ��single-use�� starch storage in black locust (Robinia pseudoacacia)

A living wood fiber (LWF) is one that retains the living protoplast. LWFs store numerous starch grains during the dormant period. In black locust (Robinia pseudoacacia), almost all wood fibers in the outer part of the annual ring are LWFs. In the outermost ring, starch is accumulated during the summer, retained in winter, and metabolized during spring. We determined the starch content of LWFs, ray parenchyma, and axial parenchyma using image analysis. More than 70% of the starch grains in the outermost ring were stored in LWFs during winter. After the breakdown of starch in spring, LWFs resulted in cell death. These results indicate that LWFs in black locust function as “single-use” large-capacity starch storage.     

Expression of normal cellular prion protein (PrP(c)) on T lymphocytes and the effect of copper ion: Analysis by wild-type and prion protein gene-deficient mice

The purpose of this report was to determine the effect of prion protein (PrP) gene disruption on T lymphocyte function. Previous studies have suggested that normal cellular prion protein (PrP(c)) binds to copper and Cu(2+) is essential for interleukin-2 (IL-2) mRNA synthesis. In this study, IL-2 mRNA levels in a copper-deficient condition were investigated using T lymphocytes from prion protein gene-deficient (PrP(0/0)) and wild-type mice. Results showed that Cu(2+) deficiency had no effect on PrP(c) expression in Con A-activated splenocytes. However, a delay in IL-2 gene expression was observed in PrP(0/0) mouse T lymphocyte cultures using Con A and Cu(2+)-chelator. These results suggest that PrP(c) expression may play an important role in rapid Cu(2+) transfer in T lymphocytes. The rapid transfer of Cu(2+) in murine T lymphocytes could be one of the normal functions of PrP(c).     

Unregulated expression of the imprinted genes H19 and Igf2r in mouse uniparental fetuses.

The present study shows that the H19 and Igf2r genes, which are imprinted and expressed solely from maternal alleles, are expressed in an unregulatable manner in mouse uniparental, androgenetic, and parthenogenetic fetuses at day 9.5 of gestation. In the androgenetic fetuses, the H19 and Igf2r genes were respectively expressed at 12 and 40% of the levels in biparental fetuses. In addition, the expression of both genes was excessive (1259 and 482%, respectively) in the parthenotes. These expressions of the imprinted genes were not regulated by methylation in the regulatory regions. Moreover, the expression of the antisense Igf2r RNA (Air) was also excessive and was not correlated with Igf2r gene expression in the uniparental fetuses. Taken together, these results indicate that the parental specific expression of imprinted genes is not maintained in particular genes in uniparental embryos, which in turn suggests that both parental genomes are required to establish maternal specific expression of the H19 and Igf2r genes by trans-acting mechanisms.