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.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

Isolation and characterization of the mouse ortholog of the Fukuyama-type congenital muscular dystrophy gene

Fukuyama-type congenital muscular dystrophy (FCMD) is a severe autosomal-recessive muscular dystrophy accompanied by brain malformation. Previously, we identified the gene responsible for FCMD through positional cloning. Here we report the isolation of its murine ortholog, Fcmd. The predicted amino acid sequence of murine fukutin protein encoded by Fcmd is 90% identical to that of its human counterpart. Radiation hybrid mapping localized the gene to 2.02 cR telomeric to D4Mit272 on chromosome 4. Northern blot analysis revealed ubiquitous expression of Fcmd in adult mouse tissues. Through in situ hybridization, we observed a wide distribution of Fcmd expression throughout embryonic development, most predominantly in the central and peripheral nervous systems. We also detected high Fcmd expression in the ventricular zone of proliferating neurons at 13.5 days post-coitum. Brain malformation in FCMD patients is thought to result from defective neuronal migration. Our data suggest that neuronally expressed Fcmd is likely to be important in the development of normal brain structure.     

Linear polyubiquitin chains: A new modifier involved in NFκB activation and chronic inflammation including dermatitis

The ubiquitin conjugation system regulates a wide variety of biological phenomena, including protein degradation and signal transduction, by regulating protein function via polyubiquitin conjugation in most cases. Several types of polyubiquitin chains exist in cells, and the type of polyubiquitin chain conjugated to a protein seems to determine how that protein is regulated. We identified a novel linear polyubiquitin chain and the ubiquitin-protein ligase complex that assembles it, designated LUBAC. Both were shown to have crucial roles in the canonical NFκB activation pathway. This year, three groups, including our laboratory, identified SHARPIN as a new subunit of LUBAC. Of great interest, Sharpin was identified as a causative gene of chronic proliferative dermatitis in mice (cpdm), which is characterized by numerous inflammatory symptoms including chronic dermatitis, arthritis and immune disorders. Deletion of SHARPIN drastically reduces the amount of LUBAC and attenuates signal-induced NFκB activation. The pleomorphic symptoms of cpdm mice suggest that LUBAC-mediated NFκB activation may play critical roles in mammals and be involved in various disorders. A forward look into the linear polyubiquitin research is also discussed.Key words: ubiquitin, linear ubiquitination, NFκB, LUBAC, SHARPIN, cpdm, chronic dermatitis, TNFα     

Identification of novel biomarker candidates by proteomic analysis of cerebrospinal fluid from patients with moyamoya disease using SELDI-TOF-MS

Background

Moyamoya disease (MMD) is an uncommon cerebrovascular condition with unknown etiology characterized by slowly progressive stenosis or occlusion of the bilateral internal carotid arteries associated with an abnormal vascular network. MMD is a major cause of stroke, specifically in the younger population. Diagnosis is based on only radiological features as no other clinical data are available. The purpose of this study was to identify novel biomarker candidate proteins differentially expressed in the cerebrospinal fluid (CSF) of patients with MMD using proteomic analysis.

Methods

For detection of biomarkers, CSF samples were obtained from 20 patients with MMD and 12 control patients. Mass spectral data were generated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) with an anion exchange chip in three different buffer conditions. After expression difference mapping was undertaken using the obtained protein profiles, a comparative analysis was performed.

Results

A statistically significant number of proteins (34) were recognized as single biomarker candidate proteins which were differentially detected in the CSF of patients with MMD, compared to the control patients (p < 0.05). All peak intensity profiles of the biomarker candidates underwent classification and regression tree (CART) analysis to produce prediction models. Two important biomarkers could successfully classify the patients with MMD and control patients.

Conclusions

In this study, several novel biomarker candidate proteins differentially expressed in the CSF of patients with MMD were identified by a recently developed proteomic approach. This is a pilot study of CSF proteomics for MMD using SELDI technology. These biomarker candidates have the potential to shed light on the underlying pathogenesis of MMD.