Neurexin-1, a presynaptic adhesion molecule, localizes at the slit diaphragm of the glomerular podocytes in kidneys

The slit diaphragm connecting the adjacent foot processes of glomerular epithelial cells (podocytes) is the final barrier of the glomerular capillary wall and serves to prevent proteinuria. Podocytes are understood to be terminally differentiated cells and share some common features with neurons. Neurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation. Although neurexin has been understood to be specifically expressed in neuronal tissues, we found that neurexin was expressed in several organs. Several forms of splice variants of neurexin-1α were detected in the cerebrum, but only one form of neurexin-1α was detected in glomeruli. Immunohistochemical study showed that neurexin restrictedly expressed in the podocytes in kidneys. Dual-labeling analyses showed that neurexin was colocalized with CD2AP, an intracellular component of the slit diaphragm. Immunoprecipitation assay using glomerular lysate showed that neurexin interacted with CD2AP and CASK. These observations indicated that neurexin localized at the slit diaphragm area. The staining intensity of neurexin in podocytes was clearly lowered, and their staining pattern shifted to a more discontinuous patchy pattern in the disease models showing severe proteinuria. The expression and localization of neurexin in these models altered more clearly and rapidly than that of other slit diaphragm components. We propose that neurexin is available as an early diagnostic marker to detect podocyte injury. Neurexin coincided with nephrin, a key molecule of the slit diaphragm detected in a presumptive podocyte of the developing glomeruli and in the glomeruli for which the slit diaphragm is repairing injury. These observations suggest that neurexin is involved in the formation of the slit diaphragm and the maintenance of its function.     

Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint

AimsTo investigate the effect of repeated stress on DNA damage in seven organs of dyslipidemic mice, and the preventive role of metallothionein (MT).Main methodsFemale adult 129/Sv wild-type and MT-null mice fed high-fat diet (HFD) were repeatedly subjected to mild stress of fasting or restraint in weeks 2 to 4 of 4-week study period. Serum cholesterol level, DNA damage in the liver, pancreas, spleen, bone marrow, kidney, lung and gastric mucosa, and other parameters were determined.Key findingsBody weights were increased in both types of mice fed HFD compared to those fed standard diet (STD), and further increased by 12 h-fasting, while they were markedly decreased by 1–3 h-restraint. Fasting accelerated accumulation of fat in the liver, and increase in serum cholesterol of both types of mice fed HFD. Feeding of HFD increased DNA damage in the pancreas, spleen and bone marrow of both types of mice, compared with those fed STD. In the wild-type mice fed HFD, 24 h-fasting increased DNA damage in the liver and spleen, while restraint increased the damage in the liver, pancreas, spleen and bone marrow. DNA damage in the cells of organs was markedly increased in the MT-null mice. Specifically, damage in the liver, pancreas, spleen and bone marrow was greatly increased with the intensity of stress increased, and the damage was much greater in the restraint mice than in the fasting mice.SignificanceMT plays a tissue-dependent preventive role against DNA damage in various murine organs induced by repeated stress.     

Effects of gender on kidney function in magnesium-deficient rats

This study investigated the gender differences in the kidney function of magnesium (Mg)-deficient rats. Male and female rats were fed a control diet or a Mg-deficient diet for 21 d. Mg-deficient diet had no significant effect on kidney calcium (Ca) or phosphorus (P) concentration in male rats, while Ca and P concentrations in female rats were significantly higher in Mg-deficient rats than in the control rats. With regard to indicators of kidney function, no significant differences in creatinine clearance and serum urea nitrogen concentration were observed among the groups. Serum albumin concentrations were significantly lower in rats fed the Mg-deficient diet than in rats fed the control diet. In both sexes, urinary albumin excretion was significantly higher in rats fed the Mg-deficient diet than in rats fed the control diet. Gender differences had no significant influence on creatinine clearance, serum urea nitrogen concentration, serum albumin concentration and urinary albumin excretion. These results suggest that gender differences have no effect on kidney function in Mg-deficient rats under the condition used.     

Genomic Motifs as a Novel Indicator of the Relationship between Strains Isolated from the Epidemic of Porcine Epidemic Diarrhea in 2013-2014

Porcine epidemic diarrhea virus (PEDV) is a positive-sense RNA virus that causes infectious gastroenteritis in pigs. Following a PED outbreak that occurred in China in 2010, the disease was identified for the first time in the United States in April 2013, and was reported in many other countries worldwide from 2013 to 2014. As a novel approach to elucidate the epidemiological relationship between PEDV strains, we explored their genome sequences to identify the motifs that were shared within related strains. Of PED outbreaks reported in many countries during 2013–2014, 119 PEDV strains in Japan, USA, Canada, Mexico, Germany, and Korea were selected and used in this study. We developed a motif mining program, which aimed to identify a specific region of the genome that was exclusively shared by a group of PEDV strains. Eight motifs were identified (M1–M8) and they were observed in 41, 9, 18, 6, 10, 14, 2, and 2 strains, respectively. Motifs M1–M6 were shared by strains from more than two countries, and seemed to originate from one PEDV strain, Indiana12.83/USA/2013, among the 119 strains studied. BLAST search for motifs M1–M6 revealed that M3–M5 were almost identical to the strain ZMDZY identified in 2011 in China, while M1 and M2 were similar to other Chinese strains isolated in 2011–2012. Consequently, the PED outbreaks in these six countries may be closely related, and multiple transmissions of PEDV strains between these countries may have occurred during 2013–2014. Although tools such as phylogenetic tree analysis with whole genome sequences are increasingly applied to reveal the connection between isolates, its interpretation is sometimes inconclusive. Application of motifs as a tool to examine the whole genome sequences of causative agents will be more objective and will be an explicit indicator of their relationship.     

Genetic identification of larvae and juveniles reveals the difference in the spawning site among Cyprininae fish species/subspecies in Lake Biwa

To understand the differences in the spawning sites among Cyprininae fishes in Lake Biwa, we conducted periodic sampling of larvae and juveniles at three sites (irrigation ditch, St. 1; river, St. 2; and satellite lake, St. 3). On the basis of species/subspecies identification by using RAPD analysis, we examined the species composition of the larvae and juveniles at these three sampling sites. The number of specimens was 616, 68, and 117 at St. 1, St. 2, and St. 3, respectively. Based on morphological and genetic identification, the specimens were found to include nine fish species/subspecies, namely, Carassius auratus grandoculis, Carassius cuvieri, Carassius auratus langsdorfii, Cyprinus carpio, Sarcocheilichthys sp., Silurus asotus, Oryzias latipes, Odontobutis obscura obscura, and Rhinogobius sp. The species composition at the three sites also differed. Among the Cyprininae fishes, C. auratus grandoculis, C. auratus langsdorfii, and Cyprinus carpio were found in abundance at St. 1; C. cuvieri was not collected from St. 1 but was found at the other two sites, particularly St. 3. Among the other fishes, Rhinogobius sp. was collected at St. 1 and St. 3, whereas the other four occurred only at St. 1. These results suggest that the selection of spawning sites by C. cuvieri differs to a certain extent from that of the other Cyprininae fishes, and the irrigation ditch in the lake is an important habitat for the larvae and juveniles of native fish species.     

Identification of catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by gamma-monofluorophosphono derivative of glutamic acid: N-terminal thr-391 in small subunit is the nucleophile

gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione metabolism and catalyzes the hydrolysis and transpeptidation of gamma-glutamyl compounds such as glutathione and its derivatives. The reaction is thought to proceed via a gamma-glutamyl-enzyme intermediate where a hitherto unknown catalytic nucleophile is gamma-glutamylated. Neither affinity labeling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile. We describe here the identification of the catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride derivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a time-dependent manner (k(on) = 4.83 x 10(4) M(-1) s(-1)). The inactivation rate was decreased by increasing the concentration of the substrate. The inactivated enzyme did not regain its activity after prolonged dialysis, suggesting that 1 served as an active-site-directed affinity label by phosphonylating the putative catalytic nucleophile. Ion-spray mass spectrometric analyses revealed that one molecule of 1 phosphonylated one molecule of the small subunit. LC/MS experiments of the proteolytic digests of the phosphonylated small subunit identified the N-terminal peptide Thr391-Lys399 as the phosphonylation site. Subsequent MS/MS experiments of this peptide revealed that the phosphonylated residue was Thr-391, the N-terminal residue of the small subunit. We conclude that the N-terminal Thr-391 is the catalytic nucleophile of E. coli gamma-glutamyltranspeptidase. This result strongly suggests that gamma-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family.     

RhoA-mediated Phospholipase D1 signaling is not required for the formation of stress fibers and focal adhesions

The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58 and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.