Effect of insulin therapy on renal changes in spontaneously diabetic Torii rats.

The spontaneously diabetic Torii (SDT) rat has recently been established as an animal model of non-obese type 2 diabetes, in which ocular complications severe occur. However, the function and morphological features of the diabetic renal lesions in SDT rats have not been reported in detail. Therefore, we evaluated changes over time in renal lesions in SDT rats. In addition, SDT rats were treated with insulin to observe whether these renal complications are caused by hyperglycemia. Renal functional parameters and renal lesions were monitored in SDT rats from 8 to 68 weeks of age. Sprague-Dawley (SD) rats of similar age were used as control animals. In the insulin-treated group of SDT rats, insulin pellets were implanted at 24 weeks of age to compare the development of renal lesions. The SDT rats began to develop hyperglycemia at 20 weeks of age. In the histopathological examination of the kidney, glycogen deposition of the renal tubular epithelium and renal tubular dilation were observed from 24 weeks of age in the untreated SDT rats, and the changes in the renal tubules markedly progressed with aging. Moreover, thickening of the glomerular basement membrane was observed from 32 weeks of age. At 50 weeks of age, the glomeruli showed increase of mesangial matrix, with predominantly diffuse lesions showing by 68 weeks of age. The mesangial proliferation gradually progressed. In the SD rats, no renal lesions were present at 50 and 68 weeks of age. SDT rats with insulin treatment remained normoglycemic throughout observation and their renal functional parameters were normal. Glycemic control in SDT rats prevented the development of renal lesions. The features of SDT rats indicate their usefulness as an animal model for investigating diabetic nephropathy.     

Role of phytoplankton size distribution in lake ecosystems revealed by a comparison of whole plankton community structure between Lake Baikal and Lake Biwa

The influence of the size distribution of phytoplankton on changes in the planktonic food web structures with eutrophication was examined using natural planktonic communities in two world-famous lakes: Lake Baikal and Lake Biwa. The size distribution of phytoplankton and the ratio of heterotrophic to autotrophic biomass (H/A ratio), indicating the balance between primary production and its consumption, were investigated in the lakes of different trophic status. The results revealed that microphytoplankton (>20μm) in mesotrophic Lake Biwa, and picophytoplankton (<2μm) or nanophytoplankton (2–20μm) in oligotrophic Lake Baikal, comprised the highest proportion of the total phytoplankton biomass. The H/A ratio was lower in Lake Biwa (<1) than in Lake Baikal (>1). The low H/A ratio in Lake Biwa appeared to be the consequence of the lack of consumption of the more abundant microphytoplankton, which were inferior competitors in nutrient uptake under oligotrophic conditions but less vulnerable to grazing. As a result, unconsumed microphytoplankton accumulated in the water column, decreasing the H/A ratio in Lake Biwa. Our results showed that food web structure and energy flow in planktonic communities were greatly influenced by the size distribution of phytoplankton, in conjunction with bottom-up (nutrient uptake) and top-down (grazing) effects at the trophic level of primary producers.     

Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3delta from association with gp78 and p97/VCP, which is accompanied by decreases in CD3delta ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3delta and misfolded Z variant of alpha-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.     

Transmission of genomic instability from a single irradiated human chromosome to the progeny of unirradiated cells

Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced. In contrast, the transferred chromosome was unstable in four out of five microcell hybrids in which an irradiated human chromosome had been introduced. The aberrations included changes in the irradiated chromosome itself and rearrangements with recipient mouse chromosomes. Thus the present study demonstrates that genomic instability can be transmitted to the progeny of unirradiated cells by a chromosome exposed to ionizing radiation, implying that the instability is caused by the irradiated chromosome itself and also that the instability is induced by the nontargeted effect of radiation.     

Sex difference in norepinephrine surge in response to psychological stress through nitric oxide in rats

Psychological stress elevates blood pressure through sympathetic nerve activation. This pressor response is supposedly associated with cardiovascular events. We investigated a sex difference in the pressor response and norepinephrine surge to cage-switch stress in rats. Wistar male and female rats were catheterized for blood pressure monitoring and blood sampling. Six days post-surgery, the rats were exposed to the cage-switch stress and blood samples were collected at rest and 30 min after the start of the stress. The stress-induced pressor response was greater in the male than in the female rats. The stress significantly increased the norepinephrine level in the male, but not in the female rats. Pre-treatment with N(G)-nitro-l-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, attenuated the norepinephrine response significantly in the male rats. There was no sex difference in the endothelial NO synthase expression in the gastrocnemius muscle. However the phosphorylation at serine 1177, a marker for eNOS activation, was higher in the male than in the female rats. These results suggest that NO is involved in the norepinephrine surge to psychological stress in the male rats, but not in the female rats. This is the first report on a sex difference in the norepinephrine surge in response to psychological stress through NO, in association with pressor response.     

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.     

Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair

One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.     

New SIRT2 inhibitors: Histidine-based bleomycin spin-off

Bleomycin is considered to exert its antitumor activity via DNA cleavage mediated by activated oxygen generated from the iron complex in its chelator moiety. Spin-offs from this moiety, HPH-1Trt and HPH-2Trt, with anti-cancer activities were recently synthesized. In this paper, we developed inhibitors of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of Sirtuin protein (SIRT2), based on HPH-1Trt/HPH-2Trt, and aimed to generate new anti-cancer drugs. HPH-1Trt and HPH-2Trt had in vitro anti-SIRT2 inhibitory activity with 50% inhibitory concentration (IC₅₀) values of 5.5 and 8.8?μM, respectively. A structural portion of HPH-1Trt/HPH-2Trt, a tritylhistidine derivative TH-1, had stronger activity (IC₅₀?=?1.7?μM), and thus, fourteen derivatives of TH-1 were synthesized. Among them, TH-3 had the strongest activity (IC₅₀?=?1.3?μM). Selective binding of TH-3 in the pocket of SIRT2 protein was confirmed with a molecular docking study. Furthermore, TH-3 strongly lowered viability of the breast cancer cell line MCF7 with an IC₅₀ of 0.71?μM. A structure-activity relationship study using cell lines suggested that the mechanism of TH-3 to suppress MCF7 cells involves not only SIRT2 inhibition, but also another function. This compound may be a new candidate anti-cancer drug.     

Ultraconserved Elements Sequencing as a Low-Cost Source of Complete Mitochondrial Genomes and Microsatellite Markers in Non-Model Amniotes

Sequence capture of ultraconserved elements (UCEs) associated with massively parallel sequencing has become a common source of nuclear data for studies of animal systematics and phylogeography. However, mitochondrial and microsatellite variation are still commonly used in various kinds of molecular studies, and probably will complement genomic data in years to come. Here we show that besides providing abundant genomic data, UCE sequencing is an excellent source of both sequences for microsatellite loci design and complete mitochondrial genomes with high sequencing depth. Identification of dozens of microsatellite loci and assembly of complete mitogenomes is exemplified here using three species of Poospiza warbling finches from southern and southeastern Brazil. This strategy opens exciting opportunities to simultaneously analyze genome-wide nuclear datasets and traditionally used mtDNA and microsatellite markers in non-model amniotes at no additional cost.     

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma

BackgroundFew driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes.MethodsWe searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort.ResultsWe found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC.ConclusionOur integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.