Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.     

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection

Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through β-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.     

Mutants of Ficus pumila produced by ion beam irradiation with an improved ability to uptake and assimilate atmospheric nitrogen dioxide

Production of novel mutants with a high ability to mitigate pollutants is important for phytoremediation. We investigated the use of ion beam irradiation to produce mutants of Ficus pumila L. with an improved ability to mitigate atmospheric nitrogen dioxide (NO2). More than 25,000 shoot explants were irradiated with an ion beam (12C5+, 12C6+, or 4He2+), from which 263 independent plant lines were obtained. The plants were analyzed for NO2 uptake by fumigation with 1 ppm 15N-labeled NO2 for 8 h in light, followed by mass spectrometric analysis. The mean NO2 uptake values of each of the 263 lines differed over a 110-fold range. Propagation was attempted using cuttings from 44 lines showing the greatest NO2 uptake; in total, 15 lines were propagated. Two of the 15 lines showed a mean NO2 uptake 1.7- to 1.8-fold greater than that of the wild-type. This increase in NO2 uptake was heritable in both lines; their progenies showed a significantly greater ability to take up and assimilate NO2 than did the wild-type. RAPD analysis demonstrated DNA variation between the progeny plants and the wild type, suggesting that the progeny were true mutants. These mutants of F. pumila may prove useful in mitigating atmospheric NO2.     

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

Crustacean insulin‐like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N‐linked glycan, was synthesized by the combination of solid‐phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N‐linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin‐like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four‐disulfide insulin‐like peptides.     

Pleckstrin-2 selectively interacts with phosphatidylinositol 3-kinase lipid products and regulates actin organization and cell spreading

Pleckstrin-2 (PLEK2) has been implicated to be regulated by phosphatidylinositol (PI) 3-kinase, while pleckstrin1 (PLEK1) has been suggested to be a major PKC substrate in platelets. In this paper, we confirmed that PLEK2 specifically bound to the PI 3-kinase products in vitro and explored its behavior. PLEK2 was found to be expressed in various adherent cell lines, while PLEK1 expression was restricted to non-adherent cells in the protein level. Expression of PLEK2 in COS1 cells induced formation of protrusive F-actin structure and enhanced the actin rearrangements induced on collagen- or fibronectin-coated plates. A PLEK2 mutant incapable of binding to the PI 3-kinase products did not show any effect on actin rearrangement. Knockdown of PLEK2 by shRNA inhibited spreading of HCC2998 adenocarcinoma cells. PLEK2 colocalized with Rac and was suggested to be oligomerized. These results suggest that PLEK2 is involved in actin rearrangement in a PI 3-kinase dependent manner.     

Immunocytochemical studies on the distribution pattern of daunomycin in rat gastrointestinal tract

The cancer drug daunomycin is used in treatment of leukemia but possesses severe side effects that involve the gastrointestinal tract. We therefore used a newly developed immunocytochemical procedure to determine the distribution of DM in the gastrointestinal tracts of rats after i.v. injection. Two hours after injection, DM was diffusely distributed in nuclei and most parts of the cytoplasm of intestinal epithelial cells. The cytoplasmic immunoreactivity for DM was most pronounced in small granules of the apical cytoplasm. Sixteen hours after injection, DM immunostaining was by and large absent in the villous epithelium but persisted in the intestinal crypts. In addition, staining was also detected in endothelial cells, scattered cells of the lamina propria and in smooth muscle cells. After 5 days, only little staining for DM remained. Similar findings were made in the colon. In the gastric mucosa, DM accumulation persisted at 16 h in some glandular cells but was lost from the surface epithelium. No staining was detected in saline-injected control rats. The distribution of DM accumulation correlated partially with the distribution of apoptotic cells as detected by the TUNEL procedure. Our results pinpoint that DM may exert prolonged effects on glandular and regenerative cells of the gastrointestinal tract—an observation that may explain the gastrointestinal toxicity of the drug. It seems possible that DM accumulation in surface epithelial cells is rapidly cleared through drug transporters.     

Involvement of splanchnic vascular bed in anaphylactic hypotension in anesthetized BALB/c mice

Using in vivo and isolated perfused liver preparations of BALB/c mice, we determined the roles of the liver and splanchnic vascular bed in anaphylactic hypotension. Intravenous injection of ovalbumin antigen into intact-sensitized mice decreased systemic arterial pressure (P(sa)) from 92 +/- 2 to 39 +/- 3 (SE) mmHg but only slightly increased portal venous pressure (P(pv)) from 6.4 +/- 0.1 cmH(2)O to the peak of 9.9 +/- 0.5 cmH(2)O at 3.5 min after antigen. Elimination of the splanchnic vascular beds by ligation of the celiac and mesenteric arteries, combined with total hepatectomy, attenuated anaphylactic hypotension. Ligation of these arteries alone, but not partial hepatectomy (70%), similarly attenuated anaphylactic hypotension. In contrast, isolated sensitized mouse liver perfused portally at constant flow did not show anaphylactic venoconstriction but, rather, substantial constriction in response to the anaphylaxis-associated platelet-activating factor, indicating that venoconstriction in mice in vivo may be induced by mediators released from extrahepatic tissues. These results suggest that splanchnic vascular beds are involved in BALB/c mouse anaphylactic hypotension. They presumably act as sources of chemical mediators to cause the anaphylaxis-induced portal hypertension, which induced splanchnic congestion, resulting in a decrease in circulating blood volume and, thus, systemic arterial hypotension. Mouse hepatic anaphylactic venoconstriction may be induced by factors outside the liver, but not by anaphylactic reaction within the liver.     

Anti-HCV activity of the Chinese medicinal fungus Cordyceps militaris

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although the sustained virologic response rate in the treatment of genotype 1 using new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has been improved by more than 70%, several severe side effects such as skin rash/ageusia and advanced anemia have become a problem. Under these circumstances, a new type of anti-HCV oral drug with few side effects is needed. Our recently developed HCV drug assay systems, including the HuH-7 cell line-derived OR6 and AH1R, and the Li23 cell line-derived ORL8 and ORL11, allow genome-length HCV RNAs (several strains of genotype 1b) encoding renilla luciferase to replicate efficiently. Using these systems as anti-HCV candidates, we have identified numerous existing medicines that can be used against HCV with few side effects, such as statins and teprenon. To obtain additional anti-HCV candidates, we evaluated a number of oral health supplements, and found that the capsule but not the liquid form of Cordyceps militaris (CM) (Ascomycotinanorth, North Chinese caterpillar fungus), which is used as a Chinese herbal medicine, exhibited moderate anti-HCV activity. In combination with interferon-α or ribavirin, CM exhibited an additive inhibitory effect. Among the main components of CM, cordycepin, but not ergosterol, contributed to the anti-HCV activity of CM. In consideration of all these results, we suggest that CM would be useful as an oral anti-HCV agent in combination with interferon-α and/or ribavirin.     

Expression of hyaluronan synthase 1 and distribution of hyaluronan during follicular atresia in pig ovaries

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.     

Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.Hepatitis C virus (HCV) infection represents a significant global healthcare burden, and current estimates suggest that a minimum of 3% of the world''s population is chronically infected (4, 19). The virus is responsible for many cases of severe chronic liver diseases, including cirrhosis and hepatocellular carcinoma (4, 16, 19). HCV is a positive-stranded RNA virus belonging to the family Flaviviridae. Its ∼9.6-kb genome is translated into a single polypeptide of about 3,000 amino acids (aa), in which the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B reside in the C-terminal half region (6, 34, 44). NS4A, a small 7-kDa protein, functions as a cofactor for NS3 to enhance NS3 enzyme activities such as serine protease and helicase activities. The hydrophobic N-terminal region of NS4A, which is predicted to form a transmembrane α-helix, is responsible for membrane anchorage of the NS3-4A complex (8, 44, 50), and the central region of NS4A is important for the interaction with NS3 (10, 44). A recent study demonstrated the involvement of the C terminus of NS4A in the regulation of NS5A hyperphosphorylation and viral replication (28).The development of HCV replicon technology several years ago accelerated research on viral RNA replication (7, 44). Furthermore, a robust cell culture system for propagation of infectious HCV particles was developed using a viral genome of HCV genotype 2a, JFH-1 strain, enabling us to study every process in the viral life cycle (27, 47, 54). RNA derived from genotype 1a, HCV H77, containing cell-culture adaptive mutations, also produces infectious viruses (52). Using these systems, it has been reported that the HCV genome replicates in a distinct, subcellular replication complex (RC) compartment, which includes NS3-5B and the viral RNA (2, 14, 33). The RC forms in a distinct compartment with high concentrations of viral and cellular components located on detergent-resistant membrane (DRM) structures, possibly a lipid-raft structure (2, 41), which may protect the RC from external proteases and nucleases. Almost all processes in viral replication are dependent on the host cell''s machinery and involve intimate interaction between viral and host proteins. However, the functional roles of host factors interacting with the HCV RC in viral genome replication remain ambiguous.To gain a better understanding of cellular factors that are components of the HCV RC and that function as regulators of viral replication, a comparative proteomic analysis of DRM fractions from HCV replicon and parental cells and subsequent RNA interference (RNAi) silencing of selected genes were performed. We identified creatine kinase B (CKB) as a key factor for the HCV genome replication. CKB catalyzes the reversible transfer of the phosphate group of phosphocreatine (pCr) to ADP to yield ATP and creatine and is known to play important roles in local delivery and cellular compartmentalization of ATP (48, 51). The findings obtained here suggest that recruitment of CKB to the HCV RC, through CKB interaction with NS4A, is essential for maintenance or enhancement of viral replicase activity.