Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.     

Human type II GnRH receptor mediates effects of GnRH on cell proliferation

GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Progress in studies on GnRH demonstrated that GnRH has both inhibitory and stimulatory effects on cell proliferation depending on the cell type, and the mechanisms of these effects have been intensively studied. However, even human GnRH receptors which mediate GnRH stimulation have not been completely identified. In the present study, we showed that the inhibitory and stimulatory effects of GnRH on colony-formation using four cell lines and have demonstrated that the inhibitory and stimulatory effects of GnRH exhibit distinctly different patterns of ligand sensitivity. This result strongly suggests that the two opposite effects of GnRH occur via different types of GnRH receptors, however expressional analyses of human GnRH receptors did not exhibit the significantly different pattern between negatively and positively responding cell lines. Then, in order to identify the GnRH receptors involved in the two opposite effects, effects of GnRH were analysed under the conditions that human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor mediates GnRH stimulation and its splice variant determines the direction of the response to GnRH. These results are the first clear evidence for the functionality of human type II GnRH receptor. Therefore our novel findings are quite noticeable and will greatly contribute to the studies on the mechanisms of the effects of GnRH on cell proliferation in the future.     

Expression of DNase gamma during Fas-independent apoptotic DNA fragmentation in rodent hepatocytes

Endonuclease-induced DNA fragmentation is a hallmark of apoptosis. DNase gamma (DNase ) was recently identified as one of the endonucleases responsible for apoptotic DNA fragmentation. In this study, immunohistochemistry for DNase was performed on paraffin sections of rodent liver in well-defined models of hepatocyte apoptosis induced by Fas antibody (Fas) or cycloheximide (CHX), and necrosis induced by lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄). DNase immunoreactivity was compared with TdT-mediated dUTP nick-end labeling (TUNEL) reactivity. Our results showed TUNEL reactivity in both apoptotic and necrotic hepatocytes. DNase immunoreactivity was not detected during LPS-induced or CCl₄-induced hepatocyte necrosis. In contrast, it was evident during CHX-induced, but not Fas-induced, apoptotic DNA fragmentation. These findings suggest that DNase plays an important role in Fas-independent apoptotic DNA fragmentation in hepatocytes.     

Cloning of a<Emphasis Type="Italic"> decapentaplegic</Emphasis> orthologue from the sawfly,<Emphasis Type="Italic"> Athalia rosae</Emphasis> (Hymenoptera), and its expression in the embryonic appendages

The cDNA of a decapentaplegic (dpp) orthologue from the sawfly, Athalia rosae (Hymenoptera), was cloned and characterized. The clone (Ar dpp) was 2,566 bp long and encoded 395 amino acids in a single open reading frame. Genomic Southern blotting showed that Ar dpp is a single copy gene. The deduced amino acid sequence can be aligned along its entire length with known insect DPPs. It shared common characteristics such as a signal sequence, a pro-domain region, and a ligand domain with seven cysteines at conserved locations. Ar dpp was expressed as a single 5.0-kb mRNA in embryos, larvae, pupae and adults. In situ hybridization showed that Ar dpp was expressed in the dorsal region proper in early embryonic stages and in the embryonic appendages of cephalic segments (labrum, antenna, mandible, maxilla, and labium), thoracic segments (thoracic legs), and all abdominal segments except the tenth segment (pleuropodia and proleg primordia). The present results indicate that Ar dpp expression reflects the primary determination of embryonic appendages.Edited by D. TautzThe sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with the accession number AB121072     

Role of follicular dendritic cells in the early HIV-1 infection: in vitro model without specific antibody

About 90% of HIV-1 RNA in the lymph nodes is reported to localize in follicular dendritic cellsnetwork (FDC-NW) as early as several days after infection and as much as that in the late stage. But the mechanism remains to be fully understood. To elucidate the role of follicular dendritic cells (FDC) in the early stage of HIV-1 infection, FDC-like cell strains (FDCLC) were established and they were characterized in the co-culture system with T cells for their effect on HIV-1 trapping and replication in p24 immunoassay, immunohistochemistry as well as confocal and electronmicroscopy. Established FDCLC were positive for CNA-42, S-100alpha and intercellular desmosome-like junctions. L-SIGN and DC-SIGN were also detected in FDCLC. Alu-HIV-1 PCR analysis showed no HIV-1 integration in FDCLC. FDCLC trapped HIV-1 and transferred them to uninfected MOLT-4 T cells (MOLT-4) efficiently in the absence of specific antibody. FDCLC also accelerated HIV-1 replication in HIV-1-pre-exposed MOLT-4. These unique FDCLC effects were explained, at least partly, by the fact that FDCLC up-regulated CD4 expression in MOLT-4 and helped T cells escape from apoptosis in the co-culture. These data suggest that FDC/FDCLC engage not only in trapping but also in active expansion of HIV-1 in the absence of specific antibody.     

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.     

Simple RNAi vectors for stable and transient suppression of gene function in rice

Since the recent sequencing of the rice genome, the functional identification of rice genes has become increasingly important. Various tagged lines have been generated; however, the number of tagged genes available is not sufficient for extensive study of gene function. To help identify the functions of genes in rice, we developed a Gateway vector, pANDA, for RNA interference of rice genes. This vector can be used for Agrobacterium transformation of rice and allows easy and fast construction of efficient RNAi vectors. In the construct, hairpin RNA derived from a given gene is transcribed from a strong maize ubiquitin promoter, and an intron is placed 5' upstream of inverted repeats to enhance RNA expression. Analysis of rice genes using this vector showed that suppression of mRNA expression was observed in more than 90% of transgenic plants examined, and short interfering RNA indicative of RNA silencing was detected in each silenced plant. A similar vector, pANDA-mini, was also developed for direct transfer into leaf cells or protoplasts. This vector can be used for transient suppression of gene function in rice. These vectors should help identify the functions of rice genes whose tagged mutants are not available at present and complement existing methods for functional genomics of rice.     

Allelotypic characteristics of thorotrast-induced intrahepatic cholangiocarcinoma: comparison to liver cancers not associated with thorotrast

To elucidate the genetic alterations that are specific to Thorotrast-induced liver cancers and their possible roles in tumorigenesis, we analyzed loss of heterozygosity (LOH) at 37 loci. Our previous study of liver cancers that were not associated with Thorotrast found LOH at 9 of these loci to be characteristic of intrahepatic cholangiocarcinoma (ICC), at 19 to be characteristic of hepatocellular carcinoma (HCC), and at 9 to be common to both ICC and HCC. LOH analysis was also performed in tissues of cholangiolocellular carcinoma, which is thought to originate from a common stem cell progenitor of hepatocytes and bile duct epithelial cells. We found frequent LOH at D4S1538, D16S2624 and D17S1303 to be common to all the subtypes of liver cancers, independent of the specific carcinogenic agent. In contrast, LOH at D4S1652 generally was not observed in Thorotrast-induced ICC. LOH analysis revealed that Thorotrast-induced ICC shares some LOH features with both ICC and HCC that were not induced by Thorotrast; however, it is more similar to ICC than to HCC in terms of genetic changes. This study could narrow down the crucial chromosomal loci whose deletions are relevant to hepatobiliary carcinogenesis irrespective of the carcinogenic agent. The study of LOH at loci other the those crucial ones may help us understand how the phenotype of liver cancers is determined.     

Role of arginine in protein refolding, solubilization, and purification

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.