Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset,a model primate

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.     

Visfatin causes endothelium-dependent relaxation in isolated blood vessels

Visfatin is a novel adipocyte-derived cytokine. We hypothesized that visfatin could directly affect vascular reactivity. To test the hypothesis, effects of visfatin on contraction of isolated blood vessels were examined. In endothelium-intact rat aorta, pretreatment with visfatin (100 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 μM)-induced contraction. In NA (100 nM)-pre-contracted aorta, visfatin (1-100 ng/ml) directly induced a relaxation. Although an N^G-Nitro-l-arginine methyl ester (300 μM, 15 min) inhibited the relaxation, an insulin receptor inhibitor, AGL2263 (10 μM, 20 min) was ineffective. Visfatin (100 ng/ml, 20 min) induced a phosphorylation of eNOS at serine 1177 and a de-phosphorylation of eNOS at threonine 495. Visfatin also induced a phosphorylation of Akt at serine 473 and a substrate of cGMP-dependent protein kinase, vasodilator stimulated phosphoprotein at serine 239. Present study revealed for the first time that visfatin has a vasodilating effect on isolated blood vessels, which is mediated via endothelium-derived NO.     

Development of mesenchymal stem cells partially originate from the neural crest

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells isolated from many adult tissues. Previous studies reported that MSCs can differentiate to both mesodermal and neural lineages by a phenomenon referred to as ‘‘dedifferentiation’’ or ‘‘transdifferentiation’’. However, since MSCs have only been defined in vitro, much of their development in vivo is still unknown. Here, we prospectively identified MSCs in the bone marrow from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. EGFP-positive MSCs formed spheres that expressed neural crest stem cell genes and differentiated into neurons, glial cells, and myofibroblasts. Interestingly, we observed MSCs both in the GFP⁺ and GFP⁻ fraction and found that there were no significant differences in the in vitro characteristics between these two populations. Our results suggest that MSCs in adult bone marrow have at least two developmental origins, one of which is the neural crest.     

Molecular Analysis of an Extrachromosomal Element Containing the C2 Toxin Gene Discovered in Clostridium botulinum Type C

Clostridium botulinum cultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including the c2I and c2II genes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were σ factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.Clostridium botulinum is a gram-positive, spore-forming, anaerobic bacterium. Cultures of this species produce poisonous botulinum neurotoxins (BoNTXs) that are lethal to humans and animals and are classified into seven types, types A to G, based on the antigenicity of the BoNTXs produced (actually, the cultures producing type G toxin were recently classified in a new species, Clostridium argentinens [40]).C. botulinum type C and D cultures produce a binary C2 toxin in addition to C (or C1) and D BoNTXs; this additional toxin consists of two nonlinked proteins, C2I and C2II (28), that occur independently in the culture supernatant and are not chemically joined to each other. The C. botulinum C2 toxin used here is a representative of the family of binary actin-ADP-ribosylating toxins, which includes, in addition to C2 toxin, the Clostridium perfringens iota toxin, Clostridium difficile toxin, Clostridium spiroforme toxin, and the vegetative insecticidal proteins from Bacillus cereus (4).The enzyme component of C2 toxin (C2I) ADP ribosylates G-actin at arginine 177 (1). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding-translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (5). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. Subsequently, C2I translocates across the membrane into the cytosol through these C2IIa pores.The production of C1 and D BoNTXs is governed by bacteriophages (12, 13, 18, 19, 26), and both toxin genes have been cloned from the corresponding phage DNAs (16, 21). Recently, we determined the whole-genome sequence of a type C toxin-converting phage (c-st) genome (31). Eklund et al. reported that C2 toxin toxigenicity (mouse lethality) became clear when strains were cultured in fortified egg-meat medium and the culture supernatants were treated with trypsin (12). They also reported that C2 toxin production was not related to the BoNTX-converting phages; some non-BoNTX-producing cells produced C2 toxin. Fujii et al. (15) and Kimura et al. (22) determined the whole nucleotide sequences of the c2I and c2II genes and speculated that these genes might be located on the bacterial chromosome (15, 22).In this study, we determined that these genes are present not on the cell chromosome but on a large plasmid; we first speculated that this was this was the case based on the results of both pulsed-field gel electrophoresis (PFGE) and Southern hybridization analysis and then confirmed it by determining the complete nucleotide sequence of the plasmid. Since the plasmid was extremely unstable, we could not purify the complete plasmid DNA; therefore, we determined the whole-genome sequence by using the inverse PCR method, which enables rapid determination of the flanking regions of unknown sequences and determination of unidentified sequences in the genome, and the primer-walking method. This is the first case in which an entire DNA sequence of a large plasmid was determined using only these two procedures. The process used to determine the whole-plasmid DNA sequence and several interesting features of the plasmid are described below.     

Nucleotide sequence analysis of the enterotoxigenic Escherichia coli Ent plasmid

We report here the complete nucleotide sequence of pEntH10407 (65 147 bp), an enterotoxigenic Escherichia coli enterotoxin plasmid (Ent plasmid), which is self-transmissible at low frequency. Within the plasmid, we identified 100 open reading frames (ORFs) which could encode polypeptides. These ORFs included regions encoding heat-labile (LT) and heat-stable (STIa) enterotoxins, regions encoding tools for plasmid replication and an incomplete tra (conjugation) region. The LT and STIa region was located 13.5 kb apart and was surrounded by three IS1s and an IS600 in opposite reading orientations, indicating that the enterotoxin genes may have been horizontally transferred into the plasmid. We identified a single RepFIIA replication region (2.0 kb) including RepA proteins similar to RepA1, RepA2, RepA3 and RepA4. The incomplete tra region was made up of 17 tra genes, which were nearly identical to the corresponding genes of R100, and showed evidence of multiple insertions of ISEc8 and ISEc8-like elements. These data suggest that pEntH10407 has the mosaic nature characteristic of bacterial virulence plasmids, which contains information about its evolution. Although the tra genes might originally have rendered pEntH10407 self-transferable to the same degree as R100, multiple insertion events have occurred in the tra region of pEntH10407 to make it less mobile. Another self-transmissible plasmid might help pEntH10407 to transfer efficiently into {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 strain. In this paper, we suggest another possibility: that the enterotoxigenic {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 strain might be formed by auto-transfer of pEntH10407 at a low rate using the incomplete tra region.     

A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.

A high-alkaline, salt-activated alginate lyase is produced by Agarivorans sp. JAM-A1m from a deep-sea sediment off Cape Nomamisaki on Kyushu Island, Japan. Purified to homogeneity, as judged by SDS-PAGE, the enzyme (A1m) had a molecular mass of approximately 31 kDa. The optimal pH was around 10 in glycine–NaOH buffer, and the activity was increased to 1.8 times by adding 0.2 M NaCl. However, when the optimal pH in the presence of 0.2 M NaCl was shifted to pH 9.0, the activity was more than 10 times compared with that at pH 9 in the absence of NaCl. A1m showed the optimal temperature at around 30°C and was stable to incubation between pH 6 and 9. The enzyme degraded favorably mannuronate–guluronate and guluronate-rich fragments in alginate. Shotgun cloning and sequencing of the gene for A1m revealed a 930-bp open reading frame, which encoded a mature enzyme of 289 amino acids (32,295 Da) belonging to polysaccharide lyase family 7. The deduced amino acid sequence showed the highest similarity to that of a Klebsiella enzyme, with only 54% identity.     

The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation

Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G₁ maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1^GT/GT) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1^GT/GT MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1^GT/GT embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1^GT/GT embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit complex that functions as an E3 ubiquitin ligase for various cell cycle proteins (19, 46). Proteins ubiquitinated by APC/C are recognized and degraded by the 26S proteasome to ensure proper cell cycle progression. APC/C activity is strictly dependent on coactivator proteins that interact with APC/C during specific phases of the cell cycle. Cdh1 (also known as Fzr, Hct1, or Srw) is one of the coactivators that maintain APC/C activity from anaphase of mitosis until the end of the G₁ phase of the cell cycle (43, 53).The role of Cdh1 (APC/C^Cdh1) on cell-cycle progression has been well studied; however, several studies have shed light into another aspect of Cdh1''s function. For example, expression of Cdh1 is not restricted to cycling cells; APC/C^Cdh1 is also present and active in quiescent cultured cells (9). Furthermore, immunohistochemical analysis has shown that Cdh1 is expressed in a wide variety of tissues that are predominantly composed of postmitotic cells, such as neurons, where APC/C^Cdh1 has a high cyclin B ubiquitination activity (1, 16). It has been reported that APC/C^Cdh1 promotes axonal growth and patterning (20) and is required for neuronal survival (1). These results highlight the importance of the APC/C activator Cdh1 in neurons. However, Cdh1 has also been shown to participate in the differentiation of tissues such as the muscle (25). Given that Cdh1 is ubiquitously expressed in organs containing quiescent cells, there might be additional roles for Cdh1.Rho GTPase proteins play a central role in the regulation of cell shape, polarity, and locomotion via their effects on actin polymerization, actomyosin contractility, cell adhesion, and microtubule dynamics (13). Small G proteins, which include Rho, act as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state. The latter form of Rho proteins interacts with and activates downstream effector proteins. The activity of Rho GTPases is controlled by three class of key regulators: (i) guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GDP to GTP for their activation (41); (ii) GTPase activating proteins (GAPs), which stimulate the intrinsic GTPase activity for their inactivation (8); and (iii) guanine nucleotide dissociation inhibitors (GDIs), which interact with GDP-bound Rho GTPases and sequester them in the cytoplasm to inhibit the exchange of GDP to GTP (33). In addition to these canonical regulations, recent studies indicate that the ubiquitination pathway is also involved in the modulation of Rho GTPase activity. Smurf1, which is a HECT domain E3 ubiquitin ligase, controls the local levels of RhoA at the cell periphery by targeting it for degradation (40, 55). Therefore, the regulatory mechanisms of Rho GTPase activity seem to be more complex than previously thought. It thus remains to be clarified whether other ubiquitin ligases also play a role in Rho signaling by targeting its components directly or indirectly.In this study, we found that the APC/C activator Cdh1 modulated actin organization. Mouse embryonic fibroblasts (MEFs) derived from a homozygous Cdh1 gene-trapped ([GT] Cdh1^GT/GT) mouse model displayed decreased numbers of stress fibers and focal adhesions (FAs). Consistent with these phenotypes, Rho activity was apparently reduced in Cdh1-deficient cells. Cdh1 regulated Rho activity via the targeting of p190 for degradation. We also found that Cdh1 knockdown cells showed decreased motility, which was rescued by codepletion of p190. Furthermore, phenotypic similarities between Cdh1^GT/GT embryos and ROCK (also known as Rho-kinase, which is the important Rho downstream effector of actin cytoskeleton formation) knockout (KO) mice (44, 49) support our notion that Cdh1 plays a role in the Rho/ROCK signaling axis. Collectively, our findings suggest an alternative role for Cdh1 other than cell cycle regulation and reveal Cdh1 as a new regulator of Rho.     

Loss of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 and reduced O-glycosylation in colon carcinoma cells selected for hepatic metastasis

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITC-PTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.