Identification in methicillin-susceptible Staphylococcus hominis of an active primordial mobile genetic element for the staphylococcal cassette chromosome mec of methicillin-resistant Staphylococcus aureus

We previously reported that the methicillin resistance gene mecA is carried by a novel type of mobile genetic element, SCCmec (staphylococcal cassette chromosome mec), in the chromosome of methicillin-resistant Staphylococcus aureus (MRSA). These elements are precisely excised from the chromosome and integrated into a specific site on the recipient chromosome by a pair of recombinase proteins encoded by the cassette chromosome recombinase genes ccrA and ccrB. In the present work, we detected homologues of the ccr genes in Staphylococcus hominis type strain GIFU12263 (equivalent to ATCC 27844), which is susceptible to methicillin. Sequence determination revealed that the ccr homologues in S. hominis were type 1 ccr genes (ccrA1 and ccrB1) that were localized on a genetic element structurally very similar to SCCmec except for the absence of the methicillin-resistance gene, mecA. This genetic element had mosaic-like patterns of homology with extant SCCmec elements, and we designated it SCC(12263) and considered it a type I staphylococcal cassette chromosome (SCC). The ccrB1 gene identified in the S. hominis strain is the first type 1 ccrB gene discovered to retain its function through the excision process as judged by two criteria: (i) SCC(12263) was spontaneously excised during cultivation of the strain and (ii) introduction of the S. hominis ccrB1 into an MRSA strain carrying a type I SCCmec whose ccrB1 gene is inactive generated SCCmec excisants at a high frequency. The existence of an SCC without a mec determinant is indicative of a staphylococcal site-specific mobile genetic element that serves as a vehicle of transfer for various genetic markers between staphylococcal species.     

Cloning and characterization of a novel RING-B-box-coiled-coil protein with apoptotic function

We have identified a novel RING-B-box-coiled-coil (RBCC) protein (MAIR for macrophage-derived apoptosis-inducing RBCC protein) that consists of an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil domain, and a B30.2 domain. MAIR mRNA was expressed widely in mouse tissues and was induced by macrophage colony-stimulating factor in murine peritoneal and bone marrow macrophages. MAIR protein initially showed a granular distribution predominantly in the cytoplasm. The addition of zinc to transfectants containing MAIR cDNA as part of a heavy metal-inducible vector caused apoptosis of the cells characterized by cell fragmentation; a reduction in mitochondrial membrane potential; activation of caspase-7, -8, and -9, but not caspase-3; and DNA degradation. We also found that the RING finger and coiled-coil domains were required for MAIR activity by analysis with deletion mutants.     

Thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) from Lenzites betulinus

A thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) was purified from the culture medium of Lenzites betulinus by ion exchange chromatography, gel filtration and isoelectric focusing chromatography. The MnP purified from L. betulinus (L-MnP) has a molecular mass of 40 kDa and its isoelectric point was determined to be 6.2. The first 19 amino acids at the N-terminal end of the L-MnP sequence were found to exhibit 74% identity with those of a Phlebia radiata MnP. L-MnP was proved to have the highest hydrogen peroxide tolerance among MnPs reported so far. It retained more than 60% of the initial activity after thermal treatment at 60°C for 60 min, and also retained more than 60% of the initial activity after exposure to 10 mM hydrogen peroxide for 5 min at 37°C.     

Thalictrum minus cell cultures and ABC-like transporter

Cultured Thalictrum minus cells produce a benzylisoquinoline alkaloid, berberine, in the presence of benzyladenine, and excrete it into the culture medium. T. minus cells excluded berberine, even if berberine was exogenously added to the medium, without benzyladenine treatment. Similarly, T. minus cells excluded a heterocyclic dye (neutral red) and calcein AM, which is used as a fluorescent probe to detect the drug efflux pump activity by ABC transporters. The addition of several inhibitors of P-glycoprotein, a representative ABC transporter, induced the accumulation in of both berberine and calcein AM ATP-dependent manner. The expression of P-glycoprotein-like ABC transporter genes was also demonstrated. The involvement of ABC transporter in the secretion of berberine in T. minus cells is discussed.     

Up-regulation of E-cadherin and β-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-α

Summary Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-α. E-cadherin expression was only up-regulated by butyrate and interferon-α (IFN-α) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, β-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-α. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that α-and γ-catenin expression was not changed, while only the expression of β-catenin increased. β-Catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type β-catenin are potentially modulated by butyrate and IFN-α, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.     

Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini

We investigated signal transduction between receptor-operated Ca(2+) influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca(2+) ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca(2+)](o), loading of the intracellular Ca(2+) chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca(2+) or Mn(2+) entry induced by CCK and carbachol, it did not affect intracellular Ca(2+) release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G(q) protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G(q)-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca(2+)](o)-dependent pancreatic exocytosis.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification,characterization, gene cloning,and applications

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.     

The role of cullin 5-containing ubiquitin ligases

The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation. Here, we review the current knowledge on the identification of Cul5 and the regulation of its expression, as well as the signaling pathways regulated by Cul5 and how viruses highjack the Cul5 system to overcome antiviral responses.