26S multicatalytic proteinase complexes decrease during the differentiation of murine erythroleukemia cells.

Changes in multicatalytic proteinase activity during differentiation were investigated using Me2SO-induced differentiation of murine erythroleukemia cells as a model. The apparent ATP-dependent multicatalytic proteinase activity decreased in the Me2SO-treated cells with ATP-dependent incorporation of [3H]diisopropyl fluorophosphate decreasing notably after Me2SO-treatment. This decrease in activity does not seem to arise from a cessation of cell-proliferation, because no significant changes in proteinase activity were observed under different culture conditions. Hydroxyapatite column chromatography was employed to analyze the form of multicatalytic proteinase. It was clearly demonstrated that the 26S form of the proteinase decrease in the differentiated cells relative to normal cells. Multicatalytic proteinase-associated proteins that bind to the proteinase in an ATP-dependent manner were purified on an anti-multicatalytic proteinase IgG conjugated column. Only a small amount of protein was recovered from the differentiated cells. These results suggest that the decrease in multicatalytic proteinase-associated proteins that occurs upon cell-differentiation abolishes the ATP-dependent activity of the proteinase.     

Antimicrobial Susceptibility of Clostridium difficile from Different Sources

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic-associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline-resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.     

Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca²⁺ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.     

The influence of oligodeoxyribonucleotide phosphorothioate pyrimidine strands on triplex stability.

The analogues of the homopyrimidine oligonucleotide dT15 has been synthesized. The analogues, contains phosphorothioate bonds of a mixture of diastereoisomers or one of the two stereoisomer (either Rp or Sp). The analogues were mixed under conditions conductive to the formation of triple stranded assemblies. The mixtures were characterized by their thermal stabilities (Tm values) and CD spectra.     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.     

Dystrophin in rod spherules; submembranous dense regions facing bipolar cell processes

 It is known that the retina contains the protein dystrophin in the ribbon synapse, but the ultrastructural analysis is not yet fully elucidated. Our previous study reported that dystrophin is localized under the rod cell membranes in rat retinas. In the present study, we have investigated the relationship between dystrophin-rich regions of rod cell membranes and other neuronal processes in mouse retinas with a monoclonal antibody raised against the human dystrophin C-terminus. Immunoblotting, immunofluorescence stainings, and immunoelectron microscopy were employed. Immunoblotting analysis indicated that mouse retinas possessed some of the dystrophin isoforms of approximately 260 kDa, 140 kDa, and 70 kDa molecular weight. Confocal images showed a punctate appearance in the outer plexiform layer, as previously described. Immunoelectron microscopy showed that dystrophin immunoreactive products were always observed at submembranous dense regions of the rod spherule abutting bipolar processes. These results suggest that retinal dystrophin may be closely involved in signal transmission from rods to bipolar cells. Accepted: 7 May 1997     

Formation of Cyclohexylamine and Cyclohexanone from Cyclamate by Microorganisms Isolated from the Feces of Guinea Pig

The assimilation of sodium cyclamate (CHS-Na) by microorganisms was studied. Fifteen strains of cyclamate-assimilating bacteria were isolated from the feces of guinea pig excreting cyclohexylamine (CHA) in urine. The majority of the strain isolated seems to belong to the genera Pseudomonas and Corynebacterium. All strains were able to assimilate CHS-Na as a sole source of carbon and nitrogen, and accumulated clearly CHA in the culture medium. It was confirmed with the cell-free extract that the strains possessed the enzyme system which formed cyclohexanone (CHnone) from CHS-Na via CHA. It seems that the desulfation of CHS-Na to CHA is catalyzed by hydrolase, and that the deamination of CHA to CHnone is catalyzed by amine oxidase depending on oxygen.     

The Nucleotide Sequence and Genome Structure of Mung Bean Yellow Mosaic Geminivirus

Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200-base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33-36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.