Separation of purine derivatives by polymer-bound nucleic acid bases

Separation of a variety of purine bases, which include 7-methyl derivatives, was studied by using polyethyleneimine-coated silicagel which bound hypoxanthine, cytosine or guanine moieties. The separation behavior seems to be related to the interaction of imidazole part of purine derivatives with the resins through hydrogen bonding.     

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.     

Persistence of the Potassium Uptake Rhythm in the Presence of Exogenous Sucrose in Lemna gibba G3

The potassium uptake rhythm in a flow medium culture of Lemnagibba G3 persisted in darkness for 3 days, when the flow mediumcontained sucrose (1%). The rhythm was damped out after thatin darkness but it persisted longer when the plants were keptunder continuous weak light (80 lux). The rhythm was not dampedout when a daily light pulse (4,200 lux for 15 min) was applied.A single light pulse (4,200 lux for 15 min) at hour 48 of theprolonged dark period caused the rhythm to start again. DCMU(1 µM) slightly reduced the amplitude of the rhythm butdid not nullify the effect of the inserted light pulse. (Received September 16, 1981; Accepted February 2, 1982)     

Effect of butyrate on glycolipid metabolism of two cell types of rat ascites hepatomas with different ganglioside biosynthesis

The effect of butyrate on glycolipid metabolism and morphological differentiation in the cell culture system of rat ascites hepatomas, AH 7974 of island-forming type and AH 7974F of free type, was studied. Both cell lines adhered to the substratum in the presence of 1 mM butyrate. In the case of AH 7974, the addition of butyrate induced a distinct morphological change but the other cell line showed no such conspicuous change. Butyrate-treated AH 7974 cells showed a 2 to 3-fold elevation of CMP-N-acetylneuraminic acid: lactosylceramide sialyltransferase activity to form N-acetylneuraminylgalactosylglucosylceramide (GM3). On the other hand, no enzyme activity could be detected in AH 7974F cells. Four glycosyltransferase activities involved in glycolipid synthesis, including sialyltransferase in AH 7974F cells, were reduced by butyrate. From these observations we concluded that sialyltransferase to form GM3, or TM3 itself, is prerequisite for the morphological alteration induced by butyrate.     

Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation,ROS generation and mitochondrial dysfunction

RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis.     

Construction of a Model Culture System of Human Colonic Microbiota to Detect Decreased Lachnospiraceae Abundance and Butyrogenesis in the Feces of Ulcerative Colitis Patients

Compositional alteration of the gut microbiota is associated with ulcerative colitis (UC). Here, a model culture system is established for the in vitro human colonic microbiota of UC, which will be helpful for determining medical interventions. 16S ribosomal RNA sequencing confirms that UC models are successfully developed from fecal inoculum and retain the bacterial species biodiversity of UC feces. The UC models closely reproduce the microbial components and successfully preserve distinct clusters from the healthy subjects (HS), as observed in the feces. The relative abundance of bacteria belonging to the family Lachnospiraceae significantly decreases in the UC models compared to that in HS, as observed in the feces. The system detects significantly lower butyrogenesis in the UC models than that in HS, correlating with the decreased abundance of Lachnospiraceae. Interestingly, the relative abundance of Lachnospiraceae does not correlate with disease activity (defined as partial Mayo score), suggesting that Lachnospiraceae persists in UC patients at a decreased level, irrespective of the alteration in disease activity. Moreover, the system shows that administration of Clostridium butyricum MIYAIRI restores butyrogenesis in the UC model. Hence, the model detects deregulation in the intestinal environment in UC patients and may be useful for simulating the effect of probiotics.     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.     

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (C_H1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between V_H-C_H1 and V_L-C_L was improved by the C_H1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.