Improved methods for detection and serotyping of coagulase from Staphylococcus aureus

Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti-coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6-amino caproic acid, polyethylene glycol 6,000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type-specifically inhibited in the presence of type-specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.     

Applying hydraulic lift in an agroecosystem: forage plants with shoots removed supply water to neighboring vegetable crops

When a plant encounters spatially heterogeneous soil moisture within its root system, usually drier surface and moister subsurface soils, water can move between these layers through the root system, a plant process known as hydraulic lift or redistribution. The water thus transferred is available not only for the plant itself but also for its neighbors. We examined application of this process as a possible biological irrigation tool. As ??donors??, we used perennial forage plants with their shoots removed to minimize the effect of light-interception by them on the ??receiver?? plants growing alongside them. In a horizontally split-root experiment, where an upper container was filled with sand and a lower one with water, superior donor species could maintain the upper sand in a fully hydrated condition for several weeks, increasing stomatal conductance in the receivers. The effects were also confirmed in a water-limited agricultural field, as significant differences were found in canopy temperature and yield in neighboring crop plants in the presence or absence of donor root systems. These results suggest that deep-rooting associate plants with their shoots removed function as an irrigation tool and improve crop production in water-scarce environments.     

Metabolic Fate of O-Ethyl S,S-Diphenyl Phosphorodithiolate (Hinosan®) in Rice Plant

Metabolism and residual fate of O-ethyl S,S-diphenyl phosphorodithiolate (Hinosan®) applied on rice plant was examined by using ³⁵S-labeled or ³²P-labeled compound. Ion exchange chromatography, thin-layer chromatography and gas-liquid chromatography with flame thermionic detector or flame photometric detector were applied for identification of water soluble and toluene soluble metabolites of Hinosan. Degradation of Hinosan at the initial stage of metabolism was mainly the cleavage of P-S linkage, and a large portion of phenyl dihydrogen phosphorothiolate and a minor portion of O-ethyl S-phenyl hydrogen phosphorothiolate were found as water soluble metabolites. Phenylthio radical released on the production of the above mentioned metabolites was recovered as diphenyl disulfide, which was finally converted to sulfuric acid through benzenesulfonic acid. Triphenyl phosphorotrithiolate and O,O-diethyl S-phenyl phosphorothiolate were produced by transesterification between molecules of Hinosan at the initial stage of metabolism. Examination of metabolites in rice grains showed that sulfur and phosphorus atoms in Hinosan were incorporated into neutral or cationic substances probably after several steps of chemical transformation.     

Circadian PER2 protein oscillations do not persist in cycloheximide-treated mouse embryonic fibroblasts in culture

It is not known whether the endogenous mammalian core clock proteins sustain measurable oscillations in cells in culture where de novo translation is pharmacologically inhibited. We studied here the mammalian core clock protein PER2, which undergoes robust circadian oscillations in both abundance and phosphorylation. With a newly developed antibody that enables tracing the endogenous PER2 protein oscillations over circadian cycles with cultured mouse embryonic fibroblast cells, we provide evidence that PER2 does not persist noticeable circadian rhythms when translation is inhibited.     

Phylogenetic Analysis of Isolates of Pasteurella pneumotropica from Laboratory Animals Based on the gyrB Gene Sequence.

Phylogenetic analysis using the gyrB sequence was performed to investigate the genetic relevance among 49 isolates of P. pneumotropica. In the phylogeny, the isolates were clearly classified into three groups as follows: group A for the isolates of biotype Jawetz derived from mice, group B for the isolates of biotype Jawetz derived from rats, and group C for the isolates of biotype Heyl. These results suggest that the gyrB sequence of P. pneumotropica differs between the isolates of two biotypes, and also between the isolates derived from mice and rats in the biotype Jawetz.     

Variation and association of fibronectin‐binding protein genes fnbA and fnbB in Staphylococcus aureus Japanese isolates

Fibronectin‐binding proteins A and B (FnBPA and FnBPB) mediate adhesion of Staphylococcus aureus to fibrinogen, elastin and fibronectin. FnBPA and FnBPB are encoded by two closely linked genes, fnbA and fnbB, respectively. With the exception of the N‐terminal regions, the amino acid sequences of FnBPA and FnBPB are highly conserved. To investigate the genetics and evolution of fnbA and fnbB, the most variable regions, which code for the 67th amino acids of the A through B regions (A67–B) of fnbA and fnbB, were focused upon. Eighty isolates of S. aureus in Japan were sequenced and 19 and 18 types in fnbA and fnbB, respectively, identified. Although the phylogeny of fnbA and fnbB were found to be quite different, each fnbA type connected with a specific fnbB type, indicating that fnbA and fnbB mutate independently, whereas the combination of both genes after recombination is stable. Hence those fnbA–fnbB combinations were defined as FnBP sequence types (FnSTs). Representative isolates of each FnST were assigned distinct STs by multilocus sequence typing, suggesting correspondence of FnST with genome lineage. Linkage disequilibrium (LD) analysis of the A67–B region revealed that subdomains N2, N3 and FnBR1 form a LD block in fnbA, whereas N2 and N3 form two independent LD blocks in fnbB. N2–N3 three‐dimensional structural models indicated that not only the variable amino acid residues, but also well‐conserved amino acid residues between FnBPA and FnBPB, are located on the surface of the protein. These results highlight a molecular process of the FnBP that has evolved by mingled mutation and recombination with retention of functions.     

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.