Requirement of protein co-factor for the DNA-binding function of the human ski proto-oncogene product.

We identified the human c-ski gene product (c-Ski) as a protein with the apparent molecular weight of 100,000, p100c-ski, by using a c-Ski-specific polyclonal antibody. p100c-ski was a nuclear protein and p100c-ski in nuclear extracts of Molt4 cells bound to calf thymus DNA cellulose, but the bacterially synthesized c-Ski did not, suggesting that Ski was associated with another protein(s) and that the Ski complex had DNA-binding activity. This hypothesis was supported by the finding that the bacterially synthesized Ski bounds to DNA cellulose after being mixed with a nuclear extract of Molt4 cells. By use of a series of deletion mutants of Ski synthesized in an in vitro translation system, two portions in Ski were found to be necessary for the DNA binding of the Ski complex: the N-proximal portion containing a cystein/histidine-rich domain and the C-terminal portion including a region rich in basic amino acids.     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.     

Reach-scale distribution dynamics of a grazing stream insect, Micrasema quadriloba Martynov (Brachycentridae, Trichoptera), in relation to current velocity and periphyton abundance

Reach-scale temporal shifts in the distribution of larvae of a grazing caddisfly, Micrasema quadriloba (Brachycentridae), were investigated in a Japanese mountain stream. The larvae showed an aggregated distribution within the reach at the beginning of the immigration, then became randomly dispersed throughout the reach as the immigration progressed. The abundance of periphyton in the reach decreased dramatically with increasing dispersal of the larvae. Simple regression analyses revealed that the stream's flow regime was the most important environmental factor that determined the reach-scale distribution of the larvae and that the relationship between the flow regime and the distribution of the larvae shifted temporally. In addition, our results suggest that only this species of grazing insect, which was dominant in the study reach, controlled the reach-scale abundance of the periphyton.     

Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.     

Estimation of mouse fetal weight by ultrasonography: application from clinic to laboratory

Ultrasonographic assessment of fetal growth to estimate fetal weight has been widely used in clinical obstetrics but not in laboratory mice. Even though it is important to assess fetal growth abnormalities for gene-targeting studies using mice, there have been no reports of accurately estimated fetal weight using fetal biometric parameters in mice. The aim of this study was to establish an accurate mouse formula using fetal biometric parameters under ultrasound imaging. Using a high-frequency ultrasound system with a 40 MHz transducer, we measured 293 fetuses of biparietal diameter and mean abdominal diameter from day 12.5 postcoitus (p.c.) until day 18.5 p.c every day. Thirteen algorithms for humans based on head and/or abdominal measurements were assessed. We established an accurate formula based on measurement of the abdomen in Jcl:ICR mice to investigate gestational complications, such as intrauterine growth restriction.     

Variation in mitochondrial DNA and maternal genetic ancestry of Ethiopian cattle populations

This study describes complete control region sequences of mitochondrial DNA (mtDNA) from 117 Ethiopian cattle from 10 representative populations, in conjunction with the available cattle sequences in GenBank. In total, 79 polymorphic sites were detected, and these defined 81 different haplotypes. The haplotype and nucleotide diversity of Ethiopian cattle did not vary among the populations studied. All mtDNA sequences from Ethiopian cattle converged into one main maternal lineage (T1) that corresponds to African Bos taurus cattle. According to the results of this study, no zebu mtDNA haplotypes have been found in Ethiopia, where the most extensive hybridization took place on the African continent.