Polymorphism of pentanucleotide repeats in the 5′ flanking region of glutathione S-transferase (GST) π gene

The upstream sequence of the glutathione S-transferase gene contains pentanucleotide (ATAAA) repeats. Analysis of the region using polymerase chain reaction indicated that the repeat sequence was polymorphic and segregation of the polymorphic alleles was codominant heredity. Heterozygosity of the new VNTR was 0.818 in healthy Japanese and 0.794 in American whites. Allelic frequencies among healthy controls and alcoholics as well as other diseases were not significantly different.     

The BIG gene is involved in regulation of sulfur deficiency-responsive genes in Arabidopsis thaliana

The Arabidopsis thaliana mutants altered sulfur response 1-1 ( asr1-1 ) and asr1-2 were isolated using the green fluorescent protein gene ( GFP ), as a marker, driven by a sulfur deficiency-responsive promoter containing the β_SR fragment, which is responsible for the induction of gene expression under sulfur deficiency. In the asr1 mutants, the expression of three sulfur deficiency-responsive genes β_SR-driven GFP , sulfate transporter 2;2 ( SULTR2;2 ) and adenosine-5'-phosphosulfate reductase 1 ( APR1 ) were induced in medium containing a normal sulfate concentration. The ASR1 locus was mapped to a 53-kb region on the upper arm of chromosome III; this is also the region of the BIG gene, which encodes a calossin-like protein necessary for the polar transport of auxin. The morphology of the asr1 mutants, i.e. reduced leaf size and inflorescence elongation, resembled that of big mutants. Using nucleotide sequence analysis of the BIG gene, we identified independent nonsense mutations in asr1-1 and asr1-2 . To confirm that ASR1 was BIG , we established lines of transgenic A. thaliana carrying a transfer DNA (T-DNA) insertion in the BIG gene. In these T-DNA insertion mutants, mRNA levels of β_SR-driven GFP and APR1 were upregulated in normal sulfate medium. The F₁ plants from crosses between asr1-1 and T-DNA insertion lines exhibited reduced leaf size and inflorescence length, indicating that ASR1 was indeed BIG . Taken together, the present results established that BIG is involved in the regulation of β_SR-driven GFP and APR1 mRNA level gene expression. Indole-3-acetic acid also upregulated β_SR-driven GFP and APR1 together with SULTR2;2 mRNA level, suggesting that the big effect on β_SR-driven GFP and APR1 is a pleiotropic aspect of the BIG gene.     

Potentiometric titration of poly(L-glutamic acid) in aqueous solutions and binding of divalent cations

The potentiometric titration of poly(L -glutamic acid) was performed under conditions of varied ionic strength and concentration of added divalent cations. From these titration curves, the amount of divalent cations, especially magnesium, bound to poly(L -glutamic acid) was determined using a new method of analysis based on polyelectrolyte theory. By comparison with the polyelectrolyte, poly(acrylic acid), it was found that there are no specific interactions between metal ion and poly(L -glutamic acid) in either the helical or random coil conformation. The effect of these divalent cations on the conformation of poly(L -glutamic acid) was also discussed.     

Physiological function and ecological aspects of fatty acid-amino acid conjugates in insects†

In tritrophic interactions, plants recognize herbivore-produced elicitors and release a blend of volatile compounds (VOCs), which work as chemical cues for parasitoids or predators to locate their hosts. From detection of elicitors to VOC emissions, plants utilize sophisticated systems that resemble the plant–microbe interaction system. Fatty acid–amino acid conjugates (FACs), a class of insect elicitors, resemble compounds synthesized by microbes in nature. Recent evidence suggests that the recognition of insect elicitors by an ancestral microbe-associated defense system may be the origin of tritrophic interactions mediated by FACs. Here we discuss our findings in light of how plants have customized this defense to be effective against insect herbivores, and how some insects have successfully adapted to these defenses.     

Variation in oxygen isotope ratio of dissolved orthophosphate induced by uptake process in natural coral holobionts

Coral Reefs - A model incubation experiment using natural zooxanthellate corals was conducted to evaluate the influence of phosphate uptake by coral holobionts on oxygen isotope ratio of dissolved...     

Role of T‐bet,the master regulator of Th1 cells,in the cytotoxicity of murine CD4+ T cells

Although CD4⁺ T cells are generally regarded as helper T cells, some activated CD4⁺ T cells have cytotoxic properties. Given that CD4⁺ cytotoxic T lymphocytes (CTLs) often secrete IFN‐γ, CTL activity among CD4⁺ T cells may be attributable to Th1 cells, where a T‐box family molecule, T‐bet serves as the “master regulator”. However, although the essential contribution of T‐bet to expression of IFN‐γ has been well‐documented, it remains unclear whether T‐bet is involved in CD4⁺ T cell‐mediated cytotoxicity. In this study, to investigate the ability of T‐bet to confer cytolytic activity on CD4⁺ T cells, the T‐bet gene (Tbx21) was introduced into non‐cytocidal CD4⁺ T cell lines and their cytolytic function analyzed. Up‐regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4⁺ T cells but not in untransfected parental cells. In one cell line, T‐bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas^? target cells. Although T‐bet was found to repress up‐regulation of CD40L (CD154), which controls FasL‐mediated cytolysis, the extent of CD40L up‐regulation on in vitro‐differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T‐bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.

     

Improved method for the determination of trimethadione and its demethylated metabolite, dimethadione, in human serum by gas chromatography

An improved gas chromatographic method, involving the use of a wide-bore capillary column, for the determination of trimethadione and its only demethylated metabolite, dimethadione, in human serum is described. The results indicate that both substances and the internal standard (maleinimide) were well separated with no tailing peak. The detection limit was 10 ng/ml for trimethadione and 50 ng/ml for dimethadione. This improved method is reliable in terms of sensitivity, selectivity and reproducibility for the simultaneous determination of both compounds in human serum.     

TaqI polymorphism in the LDL receptor gene and a TaqI 1.5-kb band associated with familial hypercholesterolemia

Summary The low density lipoprotein (LDL) receptor gene was analyzed in 67 unrelated healthy Japanese and 38 members of six consecutive families with familial hypercholesterolemia (FH) by Southern blot hybridization with TaqI, an LDL receptor cDNA fragment containing exons 1 to 8 being used as a probe. A new TaqI RFLP at the LDL receptor locus was detected with allele frequencies of 0.67 and 0.33. The data obtained with smaller cDNA subfragment probes revealed that the TaqI RFLP site is located within 1.1 kb of the 5 side of the EcoRI site of exon 5. The TaqI RFLP was in linkage disequilibrium with the PstI RFLP but showed no significant linkage disequilibrium with the RFLPs for AvaII, ApaLI/I15, PvuII, NcoI, and ApaLI/3. Among the seven RFLPs at the LDL receptor locus, the TaqI RFLP was the only useful genetic marker in one of the six families with FH. Furthermore, the association of an additional TaqI 1.5-kb band with a mutant LDL receptor gene was observed in another family with FH in which the proband was homozygous for all of the seven RFLPs. The data obtained with various restriction enzymes and smaller cDNA subfragments probes suggested that a minor change in nucleotide sequences in the region including exons 5 to 8 is present in the mutant gene. These data suggest that the TaqI RFLP is a useful genetic marker at the LDL receptor locus and that TaqI serves for the analysis of some mutant LDL receptor genes, when used with small LDL receptor cDNA probes.     

Urat1-Uox double knockout mice are experimental animal models of renal hypouricemia and exercise-induced acute kidney injury

ABSTRACT

Renal hypouricemia (RHUC) is a hereditary disease characterized by a low level of plasma urate but with normal urinary urate excretion. RHUC type 1 is caused by mutations of the urate transporter URAT1 gene (SLC22A12). However, the plasma urate levels of URAT1 knockout mice are no different from those of wild-type mice. In the present study, a double knockout mouse, in which the URAT1 and uricase (Uox) genes were deleted (Urat1-Uox-DKO), were used as an experimental animal model of RHUC type 1 to investigate RHUC and excise-induced acute kidney injury (EIAKI). Mice were given a variable content of allopurinol for one week followed by HPLC measurement of urate and creatinine concentrations in spot urine and blood from the tail. The urinary excretion of urate in Urat1-Uox-DKO mice was approximately 25 times higher than those of humans. With allopurinol, the plasma urate levels of Urat1-Uox-DKO mice were lower than those of Uox-KO mice. There were no differences in the urinary urate excretions between Urat1-Uox-DKO and Uox-KO mice administered with 9 mg allopurinol /100 g feed. In the absence of allopurinol, plasma creatinine levels of some Urat1-Uox-DKO mice were higher than those of Uox-KO mice. Consequently, hypouricemia and normouricosuria may indicate that the Urat1-Uox-DKO mouse administered with allopurinol may represent a suitable animal model of RHUC type 1. Urat1-Uox-DKO mice without allopurinol exhibited acute kidney injury, thus providing additional benefit as a potential animal model for EIAKI. Finally, our data indicate that allopurinol appears to provide prophylactic effects for EIAKI.     

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ_adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12–1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ_adjusted = 1.00–1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.