Expression and function of TETRAN, a new type of membrane transporter

In contrast to transport across basolateral membranes, the mechanism governing transport of organic anions across the luminal membranes of proximal tubules has remained unclear. We recently found Tetracycline transporter-like protein (TETRAN), a human ortholog of yeast Tpo1p that can transport anionic Non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examine the expression and function of TETRAN. TETRAN mRNA is expressed in various human tissues, including kidney. When overexpressed in cultured cells, TETRAN was predominantly localized on cytoplasmic membranes. Immunohistochemical analysis of human and mouse kidney tissue showed that TETRAN was expressed at the luminal membranes of proximal tubules. Overexpression of TETRAN in cultured cells facilitated the uptake of organic anions such as indomethacin (a NSAID) and fluorescein. The results suggest that TETRAN is a novel human organic anion transporter, and that it serves as a transporter for some NSAIDs and various other organic anions at the final excretion step.     

Modification of halogen specificity of a vanadium-dependent bromoperoxidase

The halide specificity of vanadium-dependent bromoperoxidase (BPO) from the marine algae, Corallina pilulifera, has been changed by a single amino acid substitution. The residue R397 has been substituted by the other 19 amino acids. The mutant enzymes R397W and R397F showed significant chloroperoxidase (CPO) activity as well as BPO activity. These mutant enzymes were purified and their properties were investigated. The maximal velocities of CPO activities of the R397W and R397F enzymes were 31.2 and 39.2 units/mg, and the K(m) values for Cl(-) were 780 mM and 670 mM, respectively. Unlike the native enzyme, both mutant enzymes were inhibited by NaN(3). In the case of the R397W enzyme, the incorporation rate of vanadate into the active site was low, compared with the R397F and the wild-type enzyme. These results supported the existence of a specific halogen binding site within the catalytic cleft of vanadium haloperoxidases.     

Ecm10p localizes in yeast mitochondrial nucleoids and its overexpression induces extensive mitochondrial DNA aggregations

Ecm10p was initially identified as a cell wall synthesis-related gene product [Genetics 147 (1997) 435] and also reported as a mitochondrial protein which was partially capable of compensating the phenotypic defect by SSC1 gene mutation [FEBS Lett. 487 (2000) 307]. Here we report that ecm10p is localized in mitochondrial nucleoids as its major component and the targeting signal resides between amino acid residues 161 and 240. Overexpression of ecm10p induces extensive mitochondrial DNA aggregations, which might be due to aberrant mitochondrial DNA cleavages through an altered endonuclease activity in mitochondrial nucleoids.     

Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase

Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.     

Synthesis and SAR of novel conformationally-restricted oxazolidinones possessing gram-positive and fastidious gram-negative antibacterial activity. Part 1: Substituted pyrazoles

A novel series of conformationally-restricted oxazolidinones was synthesized which possess a fused pyrazole ring substituted with various alkyl, aryl and heteroaryl substituents. A number of analogs exhibited potent activity against both gram-positive and fastidious gram-negative organisms.     

Involvement of up-regulation of PUMA in non-steroidal anti-inflammatory drug-induced apoptosis

NSAIDs such as celecoxib induce apoptosis in cancer cells. Although this apoptotic effect is involved in the anti-tumor activity associated with such drugs, the mechanism by which this occurs is not fully understood. We report here that various NSAIDs, including celecoxib, up-regulate PUMA, a Bcl-2 family protein with potent apoptosis-inducing activity, in human gastric carcinoma cell line, accompanying the induction of apoptosis. Experiments using siRNA and an intracellular Ca(2+) chelator revealed that Ca(2+)-dependent up-regulation of ATF4 and CHOP is involved in this up-regulation of PUMA. The siRNA for PUMA inhibited the celecoxib-induced activation and translocation of Bax, release of cytochrome c into the cytosol and induction of apoptosis, suggesting that PUMA plays an important role in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis.     

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.     

Control of Simian Immunodeficiency Virus Replication by Vaccine-Induced Gag- and Vif-Specific CD8+ T Cells

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8⁺ T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8⁺ T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8⁺ T-cell responses. In the first group vaccinated with Gag-expressing vectors (n = 5 animals), three animals that showed efficient Gag-specific CD8⁺ T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors (n = 6 animals), three animals that elicited Vif-specific CD8⁺ T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8⁺ T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8⁺ T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8⁺ T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8⁺ T-cell responses by prophylactic AIDS vaccines.