Phenanthraquinone inhibits eNOS activity and suppresses vasorelaxation

Diesel exhaust particles cause an impairment of endothelium-dependent vasorelaxation and are associated with cardiopulmonary-related diseases and mortality, but the mechanistic details are poorly understood. Since we reported previously that phenanthraquinone, an environmental chemical contained in diesel exhaust particles, suppresses neuronal nitric oxide synthase (nNOS) activity by shunting electrons away from the normal catalytic pathway, it was hypothesized that phenanthraquinone inhibits endothelial NOS (eNOS) activity and affects vascular tone. Therefore, the effects of phenanthraquinone on eNOS activity, endothelium-dependent relaxation, and blood pressure were examined in the present study. Phenanthraquinone inhibited NO formation evaluated by citrulline formed by total membrane fraction of bovine aortic endothelial cells with an IC(50) value of 0.6 microM. A kinetic study revealed that phenanthraquinone is a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. Endothelium-dependent relaxation of rat aortic rings by ACh was significantly inhibited by phenanthraquinone (5 microM), whereas the endothelium-independent relaxation by nitroglycerin was not. Furthermore, an intraperitoneal injection of phenanthraquinone (0.36 mmol/kg) to rats resulted in an elevation of blood pressure (1.4-fold, P < 0.01); under this condition, plasma levels of stable NO metabolites, nitrite/nitrate, in phenanthraquinone-treated rats was reduced to 68% of control levels. The present findings suggest that phenanthraquinone has a potent inhibitory action on eNOS activity via a similar mechanism reported for nNOS, thereby causing the suppression of NO-mediated vasorelaxation and elevation of blood pressure.     

Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase

We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.     

Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase

Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis. This enzyme in plants is localized in plastids and is believed to be composed of biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been purified yet. Accumulated evidence shows that pea plastidic acetyl-CoA carboxylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a redox cascade. To understand the reductive activation of carboxyltransferase at the molecular level here, we obtained the active enzyme composed of decahistidine-tagged (His tag) alpha and beta polypeptides through the expression of the pea plastidic carboxyltransferase gene in Escherichia coli. Gel filtration showed that the molecular size of the recombinant carboxyltransferase is in agreement with that of partially purified carboxyltransferase from pea chloroplasts. The catalytic activity of the recombinant enzyme was similar to that of native carboxyltransferase. These results indicate that the molecular structure and conformation of recombinant carboxyltransferase resemble those of its native counterpart and that native carboxyltransferase is indeed composed of alpha and beta polypeptides. This recombinant enzyme was activated by dithiothreitol, a known reductant of S-S bonds, with a profile similar to that of its native counterpart. The recombinant enzyme was activated by reduced thioredoxin-f, a signal transducer of redox potential in chloroplasts under irradiation. Thus, this enzyme was redox-regulated, like that of the native carboxyltransferase.     

Zinc induces mixed types of cell death, necrosis, and apoptosis, in molt-4 cells

To investigate the mode of zinc-induced cell death, the associated morphological changes, and biological events were examined in zinc-treated Molt-4 cells. Fluorescence microscope observations with double staining of zinc-treated cells with Hoechst 33342 and propidium iodide (PI) indicated that the metal induced both necrosis and apoptosis. To confirm this, cells were stained with both PI and FITC-labeled annexin V, which binds phosphatidylserine, and then analyzed by flow cytometry. The results also confirmed that zinc induces mixed types of cell death, necrosis and apoptosis, and that the former induction occurs earlier and at a greater frequency. Hallmarks of apoptosis such as abnormal chromosome condensation and release of cytochrome c, as well as the appearance of annexin-positive cells, appeared along with the expression of mitochondrial membrane protein 7A6. However, zinc did not induce increases in caspase-3 like protease and caspase-8 activities, and caused slightly hypodiploid cells. Furthermore, the induction of cell death and annexin-positive cells was not blocked by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. These results indicate that zinc induces both necrosis and apoptosis, without caspase-3 activation.     

Replacement of domain b of human protein disulfide isomerase-related protein with domain b' of human protein disulfide isomerase dramatically increases its chaperone activity

We have reported that human protein disulfide isomerase-related protein (hPDIR) has isomerase and chaperone activities that are lower than those of the human protein disulfide isomerase (hPDI), and that the b domain of hPDIR is critical for its chaperone activity [J. Biol. Chem. 279 (2004) 4604]. To investigate the basis of the differences between hPDI and hPDIR, and to determine the functions of each hPDIR domain in detail, we constructed several hPDIR domain mutants. Interestingly, when the b domain of hPDIR was replaced with the b' domain of hPDI, a dramatic increase in chaperone activity that was close to that of hPDI itself was observed. However, this mutant showed decreased oxidative refolding of alpha1-antitrypsin. The replacement of the b domain of hPDIR with the c domain of hPDI also increased its chaperone activity. These observations suggest that putative peptide-binding sites of hPDI determine both its chaperone activity and its substrate specificity.     

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.     

Preparation of DNA-modified nanoparticles and preliminary study for colorimetric SNP analysis using their selective aggregations

DNA-modified nanospheres were prepared by anchoring amino-terminated oligodeoxynucleotides (ODNs) with carboxylates onto a colored polystyrene sphere surface through amido bonds. About 220 ODN molecules were immobilized onto a nanosphere 40 nm in diameter. Preliminary studies using the microspheres with 1 μm diameter reveal that the specificity of hybridization was retained after modification. Three kinds of differently colored (RGB, red/green/blue) nanospheres bearing unique ODNs on their surface were prepared for detecting the p53 gene. Each ODN is complementary to a different part in the 45mer sample that is a part of a conservative region of the p53 gene containing one of the hot spots. In a binary system using spheres R and G, the wild-type 45mer made the aggregates with yellow emission as the result of mixing both colors. The mutant 45mer containing one nucleotide displacement did not give such aggregates with distinct colors. The study of fluorescence resonance energy transfer (FRET) showed that spheres R and G directly contact each other in the aggregates with the wild type. The RGB ternary system gave aggregates with specific colors corresponding to the added ODN samples, wild type or mutant. In addition, in the presence of both samples, all of the spheres formed aggregates with white emission as a consequence of mixing three primary colors of light. This means that the present technique should allow us to conduct an allele analysis.