Crystal structure of the sulfotransferase domain of human heparan sulfate N-deacetylase/ N-sulfotransferase 1

Heparan sulfate N-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single alpha/beta fold with a central five-stranded parallel beta-sheet and a three-stranded anti-parallel beta-sheet bearing an interstrand disulfide bond. The structural regions alpha1, alpha6, beta1, beta7, 5'-phosphosulfate binding loop (between beta1 and alpha1), and a random coil (between beta8 and alpha13) constitute the PAP binding site of NST1. The alpha6 and random coil (between beta2 and alpha2), which form an open cleft near the 5'-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5'-phosphate.     

Antifouling activity of simple synthetic isocyanides against larvae of the barnacle Balanus amphitrite

Twelve simple linear isocyanides were synthesized and examined for antifouling activity and toxicity against cyprid larvae of the barnacle, Balanus amphitrite. Larval settlement was inhibited, with EC50 values of 0.046-1.90 microg ml(-1), and they were much less toxic (LD50 values ranging over 21.28 microg ml(-1)) than CuSO4 (EC50 0.30 microg ml(-1) and LD50 2.95 microg ml(-1)). The data indicate that simple linear isocyanides are promising non-toxic antifouling agents.     

Enzymatic characterization of scytalone dehydratase Val75Met variant found in melanin biosynthesis dehydratase inhibitor (MBI-D) resistant strains of the rice blast fungus

Carpropamid ((1RS,3SR)-2,2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide) is a potent chemical against the rice blast fungus, Pyricularia oryzae. In 2001, isolates of the fungus with reduced sensitivity to this fungicide appeared in Saga Prefecture of Japan and were regarded as a potential threat to rice protection by carpropamid. The cause of the resistance has been identified genetically as a point mutation resulting in the Val75Met change in scytalone dehydratase, the primary target of the fungicide. We constructed an overexpression system of the variant enzyme and characterized the kinetics in the catalysis and the inhibition by carpropamid isomers. The variant enzyme retained a significant level of enzymatic activity. Inhibition of the variant enzyme by carpropamid was more than 200-fold reduced in comparison with that of the wild-type. Based on the results, here we propose possible mechanisms of the carpropamid-resistance of the variant enzyme in retaining the normal enzymatic activity.     

A GFP-transfected HFWT cell line,GHINK-1, as a novel target for non-RI activated natural killer cytotoxicity assay

An anchorage-dependent Wilms' tumor cell line, HFWT, has been found to be extremely susceptible to human natural killer (NK) cells. Here we established a transfectant of HFWT with the green fluorescence protein (GFP) gene, designated GHINK-1 cells, to apply to the activated NK cytotoxicity assay without radioisotope labeling. After being co-cultured with CD3 CD56+ NK cells, GHINK-1 cells released GFP into the medium. The intensity of the fluorescence from the released GFP correlated almost exactly with the radioactivity of a standard 51Cr-release assay performed with suspension-cultured K562 cells. Because it does not require separation of the remaining live target cells by centrifugation, the non-radioisotopic GFP release assay with GHINK-1 cells is a convenient alternative for monitoring human activated NK killing activity.     

Biological characteristics of cultured cells derived from various types of human brain tumors

We placed in culture brain tumors from 45 cases (7 cases of astrocytoma, 2 from oligodendrogliomas, 2 glioblastomas, 2 ependymomas, 13 meningiomas, 6 pituitary adenomas, 5 neurinomas, a malignant lymphoma, a choroid plexus papilloma, and 6 metastatic tumors) and succeeded in making a primary culture from 33, and maintained 17 in vitro over a considerable period of time (greater than three months). In the early period of the primary cultures, the astrocytoma cells had cytoplasmic processes which contacted each other, the oligodendroglioma cells were small and spindle-shaped, the glioblastoma cells were neoplastic with pleopmorphic features and possessed cytoplasmic processes, the ependymoma cells formed a rosette-like cell arrangement, the meningioma cells were spindle- or round-shaped cells and characterized as forming psammoma bodies, the pituitary adenoma cells were round- or oval-shaped cells and produced growth hormone (GH), adenocorticoid tropic hormone (ACTH), prolactin, or other hypophyseal hormones, the choroid plexus papilloma cells were round-or polygonal and showed a papillary cell arrangement, the neurinoma cells were spindle- or fibrous-shaped cells, and the malignant lymphoma cells were round and formed cell aggregates floating in the culture medium.     

Establishment and characterization of human uterine cervical epidermoid carcinoma cell line HHUS containing HPV 59 DNA

The cell line designated HHUS was established from human uterine cervical keratinizing squamous cell carcinoma. The HHUS cell line was subcultivated more than 70 times within 3 years. The cultured cells, polygonal or spindle, with neoplastic and pleomorphic features, appeared epithelial in shape, with a pavement-like arrangement and grew in multi-layers without contact inhibition. The chromosome number was varied from 40 to 88, and the modal number was stable in diploid range. The cultured cells produced keratinizing squamous cell carcinomas by heterotransplantation into the subcutis of nude mice. The HHUS cells were characterized as producing large amounts of SCC, in vitro and possessing HPV-59 DNA genomes.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Synthesis of anilino-monoindolylmaleimides as potent and selective PKCbeta inhibitors

We report herein synthesis of PKCbeta-selective inhibitors possessing the novel pharmacophore of anilino-monoindolylmaleimide. Several compounds of this series exhibited IC50's as low as 50 nM against human PKCbeta2. One of the most potent compounds, 6l, inhibited PKCbeta1 and PKCbeta2 with IC50 of 21 and 5 nM, respectively, and exhibited selectivity of more than 60-fold in favor of PKCbeta2 relative to other PKC isozymes (PKCalpha, PKCgamma, and PKCepsilon).