Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8 A resolution.

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.     

Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar).

Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells.     

Conformational feature of neuroactive domoic acid: X-ray structural comparison with isodomoic acid A and alpha-kainic acid.

As an aid for developing a new type of potent insecticide acting on the neuromuscular junction, conformational characteristics of domoic acid and isodomoic acid A, the naturally occurring glutamate agonists, were investigated by X-ray crystal analyses. Conformational comparison with a neuroactive alpha-kainic acid provides information concerning the stereochemical feature responsible for the biological activity.     

Two classes of continuous cell lines established from Syrian hamster 9 day gestation embryos: Preneoplastic cells and progenitor cells

Summary Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from unreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures. These studies were supported in part by grants from the National Institutes of Health (AG 01998), Bethesda, MD, and the U.S. Department of Energy (DE-A-C02-76-EVO-3280), Washington, DC.     

Identification of Mutations in the Pigment Cell-Specific Gene Located at the Brown Locus in Mouse

The pigment cell-specific gene, located at the brown (b)-locus in mouse, encodes the protein that determines the type of melanin synthesized. This protein is known as tyrosinase-related protein, but here we tentatively term it b-locus protein to avoid confusions with the related sequence cross-hybridizing to the tyrosinase gene. In order to identify the mutation at the b-locus, we have cloned and characterized the b-locus protein gene of BALB/c mouse (b/b, c/c). The gene is about 18 kb long and organized into 8 exons and 7 introns. Sequence analysis of the b-locus protein gene reveals four base changes within the protein-coding regions: two missense mutations and two silent mutations. Two missense mutations result in the Cys to Tyr substitution at position 86 (codon 110) and the Arg to His substitution at position 302 (codon 326) of a b-locus protein molecule. Using allele-specific amplification, we confirmed that these missense mutations are actually present in the genomic DNA of two b-mutant strains examined, BALB/c and DBA/2 (b/b, C/C) mice, suggesting that these mutations are specific for the mutant mice at the b-locus. Moreover, we are able to show that the b-locus protein containing Tyr 86 is not reactive with the anti-b-locus protein monoclonal antibody, TMH-1, in transient expression assays.     

Increased in vitro responses of tracheal smooth muscle from hyperresponsive guinea pigs

Repeated aerosol antigen challenge of previously sensitized guinea pigs induces airway hyperresponsiveness to inhaled acetylcholine. To determine the mechanism producing these airway changes and assuming that changes in the trachealis muscle reflect changes in muscle of the entire tracheobronchial tree, we examined the in vitro smooth muscle mechanics and morphometric parameters of tracheae from guinea pigs demonstrating hyperresponsiveness in vivo vs. tracheae from control guinea pigs. No differences between these groups were found in luminal volume at zero transmural pressure, passive pressure-volume characteristics, or area of airway wall. Smooth muscle areas were slightly less in tracheae from hyperresponsive guinea pigs. Tracheae from hyperresponsive guinea pigs had both significantly increased isovolumetric force generation and isobaric shortening compared with tracheae from controls when evaluated over the range of transmural pressures from -40 to 40 cmH2O. We conclude that the in vivo airway hyperresponsiveness induced with repeated antigen challenge is associated with both increased force generation and shortening of tracheal smooth muscle without increased muscle mass, suggesting enhanced contractile activity.     

biparous:A Novel bHLH Gene Expressed in Neuronal and Glial Precursors inDrosophila

Genetic studies have uncovered many genes that are involved in the first steps of neuronal development inDrosophila.Less is known about the intermediate steps during which individual precursor cells follow either the neuronal pathway or the glial pathway. We report the identification of a novel bHLH gene,biparous,expressed in neuronal and glial precursors inDrosophila.Unlike most bHLH genes,biparousexpression continues to the final stages of neurogenesis in the embryo. Expression ofbiparousis not observed in end stage postmitotic neurons and precedes the expression ofrepo,a gene activated in later stages of glial differentiation. The bHLH domain is sufficiently different from previously described bHLH domains to imply a novel function.     

Biochemical systematics of Japanese fireflies of the subfamily Luciolinae and their flash communication systems

Japanese fireflies of the subfamily Luciolinae are biochemically analyzed using 13 allozymes, and the phylogenetic relationships obtained from this analysis are compared with their flash communication systems. As a result, the Japanese Luciolinae can be divided into three groups.Hotaria parvula andH. tsushimana together withLuciola yayeyamana andL. kuroiwae from the first group, and they use the same communication system.L. lateralis, Curtos okinawana, andC. costipennis make up the second group, and their communication systems are also the same.L. cruciata makes up the last one, and its communication system is different from the other fireflies of Luciolinae. Therefore, their taxonomical arrangement and communication systems are not congruent. However, the genetic similarity deduced by allozymic analysis of the members of the Japanese Luciolinae is highly consistent with their flash communication systems.     

Urocystis tranzscheliana,a newly recorded smut fungus onPrimula sieboldii from Japan

A smut fungus onPrimula sieboldii was newly found in Japan and identified asUrocystis tranzscheliana by comparative morphology. This species causes systemic infection ofP. sieboldii and produces sori in its ovaries.Contribution No. 121, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.