Chemical modification of alpha-thrombin and in vitro characterization of its anticoagulant activity

Acetylthrombin, succinylthrombin, maleylthrombin, polyethylene glycol (PEG)-thrombin, and acetylated PEG-thrombin were prepared and characterized. Although clotting activities could be reduced remarkably by the modification, the potencies of all the modified thrombins for protein C activation also decreased. However, in the case of acetylthrombin, succinylthrombin, and acetylated PEG-thrombin, the remaining potencies for protein C activation exceeded largely the remaining clotting activities. Moreover, by the addition of thrombodulin, the potencies for protein C activation were enhanced 16 - 60 fold for succinylthrombin, maleylthrombin, and PEG-thrombin.     

Molecular Cloning and Characterization of the cDNA for Discoidin II of Dictyostelium discoideum

By using monoclonal antibodies directed against discoidin II,we have isolated cDNA clones from axenically grown Ax-2 cells.On cDNA clone (D2) condtained a 1.2-k.b insert encoding theentire discoidin II protein, which is conposed of 257 aminoacid residuces and has a calculated molecular mass of 28,574.The amino acid sequences, determined by Edman degradation ofsix tryptic peptides of discoidin II, were identical to thosededuced from the cDNA sequences. The protein bears no resemblanceto any proteins in the data banks, except that its sequenceis 49% identical with the amino acid sequence of discoidin I.Discoidin II shares with discoidin I both a carbohydratebindingsite and an Arg-Gly-Asp (RGD) sequence, which has been foundin fibronectin in mammalian cells. With the onset of aggregation(8 h of development), a 1.3-kb discoidin II mRNA begins to accumulate.A similar pattern of regulation occurs at the protein level. ¹Present address: MRC Laboratory for Molecular Cell Biology,University College London, Gower Street, London, WC1E 6BT UnitedKingdom     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.     

Low cost multi user multi platform accessible HPLC data acquisition system

Summary A low cost multi user multi platform accessible HPLC data acquisition system has been designed for use in a laboratory environment. This system uses available HPLC measurement systems that lack modern network communication tools and a low cost computer with reliable software. HPLC data are portable to any other computer by means of File Transport Protocol (FTP) communication and can then be used for data analysis. Off line analysis of ethanol data showed a substantial improvement over the old system in terms of data accuracy and skewness. Furthermore, off line data analysis could resolve hidden acetaldehyde peaks which revealed to be oscillatory.     

The Nucleotide Sequence of Human Acylamino Acid-Releasing Enzyme

The nucleotide sequence of a cDNA coding for the human acylaminoacid-releasing enzyme (AARE, also known as acylpeptide hydrolase)[EC 3.4.19.1] subunit has been determined. The amino acid sequenceof human AARE subunit deduced from its cDNA nucleotide sequenceshowed a high degree of identity (91.5%) with both the correspondingproteins from the pig and the rat. The AARE cDNA shows 99.2%identity with a 3.3 kb cDNA transcribed from a locus (DNF15S2)on the short arm of human chromosome 3, whose deletion is associatedwith small cell lung cancer, taking into consideration thatthe sequence of the 3.3-kb cDNA previously reported was causedby misreading.     

Cloning and expression of a porcine UDP-GalNAc: polypeptideN-acetylgalactosaminyl transferase

By employing a bovine UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyl transferase (O-GalNAc transferase) cDNA as a probe, we isolated four overlapping cDNAs from a porcine lung cDNA library. Both the nucleotide sequence of the porcine cDNA and the predicted primary structure of the protein (559 amino acids) proved to be very similar to those of the bovine enzyme (95% and 99% identity, respectively). Transient expression of the clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes a porcine O-GalNAc transferase. The intracellular O-GalNAc transferase activity was increased approximately 100-fold by transfecting cells with the porcine cDNA.Abbreviations O-GalNAc transferase UDP-N-acetylgalactosamine: polypeptideN-acetylgalactosaminyltransferase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - GnT-III UDP-N-acetylglucosamine: -mannoside -1,4N-acetylglucosaminyltransferase III     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Formation of bioorganic compounds in aqueous solution induced by flames

Flames of flammable gases, when blown against a surface of an aqueous solution of organic compounds, were found to induce oxidation as well as other reactions in the solution. This reaction would be regarded as a new model for formation of bioorganic molecules in the primitive hydrosphere exposed to some radical-containing atmosphere.