Restriction fragment length polymorphism detected by human salivary amylase cDNA

Summary The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7kb and 6.5kb fragment alleles, are 0.55 and 0.45, respectively.     

The hemolymph coagulation system in invertebrate animals

A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 3)--d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade.     

Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Inactivation of bleomycin by an N-acetyltransferase in the bleomycin-producing strain Streptomyces verticillus

Abstract Bleomycin-producing Streptomyces verticillus ATCC15003 possesses a bleomycin acetyltransferase which inactivates the drug in the presence of acetyl coenzyme A. The site of acylation in enzymically prepared acetylbleomycin A2 was determined by nuclear magnetic resonance analysis; the primary amino group of the β-aminoalanine moiety of bleomycin was acetylated. Acetylbleomycin A2 had no detectable antibacterial activity and did not induce in vitro DNA degradation.     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.     

Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.     

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of β-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon

Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of β-carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.     

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