Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Detection of Short Protein Coding Regions within the Cyanobacterium Genome: Application of the Hidden Markov Model

The gene-finding programs developed so far have not paid muchattention to the detection of short protein coding regions (CDSs).However, the detection of short CDSs is important for the studyof photosynthesis. We utilized GeneHacker, a gene-finding programbased on the hidden Markov model (HMM), to detect short CDSs(from 90 to 300 bases) in a 1.0 mega contiguous sequence ofcyanobacterium Synechocystis sp. strain PCC6803 which carriesa complete set of genes for oxygenic photosynthesis. GeneHackerdiffers from other gene-finding programs based on the HMM inthat it utilizes di-codon statistics as well. GeneHacker successfullydetected seven out of the eight short CDSs annotated in thissequence and was clearly superior to GeneMark in this rangeof length. GeneHacker detected 94 potentially new CDSs, 9 ofwhich have counterparts in the genetic databases. Four of thenine CDSs were less than 150 bases and were photosynthesis-relatedgenes. The results show the effectiveness of GeneHacker in detectingvery short CDSs corresponding to genes.     

Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis

The biosynthetic pathway for production of the antibiotic fosfomycin by Streptomyces wedmorensis consists of four steps including the formation of a C-P bond and an epoxide. Fosfomycin production genes were cloned from genomic DNA using S. wedmorensis mutants blocked at different steps of the biosynthetic pathway. Four genes corresponding to each of the biosynthetic steps were found to be clustered in a DNA fragment of about 5 kb. Nucleotide sequencing of a large fragment revealed the presence of ten open reading frames, including the four biosynthetic genes and six genes with unknown functions.     

Isolation and Characterization of Hardening-Induced Proteins in Chlorella vulgaris C-27: Identification of Late Embryogenesis Abundant Proteins

Hardening-induced soluble proteins of Chlorella vulgaris BeijerinkIAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) wereisolated and purified by two-dimensional high-performance liquidchromatography (2D-HPLC) on an anion-exchange column, with subsequentreversed-phase chromatography. Some of the proteins were resolvedby SDS-PAGE, characterized by amino-terminal sequencing andidentified by searching for homologies in databases. Separationof the soluble proteins during the hardening of Chlorella bya combination of 2D-HPLC and SDS-PAGE revealed that at least31 proteins were induced or increased in abundance. Of particularinterest was the induction after 12 h of a 10-kDa protein withthe amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVGand the induction after 6 h of a 14-kDa protein with the amino-terminalsequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminalsequences of these proteins indicated that they were homologousto late embryogenesis abundant (LEA) proteins. Furthermore,the level of a 22-kDa protein also increased after 12 h. Theamino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD,indicated that it was homologous to thioredoxin peroxidase. (Received June 9, 1995; Accepted September 12, 1995)     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid     

Spectral and electrochemical properties of vanadyl hexadecafluorophthalocyanine

A vanadyl complex with perfluorinate phthalocyanine, VOPcF₁₆, was prepared. The monomer-dimer solvent dependence was confirmed based on the solvent effect for the Q-band position-that is, VOPcF₁₆ exists as a monomer in a nonpolar solvent such as benzene, but dimerizes in a polar solvent such as acetone. Electron spin resonance data also supported the solvent dependence found. In addition, the substituent effect of fluorine atoms on the redox properties was investigated by measuring the cyclic voltammograms in dichloromethane. On the reduction side, three redox couples were observed, the first two of which were assigned as being due to the reduction of the phthalocyanine ring (to LUMO), whose potentials are 0.4–0.5 V higher than those of the tetra-t-butyl and octabutoxy derivatives, VOPc(t-Bu)₄ and VOPc(O-n-Bu)₈.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

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