Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of beta-migrating endothelial-type plasminogen activator inhibitor (PAI-1) from that of placental-type plasminogen activator inhibitor (PAI-2). These assays are based upon the binding of PAI-1 and PAI-2 to immobilized single-chain tissue-type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI-PA complex first with rabbit antiserum specific for the individual PAI and then with 125I-labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose-dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI-1 nor PAI-2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U-937) produced only PAI-2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT-1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI-1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI-2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.     

Immunological profiles in workers of a power plant burning coal rich in arsenic content

A group of 47 male adults working in a thermal power plant burning coal containing 900 to 1,500 g of arsenic per ton dry weight was examined on the blood serum immunoglobulins IgG, IgA and IgM content and levels of acute reactants alpha-1-antitrypsin (A1AT), alpha-2-macroglobulin (A2M), transferrin (TRF), orosomucoid (ORO) ceruloplasmin (CPL), and lysozyme (LYS). Investigations in the control group comprising 27 workers from another power plant in the same district where the coal content of arsenic was more than 10 times lower were analogous. The inter-group differences in means were evaluated by t-test, differences in the association of values by F-test, and the correlations with age and the length of exposure were assessed using the regression analysis method. The differences in mean IgG, IgA, IgM, LYS and A2M levels between the exposed and control groups of workers were insignificant or of borderline significance only. In contrast, differences in TRF, ORO and particularly CPL levels were statistically highly significant, in all instances P less than 0.001. In the control group, persons with abnormal values in at least two immunobiochemical tests used accounted for 3.7%, in the group of the exposed for 51% (P less than 0.002). All these findings, especially the rise in CPL concentration levels in the exposed group are discussed on the background of the rise in cancer mortality rates found previously in this group of power plant workers.     

Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of [Cd7]-metallothionein-2 from rat liver was determined in aqueous solution, using nuclear magnetic resonance spectrometry and distance geometry calculations. The experimental data provided proton-proton distance constraints from measurements of nuclear Overhauser effects, constraints on the geometry of the metal-cysteine clusters determined by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calculations were performed separately for the alpha and beta-domains containing the 4 and 3-metal clusters, respectively, since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-sulfur cluster and the local chirality of the metal co-ordination are very similar to the solution structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.     

Somatic embryogenesis and plant regeneration from immature embryos of western larch

Somatic embryogenesis was initiated from immature embryos of western larch (Larix occidentalis Nutt.) on media containing 2,4-dichlorophenoxyacetic acid and N6- benzyladenine. The effects of explant type and ammonium nitrate and glutamine concentrations on initiation were tested. Although 21–93% of explants rendered cultures in various experiments, only 3% yielded sustainable embryogenic lines. Excised embryos at the early cotyledonary stage were optimal for initiation. Maturation of somatic embryos was promoted by abscisic acid. Response to abscisic acid concentrations and duration of exposure to abscisic acid varied with genotype. Maximal results were obtained with 0.025 M abscisic acid for 1 to 2 weeks followed by individual culture on medium without growth regulators. Mature somatic embryos developed into shoots with roots. Plantlets have been established in peat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - IBA indole-3-butyric acid - ABA abscisic acid     

Analysis of the human regulators of complement activation (RCA) gene cluster with yeast artificial chromosomes (YACs).

The human regulators of complement activation gene cluster (RCA cluster) have been partially characterized with yeast artificial chromosomes (YACs). While the data confirm many points previously elucidated, the finer resolution of YAC mapping has allowed the discovery and/or localization of partial gene duplications, the determination of gene orientations, and the measurement of gaps between known genes. Here nine overlapping YACs that encompass a genomic region of 800 kb, encoding four RCA genes and three gene-like elements, are described. The encoded genes and two of the gene-like elements share the same orientation and are ordered (5' to 3') DAF, CR2, CR1, MCP-like, CR1-like, and MCP. A C4bp-like region lies upstream from DAF and is likely to correspond to one recently observed by F. Pardo-Manuel, J. Rey-Campos, A. Hillarp, B. Dahlback, and S. Rodriguez de Cordoba (1990, Proc. Natl. Acad. Sci. USA 87: 4529-4533). MCP-like, a new genetic element, was discovered and found to be homologous to the 5' portion of the MCP gene. Two large gaps of 85 kb (between CR2 and DAF) and 110 kb (between DAF and the C4bp-like element) could carry additional RCA genes. The arrangement of CR1, MCP-like, CR1-like, and MCP, in that order, strongly suggests that this region was generated by a single duplication of neighboring CR1/CR1-like and MCP/MCP-like forerunners. The RCA YACs will now serve as convenient DNA sources for the subcloning and further characterization of this region.