The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products

Three forms of protein phosphatase-1 were isolated from rabbit skeletal muscle that had Mr values of 37 000, 34 000 and 33 000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the beta-subunit of phosphorylase kinase very much faster than the alpha-subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase-3 and Mg-ATP for activity by incubation with inhibitor-2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr-37 000 and Mr-34 000 species were converted to the Mr-33 000 form by incubation with chymotrypsin. It is concluded that the Mr-33 000 and Mr-34 000 forms are derived from the Mr-37 000 component by limited proteolysis. Conversion of the Mr-37 000 to the Mr-33 000 form was accompanied by a two-fold increase in activity, indicating that an Mr-4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36 000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr-37 000 (15-20 kU/mg) or Mr-33/34 000 (40-50 kU/mg) forms of protein phosphatase-1. It dephosphorylated the alpha-subunit of phosphorylase kinase 4-5-fold faster than the beta-subunit, was unaffected by inhibitor-1 or inhibitor-2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase-3 and Mg-ATP-dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr-37 000 form of protein phosphatase-1 to the Mr-33 000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observed with protein phosphatase 1. Antibody to protein phosphatase-2A raised in sheep did not cross-react with any of the forms of protein phosphatase-1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase-1 and protein phosphatase-2A are distinct gene products.     

Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells

The ability of endothelin to promote phospholipid hydrolysis has been studied in myo-[2-3H]-inositol-, [3H]-arachidonic acid- or methyl-[3H]choline chloride-prelabelled cultured vascular smooth muscle cells (VSMC) from rat and bovine thoracic aortae and human omental vessels. The biochemical responses to endothelin were comparable between the different VSMC isolates. Endothelin promoted the accumulation of glycerolphospho[3H]inositol and concomitant loss of [3H]-inositol label from phosphatidylinositol. Exposure of [3H]choline-labelled VSMC to endothelin resulted in a loss of radioactivity from phosphatidylcholine that was inversely parallelled by an increase in water-soluble [3H]-choline metabolites. In [3H]-arachidonic acid ([3H]-AA)-labelled VSMC, endothelin induced extracellular release of [3H]-AA which derived from both phosphatidylcholine and phosphatidylinositol. Half-maximally effective concentrations of endothelin for all these responses were approximately 2-7 nM and did not vary between VSMC types. Endothelin-induced release of [3H]-AA into VSMC medium-overlay was inhibited by quinacrine and nordihydroguaiaretic acid but not by neomycin or indomethacin. The data herein implicate activation of phospholipase A2 by endothelin with subsequent metabolism of arachidonic acid via the lipoxygenase pathway.     

Decreased susceptibility of cultured smooth muscle cells from SHR rats to growth inhibition by heparin

Smooth muscle cell proliferation is regulated through the coordinated action of growth inhibitors and growth factors/mitogens; a specific heparin-epidermal growth factor (EGF) complementation has been proposed (Reilly et al., 1987, J. Cell. Physiol., 131:149-157). In culture, vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate more rapidly than VSMC from control Wistar Kyoto rats (WKY). We observed that, compared with WKY-derived VSMC, cells from SHR were markedly less susceptible to growth inhibition both by heparin and its homopolysaccharide analogue pentosan polysulphate (PPS). SHR-derived VSMC exhibited a reduced capacity for binding of [3H]heparin to specific extracellular surface receptors, whereas affinities for heparin were comparable between both VSMC isolates. The early (0-2 hr at 37 degrees C) kinetics of internalization did not differ between SHR- and WKY-derived VSMC, but both internalized equivalent proportions (approximately 10%) of initially surface-bound heparin. VSMC from SHR exhibited a greater capacity, without a changed affinity, for [I125]EGF binding than VSMC from WKY. Pre-exposure of VSMC to heparin or PPS decreased, in a time-dependent manner, the EGF binding capacity for both SHR and WKY (by 40-50% after 72 hr). However, in absolute terms, the EGF-binding capacity of VSMC from SHR exposed to heparinoids was similar to that of nonexposed VSMC from WKY.     

Isolation of a mutant LLC-PK1 cell line defective in hormonal responsiveness. A pleiotropic lesion in receptor function

A mutant LLC-PK1 cell line, M18, was isolated after a single treatment of the parent culture with N-methyl-N'-nitro-N-nitroso-guanidine. In contrast to LLC-PK1 cells, the mutant did not exhibit production of urokinase-type plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor-independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8-bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP-dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non-hydrolyzable GTP analogue guanosine 5'-[beta, gamma-imino]triphosphate, but were unresponsive to calcitonin or vasopressin. The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC-PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.     

Transformation of Saccharopolyspora erythraea by electroporation of germinating spores: construction of propionyl Co-A carboxylase mutants

The introduction of plasmid DNA into germinating spores of an industrially improved strain of Saccharopolyspora erythraea was accomplished by electroporation. Various parameters affecting the efficiency of electroporation were examined. The most critical factor was the extent of spore germination. Electrocompetence was limited to a 4-h period following the initial emergence of the germ tube. Electroporation efficiencies as high as 2 × 10⁵ CFU μg⁻¹ of plasmid DNA were obtained using electrocompetent germlings. The optimal field strength was 12–14 kV cm⁻¹ with a pulse duration of 15–20 ms. Electrocompetent germlings were stored at −80°C without a significant decrease in transformation efficiency. The utility of this protocol was demonstrated by isolating a propionyl-CoA carboxylase mutant through targeted gene disruption and replacement. Received 3 April 1998/ Accepted in revised form 28 September 1998     

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.      

LDL induces intracellular signalling and cell migration via atypical LDL-binding protein T-cadherin

Cadherins are a superfamily of adhesion molecules that mediate Ca²⁺-dependent cell–cell adhesion. T-cadherin (T-cad), a unique glycosylphosphatidylinositol-anchored member of the cadherin superfamily, was initially identified by immunoblotting of vascular cell membranes as an atypical low affinity low density lipoprotein (LDL)-binding protein. It is not known whether this heterophilic interaction is physiologically relevant. Expression of T-cadherin is upregulated in vascular cells during atherosclerosis, restenosis and tumour angiogenesis, conditions characterized by enhanced cell migration and growth. Elevated levels of serum low density lipoproteins (LDL), which result in cholesterol accumulation in vascular wall, is a widely accepted risk factor in atherosclerosis development. Additionally to its metabolic effects, LDL can produce hormone-like effects in a number of cell types. This study has utilized HEK293 cells and L929 cells stably transfected with T-cadherin cDNA to investigate T-cad-dependent responses to LDL. Stable expression of T-cad in both HEK293 and L929 cells results in significantly (p < 0.05) elevated specific surface binding of [I¹²⁵]-LDL. Compared with mock-transfectants, cells expressing T-cad exhibit significantly (p < 0.01) enhanced LDL-induced mobilization of intracellular Ca²⁺-stores and a significantly (p < 0.01) increased migration toward an LDL gradient (0.1% BSA + 60 g/ml LDL) in Boyden chamber migration assay. Thus LDL-binding to T-cad is capable of activating physiologically relevant intracellular signaling and functional responses.     

The involvement of interleukin-1 and interleukin-4 in the response of human annulus fibrosus cells to cyclic tensile strain: an altered mechanotransduction pathway with degeneration

Introduction  

Recent evidence suggests that intervertebral disc (IVD) cells derived from degenerative tissue are unable to respond to physiologically relevant mechanical stimuli in the 'normal' anabolic manner, but instead respond by increasing matrix catabolism. Understanding the nature of the biological processes which allow disc cells to sense and respond to mechanical stimuli (a process termed 'mechanotransduction') is important to ascertain whether these signalling pathways differ with disease. The aim here was to investigate the involvement of interleukin (IL)-1 and IL-4 in the response of annulus fibrosus (AF) cells derived from nondegenerative and degenerative tissue to cyclic tensile strain to determine whether cytokine involvement differed with IVD degeneration.