Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming fibrosarcoma (HT-1080) cells.

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.     

The pathway of myo-inositol 1,3,4-trisphosphate dephosphorylation in liver.

We studied the dephosphorylation pathway for Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) by liver homogenates and soluble and particulate subfractions incubated in media resembling physiological ionic strength and pH. Ins(1,3,4)P3 was dephosphorylated to two InsP2 (inositol bisphosphate) isomers, one of which is Ins(3,4)P2 [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. The second InsP2 is the 1,3 isomer. Ins(3,4)P2 is dephosphorylated to inositol 3-phosphate by an enzyme activity located in both soluble and particulate fractions. The phosphatase(s) that attacks Ins(1,3)P2 was largely soluble, but we have not determined which phosphate(s) is removed. When the initial substrate concentration was 1 nM, the rate of dephosphorylation of Ins(1,4)P2 greater than Ins(1,3)P2 greater than Ins(3,4)P2. None of these bisphosphates was phosphorylated when incubated with liver homogenates and 5 mM-ATP, but their rates of dephosphorylation were then decreased.     

Expression of UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase in functionally defined T-cell clones.

To measure UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase (beta 1,4-GalNActransferase) in crude cell and tissue extracts we designed an assay containing UDP-[3H]N-acetylgalactosamine as donor and biotinylated human glycophorin A as an acceptor. After incubation the labelled acceptor was separated by the use of avidin-agarose from extract-derived endogenous acceptors. This assay permitted one to measure specifically the beta 1,4-GalNActransferase in crude extracts. This glycosyltransferase has previously been shown to be involved in the biosynthesis of Vicia villosa (hairy winter vetch)-lectin (VV)-binding sites of the murine cytotoxic T-cell line B6.1. Since VV-binding sites are a distinct marker for the cytotoxic subclass of murine T-lymphocytes, we used this assay to determine enzyme levels in a panel of functionally defined murine T-cell clones. Non-cytolytic T-cell lines generally have low activity, whereas most cytotoxic lines have high levels of activity. However, one cytotoxic T-cell line does not express the enzyme, although it has large numbers of VV-binding sites. This suggests the existence of another type of VV-binding sites which is independent of the beta 1,4-GalNActransferase in some cytotoxic-T-lymphocyte lines. The enzyme was also assayed in a variety of other tissues and found to have a very high activity in the intestine but a low activity in most other tissues. This was in considerable contrast with the ubiquitously high expression of UDP-GalNAc:peptide alpha 1-GalNActransferase. Therefore, the beta 1,4-GalNActransferase seems to be regulated during differentiation.     

Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

Phospholipid and fatty acid composition in mitochondria from spinach (Spinacia oleracea) leaves and petioles. A comparative study.

Essentially chlorophyll-free mitochondria from photosynthetic (leaf) and non-photosynthetic tissue (petiole) were isolated from spinach (Spinacia oleracea). Leaf mitochondria were found to contain more phosphatidylcholine than phosphatidylethanolamine compared with petiole mitochondria. Galactolipids were found in small and equal amounts (5 mol of galactolipids/100 mol of galactolipids and phospholipids) in both leaf and petiole mitochondria. Fatty acid composition showed a significant difference in the amounts of C18:2 and C18:3 acids. The C18:2/C18:3 ratio was more than twice as high in all of the phospholipids studied from petiole mitochondria compared with the ratio in leaf mitochondria. More than 50% (mol/100 mol) of the fatty acids in the major lipids (phosphatidylcholine, phosphatidylethanolamine and cardiolipin) in petiole mitochondria were C18:2. In the minor lipids (phosphatidylinositol and phosphatidylglycerol), C16:0 dominated in both leaf and petiole mitochondria.     

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro.

The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.     

Effects of the haem precursor 5-aminolaevulinate on tryptophan metabolism and disposition in the rat.

5-Aminolaevulinate administration to rats inhibits cerebral 5-hydroxytryptamine synthesis by decreasing tryptophan availability to the brain secondarily to activation of hepatic tryptophan pyrrolase. The results show that tryptophan metabolism and disposition can be influenced by changes in liver haem concentration, and are discussed briefly in relation to mood disorders in the hepatic porphyrias.     

Conformational changes induced in lens alpha- and gamma-crystallins by modification with glucose 6-phosphate. Implications for cataract.

There is good evidence that the non-enzymic chemical modification of proteins plays a role in the aetiology of cataract and diabetic sequelae. This paper presents new evidence that glycosylation of two major lens structural crystallins, alpha- and gamma-crystallins, by glucose 6-phosphate (G6P) induces conformational changes in the proteins. In addition the surface charge on the molecules is altered. These changes would affect protein-protein and protein-water interactions within the lens and could lead to disruption of the short-range order of the lens proteins which is essential for lens transparency. Conformational changes to lens proteins are known to occur in human cataractous lenses but their cause in vivo is not established. Cumulative chemical modification of proteins, over a period of decades, is a strong candidate as a causal agent.