Protection of transforming growth factor β activity by heparin and fucoidan

The transforming growth factor-β (TGF-β) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-β activity in vitro or in vivo. Our previous work indicated that (1) TGF-β1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and (2) heparin, and certain other polyanions, could block the binding of TGF-β1 to α₂-macroglobulin (α₂-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-β1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between ₁₂₅I-TGF-β1 and activated α₂-M. Electrophoresis of ₁₂₅I-TGF-β1 showed that fucoidan protects TGF-β1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-β derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-β, and purified TGF-β1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of ₁₂₅I-TGF-β1 and doubled the amount of cell-associated ₁₂₅I-TGF-β1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-β activity. © 1994 wiley-Liss, Inc.     

Exposure of cryptic domains in the alpha 1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that alpha1:Ser(2091)-Arg(2108), a peptide derived from the alpha1-chain of laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that alpha1:Ser(2091)-Arg(2108) is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparin-binding fragments stimulated macrophages' proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the alpha1-chain including alpha1:Ser(2091)-Arg(2108) and the globular domain. A peptide from the first loop of the globular domain (alpha1:Ser(2179)-Ser(2198)) triggered the phosphorylation of MAPK(erk1/2) and stimulated the expression of macrophage urokinase type plasminogen activator and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of alpha1-chain and stimulated macrophages' proteinase expression. Based on these data, we conclude that cryptic domains in the COOH-terminal portion of the alpha1-chain of laminin are exposed by proteolysis and stimulate macrophages' proteinase expression.     

Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPK(erk1/2) leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E(2) synthesis. The addition of PGE(2) to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE(2) and MMP-9 expression by elicited COX-2(-/-) macrophages is markedly reduced when compared with the response of either COX-2(+/-) or COX-2(+/+) macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.     

The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria

Mutations in mitochondrial small subunit ribosomal proteins MRPS16 or MRPS22 cause severe, fatal respiratory chain dysfunction due to impaired translation of mitochondrial mRNAs. The loss of either MRPS16 or MRPS22 was accompanied by the loss of most of another small subunit protein MRPS11. However, MRPS2 was reduced only about 2-fold in patient fibroblasts. This observation suggests that the small ribosomal subunit is only partially able to assemble in these patients. Two large subunit ribosomal proteins, MRPL13 and MRPL15, were present in substantial amounts suggesting that the large ribosomal subunit is still present despite a non-functional small subunit.     

Impact of anaerobic glycolysis and oxidative substrate selection on contractile function and mechanical efficiency during moderate severity ischemia

The role of anaerobic glycolysis and oxidative substrate selection on contractile function and mechanical efficiency during moderate severity myocardial ischemia is unclear. We hypothesize that 1) preventing anaerobic glycolysis worsens contractile function and mechanical efficiency and 2) increasing glycolysis and glucose oxidation while inhibiting free fatty acid oxidation improves contractile function during ischemia. Experiments were performed in anesthetized pigs, with regional ischemia induced by a 60% decrease in left anterior descending coronary artery blood flow for 40 min. Three groups were studied: 1) no treatment, 2) inhibition of glycolysis with iodoacetate (IAA), or 3) hyperinsulinemia and hyperglycemia (HI + HG). Glucose and free fatty acid oxidation were measured using radioisotopes and anaerobic glycolysis from net lactate efflux and myocardial lactate content. Regional contractile power was assessed from left ventricular pressure and segment length in the anterior wall. We found that preventing anaerobic glycolysis with IAA during ischemia in the absence of alterations in free fatty acid and glucose oxidation did not adversely affect contractile function or mechanical efficiency during myocardial ischemia, suggesting that anaerobic glycolysis is not essential for maintaining residual contractile function. Increasing glycolysis and glucose oxidation with HI + HG inhibited free fatty acid oxidation and improved contractile function and mechanical efficiency. In conclusion, these results show a dissociation between myocardial function and anaerobic glycolysis during moderate severity ischemia in vivo, suggesting that metabolic therapies should not be aimed at inhibiting anaerobic glycolysis per se, but rather activating insulin signaling and/or enhancing carbohydrate oxidation and/or decreasing fatty acid oxidation.     

Peroxomanganese complexes as an aid to understanding redox-active manganese enzymes

Over the past 7 years, there have been a significant number of studies describing the structural and electronic properties, as well as the chemical reactivity, of synthetic peroxomanganese adducts. Many redox-active manganese enzymes, including manganese-containing superoxide dismutases, extradiol catechol dioxygenases, and ribonucleotide reductases, are proposed to feature peroxomanganese intermediates in their catalytic cycles. The recent efforts to model these intermediates using synthetic complexes have thus provided a strong complement to mechanistic studies of the enzymes. This review provides both a summary and a perspective of work in this area, with an emphasis on the relationship between geometric and electronic structure and chemical reactivity for η²-peroxomanganese(III) and η¹-alkylperoxomanganese(III) adducts.     

Changes in stroke volume with beta -blockade before and after 10days of exercise training in men and women

Mier, Constance M., Melissa A. Domenick, and Jack H. Wilmore. Changes in stroke volume with -blockade before andafter 10 days of exercise training in men and women.J. Appl. Physiol. 83(5):1660-1665, 1997. We sought to determine whether 10 days oftraining would be a sufficient stimulus for cardiac adaptations thatwould allow a greater compensatory stroke volume during -blockade. We also sought to determine whether men and women had a similar cardiacreserve capacity for increasing stroke volume with -blockade duringsubmaximal exercise. Eight men (age 29 ± 2 yr, mean ± SE) andeight women (25 ± 2 yr) cycled at 65% of peakO₂ consumption (unblocked) underplacebo-control and -blockade (100 mg atenolol) conditions performedon separate days. These tests were repeated at the same power outputafter training (10 consecutive days, 1 h of cycling per day). Beforetraining, -blockade significantly (P < 0.05) decreased heartrate (HR) and cardiac output and increased stroke volume in both menand women. After training, the increase in stroke volume and decreasein HR with -blockade was significantly less while cardiac output wasreduced more. There were no gender differences in the effects of-blockade on HR, stroke volume, or cardiac output. These dataindicate that, during exercise with -blockade, exercise training for10 days does not enhance the compensatory increase in stroke volume andthat men and women have a similar cardiac reserve capacity forincreasing stroke volume.

     

Trisporic Acid Biosynthesis in Separate Plus and Minus Cultures of Blakeslea trispora: Identification by Mucor Assay of Two Mating-Type-Specific Components

Separate plus and minus cultures of Blakeslea trispora synthesize small amounts of trisporic acids under specific conditions. These amounts are expressed as a percentage of the trisporic acids (50 mg/liter of medium) synthesized by mixed plus-minus cultures in 5 days. Plus cultures, without additives from minus cultures, synthesize 0.1% trisporic acids. Plus cultures synthesize 0.4% trisporic acids when stimulated by M-factor, a mating-type-specific component synthesized by minus cultures. Minus cultures, without additives from plus cultures, do not synthesize even 0.0001% trisporic acids. Minus cultures synthesize 1% trisporic acids when stimulated by P-factor, a mating-type-specific component synthesized by plus cultures. Minus cultures synthesize M-factor when stimulated by pi, a component synthesized by plus cultures. We speculate that (i) minus cultures synthesize a component, mu, which stimulates P-factor synthesis in plus cultures, and (ii) both M-factor and P-factor are precursors of trisporic acids.     

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt

Mammalian mitochondrial initiation factor 3 (IF3_mt) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3_mt to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K_d for the binding of IF3_mt to 28S subunits is ~30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3_mt has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K_d = 1.5 μM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K_d = 0.12−0.25 μM). IF3_mt does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3_mt is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3_mt from the 28S subunits upon 39S subunit joining.