The role of histidyl residues in zinc-induced precipitation of alpha 2-macroglobulin-proteinase complexes

When alpha 2-macroglobulin (alpha 2M) is reacted with proteinases including trypsin, plasmin, alpha-thrombin, or with CH3NH2, each resulting alpha 2M derivative is precipitated by Zn2+ in a similar manner. By contrast, unreacted alpha 2M is not precipitated over the same Zn2+ concentration range. Zn2+-induced precipitation of alpha 2M-CH3NH2 or alpha 2M-trypsin is prevented by acylation of the protein employing the histidine-specific reagent diethylpyrocarbonate (DEP). The Zn2+-induced precipitation of alpha 2M-trypsin is prevented by acylation of the preformed alpha 2M-trypsin complex or by the reaction of acylated native alpha 2M with trypsin. Acylation of alpha 2M by treatment with DEP results in the modification of 13.5 histidyl residues per subunit of either native alpha 2M or alpha 2M-CH3NH2. Subsequent treatment with hydroxylamine reverses the modification of 10.5 histidyl residues per subunit in each protein preparation. These results indicate that histidyl residues are involved in the Zn2+-induced precipitation of alpha 2M-proteinase or alpha 2M-CH3NH2 complexes, and that these residues are accessible to extensive protein-metal interactions only after alpha 2M has undergone a major conformational change. These appear to be the same histidyl residues which, upon acylation by DEP, are responsible for recognition of alpha 2M-proteinase complexes by the acyl-low-density-lipoprotein cell surface receptor (S. V. Pizzo, P. A. Roche, S. R. Feldman, and S. L. Gonias (1986) Biochem. J. 238, 217-225).     

cis-dichlorodiammineplatinum (II) as a selective modifier of the oxidation-sensitive reactive-center methionine in alpha 1-antitrypsin

Methionine 358 in the plasma protein alpha 1-antitrypsin (alpha 1AT) is an oxidation-sensitive reactive-center residue critical for proteinase-inhibitory activity. Reaction of alpha 1AT with 20 microM to 1.67 mM cis-dichlorodiammineplatinum (II) (cis-DDP) or trans-DDP afforded concentration-dependent loss of trypsin-inhibitory activity. This effect, studied by gel electrophoresis and activity assays, is essentially independent of pH over the range 4.9-8.6. Binding assays showed covalent incorporation of 1 mol of cis-DDP into each mol of alpha 1AT. cis-DDP protected a single methionine residue from oxidation and made alpha 1AT resistant to degradation by papain, which cleaves alpha 1AT at Met358. These findings strongly suggest that cis-DDP inactivates alpha 1AT by binding exclusively to its reactive-center methionine. alpha 1AT bound twice as much platinum when reacted with trans-DDP. Because carboxamidomethylated alpha 1AT incorporated nearly 1 mol of both cis- and trans-DDP, the trans isomer apparently binds to both the reactive-center methionine and to the single cysteine residue of alpha 1AT. Because of its greater selectivity, cis-DDP is the superior reagent for modification of the alpha 1AT reactive-center methionine.     

Analysis of inter-alpha-trypsin inhibitor and a novel trypsin inhibitor, pre-alpha-trypsin inhibitor, from human plasma. Polypeptide chain stoichiometry and assembly by glycan

The polypeptide chain composition of protein material referred to in the literature as "inter-alpha-trypsin inhibitor" was investigated. The material was found to consist of distinct proteins of 125,000 and 225,000 Da, each of which contained more than one polypeptide chain. The links that assemble each protein were found to be stable to various strong denaturants, but susceptible to treatment with trifluoromethanesulfonic acid or hyaluronidase, indicating a glycan nature. The 225,000-Da protein migrated with inter-alpha mobility on agarose gel electrophoresis and is designated inter-alpha-trypsin inhibitor, whereas the 125,000-Da protein migrated with pre-alpha mobility, and we designate it pre-alpha-trypsin inhibitor. Analysis of the proteins, the separated chains, and proteolytic derivatives thereof revealed that each protein contained a single, identical, trypsin-inhibitory chain of 30,000 Da. Inter-alpha-trypsin inhibitor contains noninhibitory heavy chains of 65,000 and 70,000 Da, whereas pre-alpha-trypsin inhibitor contains a heavy chain of 90,000 Da. Our data allow identification of several recently reported cDNA clones and clarify the confusion surrounding the composition of plasma proteins referred to as inter-alpha-trypsin inhibitor.     

Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha 2-macroglobulin.

The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.     

Anti-Inflammatory and Vasoprotective Activity of a Retroviral-Derived Peptide,Homologous to Human Endogenous Retroviruses: Endothelial Cell Effects

Malignant and inflammatory tissues sometimes express endogenous retroviruses or their proteins. A highly-conserved sequence from retroviral transmembrane (TM) proteins, termed the “immunosuppressive domain (ID)”, is associated with inhibition of immune and inflammatory functions. An octadecapeptide (MN10021) from the ID of retroviral TM protein p15E inhibits in vitro release of pro-inflammatory cytokines and increases synthesis of anti-inflammatory IL-10. We sought to determine if MN10021 has significant in vivo effects. MN10021, prepared by solid-phase synthesis, was dimerized through a naturally-occurring, carboxy-terminal cysteine. In vivo anti-inflammatory activity was determined using a murine model of sodium periodate (NaIO₄)-induced peritonitis. In vivo vasoprotective effects were determined using: (1) a carrageenan-induced model of disseminated intravascular coagulation (DIC) in mice; (2) a reverse passive Arthus model in guinea pigs; and (3) vasoregulatory effects in spontaneously hypertensive rats (SHR). In vitro studies included: (1) binding/uptake of MN10021 using human monocytes, cultured fibroblasts, and vascular endothelial cells (VEC); (2) gene expression by RT-PCR of MN10021-treated VEC; and (3) apoptosis of MN10021-treated VEC exposed to staurosporine or TNF-α. One-tenth nmol MN10021 inhibits 50 percent of the inflammatory response in the mouse peritonitis model. Furthermore, 73 nmol MN10021 completely protects mice in a lethal model of carrageenan-induced DIC and inhibits vascular leak in both the mouse DIC model and a guinea pig reverse passive Arthus reaction. MN10021 binds to and is taken up in a specific manner by both human monocytes and VEC but not by cultured human fibroblasts. Surprisingly, orally-administered MN10021 lowers blood pressure in SHR rats by 10–15% within 1 h suggesting a direct or indirect effect on the vascular endothelium. MN10021 and derived octapeptides induce iNOS (inducible nitric oxide synthase) mRNA in VEC and nitrate in VEC cell culture supernatants and protect VEC from induced apoptosis or necrosis. However, pretreatment of VEC with nitro-L-arginine methyl ester (L-NAME), while inhibiting the release of nitrate, does not block the anti-apoptotic effect of MN10021 and derived octapeptides suggesting that their potent vasoprotective and anti-inflammatory activity is not nitric oxide dependent.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Mitochondria, calcium and cell death: A deadly triad in neurodegeneration

Mitochondrial Ca²⁺ accumulation is a tightly controlled process, in turn regulating functions as diverse as aerobic metabolism and induction of cell death. The link between Ca²⁺ (dys)regulation, mitochondria and cellular derangement is particularly evident in neurodegenerative disorders, in which genetic models and environmental factors allowed to identify common traits in the pathogenic routes. We will here summarize: i) the current view of mechanisms and functions of mitochondrial Ca²⁺ homeostasis, ii) the basic principles of organelle Ca²⁺ transport, iii) the role of Ca²⁺ in neuronal cell death, and iv) the new information on the pathogenesis of Alzheimer's, Huntington's and Parkinson's diseases, highlighting the role of Ca²⁺ and mitochondria.     

Human CD34+/CD90+ ASCs Are Capable of Growing as Sphere Clusters,Producing High Levels of VEGF and Forming Capillaries

Background

Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.

Methodology/Principal Findings

In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34⁺/CD90⁺ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34⁺/CD90⁺ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31⁺/VEGF⁺/Flk-1⁺) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.

Conclusions/Significance

Our results demonstrate, for the first time, that CD34⁺/CD90⁺ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.