Proteolytic inactivation of alpha-1-anti-chymotrypsin. Sites of cleavage and generation of chemotactic activity.

The effect of several microbial and mammalian proteinases on the inhibitory activity of human plasma alpha-1-anti-chymotrypsin (alpha-1-Achy) has been tested. Most of these enzymes caused rapid inactivation of the inhibitor by cleavage at single sites within the reactive-site loop between P5 Lys and P3' Leu, with additional cleavages also being detected in some cases near the NH2 terminus of the native protein. In contrast, two of the enzymes tested failed to inactivate alpha-1-Achy, although they could cause removal of peptides near the NH2 terminus. Studies of neutrophil chemotaxis revealed that native or NH2-terminally truncated alpha-1-Achy was not stimulatory. However, testing of two enzymatically inactivated forms of the inhibitor (alpha-1-Achy), cleaved at widely different positions within the reactive-site loop, indicated that they had become potent chemoattractants at concentrations within the nanomolar range. Competition studies using proteolytically inactivated alpha-1-proteinase inhibitor suggested that the chemotactic activity of the two inactivated serpins was probably mediated by the same mechanism. The physiological relevance of this chemotactic activity is underscored by the results of plasma elimination studies which indicate that alpha-1-Achy is eliminated at approximately the same rate as native alpha-1-Achy, thus prolonging chemotactic stimuli within the tissues.     

Polymorphism,genetic exchange and intragenic recombination of the aureolysin gene among <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>strains

Background  

Staphylococcus aureus expresses several proteases, which are thought to contribute to the virulence of this bacterium. Here we focus on aureolysin, the major thermolysin-like metalloprotease. Despite the importance of aureolysin in the physiology and pathogenesis of S. aureus, relatively little information was so far available concerning the aur gene diversity and mobility within and between the major subdivisions of the S. aureus population. Therefore, an epidemiologically and genetically diverse collection of S. aureus strains was used to determine the range of aureolysin (aur) gene polymorphism.     

α1-Antichymotrypsin inactivates staphylococcal cysteine protease in cross-class inhibition

Staphylococcal cysteine proteases are implicated as virulence factors in human and avian infections. Human strains of Staphylococcus aureus secrete two cysteine proteases (staphopains A and B), whereas avian strains express staphopain C (ScpA2), which is distinct from both human homologues. Here, we describe probable reasons why the horizontal transfer of a plasmid encoding staphopain C between avian and human strains has never been observed. The human plasma serine protease inhibitor α₁-antichymotrypsin (ACHT) inhibits ScpA2. Together with the lack of ScpA2 inhibition by chicken plasma, these data may explain the exclusively avian occurrence of ScpA2. We also clarify the mechanistic details of this unusual cross-class inhibition. Analysis of mutated ACHT variants revealed that the cleavage of the Leu383-Ser384 peptide bond results in ScpA2 inhibition, whereas hydrolysis of the preceding peptide bond leads to ACHT inactivation. This evidence is consistent with the suicide-substrate-like mechanism of inhibition.     

Solanesyl Diphosphate Synthase,an Enzyme of the Ubiquinone Synthetic Pathway,Is Required throughout the Life Cycle of Trypanosoma brucei

Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target.     

Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10 ng/ml) and IL-6 (10 ng/ml). After 24 h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera.     

The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

The use of liposuction to contour cherubism

Yet another application of suction lipectomy equipment is presented to remove the pathologic tissue in cherubism. Owing to the variations of consistency of the material in this condition, this technique may not be successful in all patients, but it is certainly recommended when indicated.     

Isolation of nine human plasma proteinase inhibitors by sequential affinity chromatography.

Purification of nine plasma proteinase inhibitors and one zymogen from a single batch of human plasma, using affinity chromatography has been accomplished. Those isolated were plasminogen (lysine-Sepharose), alpha-2-antiplasmin (plasminogen-Sepharose), high and low molecular weight kininogens (CM-papain-Sepharose), alpha-2-macroglobulin (Zn++ chelate-Sepharose), alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, Cl-inhibitor, inter-alpha-trypsin inhibitor (Blue-Sepharose) and antithrombin III (heparin-Sepharose). Alpha-2-macroglobulin and alpha-1-proteinase inhibitor required gel filtration as additional purification steps. Each protein was recovered in both high yield and purity.