Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae).

A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.     

Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis.

The dynamic mechanical properties of lung tissue and its contents of collagen and elastic fibers were studied in strips prepared from mice instilled intratracheally with saline (C) or silica [15 (S15) and 30 days (S30) after instillation]. Resistance, elastance, and hysteresivity were studied during oscillations at different frequencies on S15 and S30. Elastance increased from C to silica groups but was similar between S15 and S30. Resistance was augmented from C to S15 and S30 and was greater in S30 than in S15 at higher frequencies. Hysteresivity was higher in S30 than in C and S15. Silica groups presented a greater amount of collagen than did C. Elastic fiber content increased progressively along time. This increment was related to the higher amount of oxytalan fibers at 15 and 30 days, whereas elaunin and fully developed elastic fibers were augmented only at 30 days. Silicosis led not only to pulmonary fibrosis but also to fibroelastosis, thus assigning a major role to the elastic system in the silicotic lung.     

Sensitization of mice with wild-type and cold-adapted influenza virus variants: immune response to two H1N1 and H3N2 viruses

Two A strain influenza viruses, A/Hong Kong/123/77 (A/HK/123/77) (H1N1) and A/Queensland/6/72 (A/Qld/6/72) (H3N2), and the two cold-adapted reassortants which possess the surface antigens of these strains (CR35 and CR6, respectively) were tested for their ability both to induce primary cytotoxic T-cell (Tc cell) responses in mice and to sensitize mice for a second Tc cell response when challenged with a distantly related A strain virus, A/Shearwater/72 (H6N5). After intranasal inoculation, A/Qld/6/72 replicated to higher titers in the lung (1 to 2 log10 50% egg infective doses) than did A/HK/123/77 or either of the reassortants. A/Qld/6/72 induced higher Tc cell responses in the lung than did CR6, and both were more effective than either A/HK/123/77 or CR35 in this respect. When similar doses (10 or 10(3) hemagglutinin units) of each virus were injected intravenously into mice and the spleens were tested for Tc cell activity 6 days later, both A/Qld/6/72 and CR6 were ca. 100-fold better at inducing a primary Tc cell response than A/HK/123/77 or CR35. In contrast, the H1N1 and H3N2 viruses gave rather similar anti-hemagglutinin antibody titers (after intravenous injection) and delayed-type hypersensitivity reactions (after subcutaneous injection). If mice were primed with a low dose of these viruses (10(4) 50% egg infective doses intranasally), A/Qld/6/72 and CR6 were more effective than A/HK/123/77 or CR35 at sensitizing for a secondary Tc cell response when challenged with A/Shearwater/72, but if larger doses were given either intranasally (10(6) 50% egg infective doses) or intravenously (10 to 10(3) hemagglutinin units), all viruses sensitized the mice equally well, despite the fact the A/Shearwater/72 gives a poor primary Tc cell response in mice. Thus, the viral glycoprotein antigens can be important in determining the immunogenicity of the virus and, particularly, the class I antigen-restricted Tc cell response of the host.     

Ca2+ channel antagonist actions in bladder smooth muscle: comparative pharmacologic and [3H]nitrendipine binding studies

The actions of a series of 15 Ca2+ channel antagonists including D-600, nifedipine, and diltiazem were examined against K+ depolarization and muscarinic receptor induced responses in guinea pig bladder smooth muscle. Responses of bladder are very dependent upon extracellular Ca2+ and sensitive to the Ca2+ channel antagonists, the tonic component more than the phasic component of response. Regardless of stimulant, K+ or methylfurmethide (MF), or component of response, the same rank order of antagonist activities is expressed, suggestive of a single structure-activity relationship and the existence of a single category of binding site which may, however, exist in several affinity states. High affinity binding of [3H]nitrendipine (KD = 1.1 X 10(-10) M) occurs in bladder membranes, and similar high affinity binding was found in microsomal preparations from other smooth muscles including guinea pig and rat lung, rat vas deferens, uterus, and stomach. [3H]nitrendipine binding in the bladder was sensitive to displacement by other 1,4-dihydropyridines, paralleling their pharmacologic activities and showing excellent agreement with binding data previously obtained for guinea pig ileal smooth muscle. Comparison of pharmacologic data for inhibition of K+- and MF-induced responses by a common series of Ca2+ channel antagonists in bladder and ileum revealed excellent correlations. Neither pharmacologic nor binding studies suggest significant differences in Ca2+ channel antagonist properties in smooth muscle from bladder and intestine.     

Protein metal-binding sites.

Metal ions have a role in a variety of important functions in proteins including protein folding, assembly, stability, conformational change, and catalysis. The presence or absence of a given metal ion is crucial to the conformation or activity of over one third of all proteins. Recent developments have been made in the understanding and design of metal-binding sites in proteins, an important and rapidly advancing area of protein engineering.     

Maleylated-BSA suppresses IFN-gamma-mediated Ia expression in murine peritoneal macrophages

Maleylated bovine serum albumin (maleyl-BSA) and other polyanionic polymers that are recognized by cell surface receptors on macrophages have been shown to induce chemotaxis, protease secretion, and tumoricidal function in this cell type. In this paper the effect of maleyl-BSA on Ia antigen expression has been evaluated. In a fashion similar to LPS, maleyl-BSA suppressed IFN-gamma-induced expression of Ia in a time- and dose-dependent manner. Also like LPS, maleyl-BSA stimulated the production and secretion of substantial amounts of PGE2 over a 24-hr period. This did not, however, appear to be the primary mechanism by which expression of Ia was suppressed, because co-treatment of the cells with indomethacin, which totally inhibited the production of PGE2, only minimally affected the suppressive activity. Surprisingly, the suppressive activity of both maleyl-BSA and LPS could be largely abrogated by co-treatment of the cells with cyclohexamide during the time period when Ia expression was sensitive to suppression. This effect was selective in that PGE2- or dibutyryl cyclic AMP-induced suppression of Ia expression was not affected by cyclohexamide treatment. The data support the concept that there are multiple molecular mechanisms involved in the negative regulation of IFN-gamma-induced Ia expression in macrophages. Such mechanisms may include, in addition to the synthesis of PGE2 and consequent elevation in intracellular levels of cyclic AMP, one or more proteins made early after treatment with either maleyl-BSA or LPS. Thus the function of some of these early gene products may be to regulate expression of functional genes such as that encoding Ia antigen.     

Interrelation of the changes in the blood rheological properties and the microcirculatory disorders in the early and late stages of experimental hyperlipoproteinemia

Experiments were made on rabbit fed an atherogenic diet (0.5 g/kg cholesterol) singly for 15 and 24 h and repeatedly for 3, 9 and 30 days. At early stages of lipid metabolism distress the interrelationship was established between blood rheological disorders and microcirculatory abnormalities. The dependence of the initial reaction of some rheological characteristics on their initial level was marked.