We used cardiopulmonary bypass (CPB) as a model of activation of the contact system and investigated the involvement of the plasma and tissue kallikrein-kinin systems (KKS) in this process. Circulating levels of bradykinin and kallidin and their metabolites, plasma and tissue kallikrein, low and high molecular weight kininogen, and kallistatin were measured before, during, and 1, 4, and 10 h after CPB in subjects undergoing cardiac surgery. Bradykinin peptide levels increased 10- to 20-fold during the first 10 min, returned toward basal levels by 70 min of CPB, and remained 1.2- to 2.5-fold elevated after CPB. Kallidin peptide levels showed little change during CPB, but they were elevated 1.7- to 5.2-fold after CPB. There were reductions of 80 and 60% in plasma and tissue kallikrein levels, respectively, during the first minute of CPB. Kininogen and kallistatin levels were unchanged. Angiotensin-converting enzyme inhibition did not amplify the increase in bradykinin levels during CPB. Aprotinin administration prevented activation of the KKS. The changes in circulating kinin and kallikrein levels indicate activation of both the plasma and tissue KKS during activation of the contact system by CPB.

Profound screening and evaluation methods for biocide-releasing polymer films are crucial for predicting applicability and therapeutic outcome of these drug delivery systems. For this purpose, we developed an agar overlay assay embedding biopolymer composite films in a seeded microbial lawn. By combining this approach with model-dependent analysis for agar diffusion, antimicrobial potency of the entrapped drug can be calculated in terms of minimum inhibitory concentrations (MICs). Thus, the topical antiseptic 4-hexylresorcinol (4-HR) was incorporated into poly(lactic-co-glycolic acid) (PLGA) films at different loadings up to 3.7 mg/cm2 surface area through a solvent casting technique. The antimicrobial activity of 4-HR released from these composite films was assessed against a panel of Gram-negative and Gram–positive bacteria, yeasts and filamentous fungi by the proposed assay. All the microbial strains tested were susceptible to PLGA-4-HR films with MIC values down to 0.4% (w/w). The presented approach serves as a reliable method in screening and quantifying the antimicrobial activity of polymer composite films. Moreover, 4-HR-loaded PLGA films are a promising biomaterial that may find future application in the biomedical and packaging sector.

Secretory leukoprotease inhibitor (SLPI) is one of the major physiological inhibitors protecting respiratory epithelium from attack by excess human leukocyte elastase (HLE), a serine protease released by neutrophils upon activation in response to inflammatory stimuli. Reaction with N-chlorotaurine, a major long-lived oxidant generated by activated neutrophils, oxidized all four methionine residues, but no other amino acids, in SLPI, resulting in substantial diminution of its elastase inhibitory activity. Oxidation of the P1' residue, Met73, accounted for most of the diminution in activity since a site-directed mutant of SLPI with leucine at the P1' position retained much higher residual activity after reaction with N-chlorotaurine. The diminished activity of oxidized SLPI could be almost completely restored when an iduronate-containing glycosaminoglycan, such as heparin, heparan sulfate, or dermatan sulfate, was added to the reaction medium. Addition of a sulfated glucuronate-containing glycosaminoglycan, chondroitin 4- or 6-sulfate, to the medium resulted in smaller but significant restoration of the lost activity, whereas the effects of hyaluronic acid and keratan sulfate were negligible. Kinetic analysis revealed that glycosaminoglycans greatly accelerated the association of oxidized SLPI and HLE, whereas iduronate-containing glycosaminoglycans also stabilized the enzyme-inhibitor complex formed. Based on these findings, we suggest that oxidized SLPI is a functionally active form of the inhibitor but that expression of its elastase inhibitory activity is regulated by sulfated uronate-containing glycosaminoglycans. Because its methionine residues have already been oxidized, this form of SLPI is resistant to the oxidant species that selectively attacks methionine residues in proteins. These findings indicate that SLPI may play a previously unexpected role in elastase inhibitory function in the lungs when significant inflammation is present.

The seminal vesicles synthesize in an androgen-dependent manner a neutral protein of 13.5 kDa molecular weight that makes up about 40% of their secretion ("major protein"). An antiserum against this protein raised in rabbits was used to localize the antigen within the seminal vesicles. In addition to intraluminal secretion of the seminal vesicles and the ampulla of the vas deferens, ejaculated and ampullary spermatozoa revealed an intense immunoreaction, which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Comparison of spermatozoa taken from the tail of the epididymis with ampullary spermatozoa showed that about 90% of the latter, but only 10-20% of the former presented this distributional pattern of immunoreactive sites. Epididymal epithelium as well as calf seminal vesicle epithelium showed no immunoreactivity with major protein antiserum. Using a pre-embedding staining technique with gold-labeled primary or secondary antibodies, respectively, no immunostaining could be achieved at the ultrastructural level. Incubation experiments of epididymal spermatozoa in EGTA-containing solutions in the absence of calcium resulted in a gradual labilization and eventual loss of the plasma membrane of the sperm middle piece. After removal of (at least part of) the plasma membrane, bound major protein could be visualized immunohistochemically close to the mitochondria of the middle piece using a gold-labeled primary or secondary antibody. The acceptor site for major protein therefore seems to reside inside the plasma membrane of the sperm middle piece.(ABSTRACT TRUNCATED AT 250 WORDS)