Proteolytic Damage Research Articles

LIVER INJURY IN ALPHA 1-ANTITRYPSIN DEFICIENCY

Alpha 1-antitrypsin deficiency is the most common genetic cause of liver disease in children. It is also associated with chronic liver disease, hepatocellular carcinoma, and pulmonary emphysema in adults. Liver injury is caused by hepatotoxic effects of retention of the mutant alpha 1-antitrypsin molecule within the endoplasmic reticulum of liver cells, and emphysema is caused by uninhibited proteolytic damage to elastic tissue in the lung parenchyma. Recent studies of the biochemistry and cell biology of the mutant alpha 1-antitrypsin molecule have led to advances in understanding susceptibility to liver injury and in developing new strategies for prevention of both liver and lung disease.

Amniotic Membrane Patching Promotes Healing and Inhibits Proteinase Activity on Wound Healing Following Acute Corneal Alkali Burn

Amniotic membrane (AM) contains basement membrane components and various proteinase inhibitors. Furthermore, when used as a graft, the basement membrane of AM could block inflammatory insults to a damaged corneal surface. Thus, we evaluated whether amniotic membrane patching could promote the healing process by inhibiting proteolytic damage. Alkali wounds were inflicted on the central corneas of rabbits by applying a round filter paper, 6.0 mm in diameter, soaked in 1 N NaOH for 30 sec. Amniotic membrane patching was performed over the perilimbal sclera immediately after wounding. A total of 115 rabbits were divided into four groups: (1) immediately covered by AM with the amnion cell side down up to the perilimbal sclera (n =26); (2) covered by AM with the stromal side down up to the perilimbal sclera (n =19); (3) anchored to the fornix (n =29); and (4) uncovered as a control (n =41). AM was removed 3 days postoperatively. During follow-ups, epithelial defects, corneal thickness and its opacity of each eye were measured. Some corneas were removed for histopathologic studies and for proteinase activity assay and zymography. The epithelial healing was faster and the corneal thickness was thicker in all three AM-covered groups than in the control (P<0.05). No significant difference was found between covered and anchored groups (P>0.05). Corneal opacity was least in the amnion cell side down group. Infiltration of polymorphonuclear leukocytes (PMNs) was much less in AM-covered groups than in the control. Pathological results were associated with zymographic findings, which revealed much higher proteinase activity in uncovered group than AM-covered groups. Immediate intervention for acute alkali burns with AM as a temporary patch promotes wound healing by inhibiting proteinase activity and PMNs infiltration.

Therapy for ??1-Antitrypsin Deficiency

α1-Antitrypsin (A1AT) deficiency is inherited as an autosomal codominant disorder characterised by reduced levels of A1AT in the serum. Low levels of A1AT in blood perfusing the lung cause low levels in the lung interstitium, making it susceptible to proteolytic damage from resident neutrophil elastase. A ‘protective threshold’ serum A1AT level of 11 μmol/L has been identified by epidemiological studies as a minimum value below which there is an increased risk of emphysema. Intravenous augmentation therapy for patients with severe deficiency of A1AT has been shown to have biochemical efficacy. Although the clinical efficacy of intravenous augmentation therapy has not been demonstrated in a randomised clinical trial, available studies suggest that augmentation therapy is associated with a slowed rate of decline of lung function and enhanced survival. The criteria for patient selection include: age >18 years, serum A1AT level ≤11 μmol/L, a high-risk phenotype (usually PI*ZZ), and documented fixed airflow obstruction (consistent with chronic obstructive pulmonary disease). Although intravenous augmentation is currently the only form of specific therapy approved in the US, active research in the fields of aerosol and gene therapy promise to offer new treatment prospects. In this article, we review the available literature on A1AT augmentation therapy and discuss our recommendations.

Production of Fab Fragments of Monoclonal Antibodies Using Polyelectrolyte Complexes

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen–polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen–polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen–polyelectrolyte complex was less damaging, suggesting that binding to the antigen–polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.

Functional protective role for mucin glycosylated repetitive domains.

Mucins carry out a number of protective roles, some of which are more easily studied than others. One mucin function is believed to be the protection of the mucosal epithelium against acidic and proteolytic damage in the stomach and intestines. In the present work, a portion of stomach mucin tandem repeat sequence (Muc6) was joined to the catalytic domain of a reporter enzyme [human milk cholesterol esterase (CE)] to determine whether the former can protect the latter protein from damage. This Muc6 domain replaced a unique series of glycosylated C-terminal repeats normally present in CE. The chimeric protein (CE/Muc6) was expressed in two different cell lines and its properties compared to recombinant full-length CE and a truncated version of CE which contained only the catalytic domain (CE/trunc). Results showed that both CE and CE/Muc6 were resistant to denaturation by acid and to proteolysis by pepsin at low pH values or by pancreatic proteases compared to CE/trunc. Thus, a stomach Muc6 domain is sufficient to confer stability on the CE catalytic domain, demonstrating a protective effect by a glycosylated mucin sequence.

Functionally inactive protein C inhibitor in seminal plasma may be associated with infertility.

Protein C inhibitor (PCI) has been found in seminal plasma and is considered to protect intact surrounding cells and seminal plasma proteins from possible proteolytic damage. In the present study, we showed that although the antigenic levels of PCI in two seminal plasma samples from patients with infertility were normal or slightly elevated, their inhibitory activities toward urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) were absent. In contrast, uPA and tPA proteolytic activities in these two samples were 20-60-fold higher than that from normal volunteers. A time-course analysis of PCI-uPA complex formation showed that >80% of the complex had been formed within 15 min in normal seminal plasma in the presence of heparin, compared with the total complex formed after 150 min incubation, whereas no response to heparin stimulation was observed in the assays with the two patient samples. Similarly, >90% of PCI-tPA complex was formed after 30 min of heparin stimulation in normal seminal plasma but no response was observed in the two patient samples. Kinetic assays of PCI inhibitory function in the presence of activated protein C (APC) showed that PCI inhibitory activity in the two patient samples was absent and not stimulated by heparin. Western blotting also showed that most of the intact PCI molecules, in normal samples, formed complexes with either uPA or tPA but there was no complex formed in one of the two patient samples and very little complex was observed in the other, suggesting that PCI in the two patient samples is inactive. These results suggest that the presence of functionally inactive PCI in seminal plasma may be associated with infertility.

Molecular characterisation of the defective α1-antitrypsin alleles PI Mwürzburg (Pro369Ser), Mheerlen (Pro369Leu), and Q0lisbon (Thr68lle)

Deficiency of the serine proteinase inhibitor (serpin) alpha 1-antitrypsin (alpha 1AT) is the most common autosomal recessive genetic disorder in Northern Europe. alpha 1AT is the physiological regulator of the proteolytic enzyme neutrophil elastase and severe deficiency states are associated with an increased risk of developing chronic obstructive pulmonary disease (COPD) as a consequence of chronic proteolytic damage to the lungs. Among the known mutations of the alpha 1AT gene causing severe alpha 1AT deficiency and COPD a few alleles are also associated with liver disease. When expressed in cell cultures, all these particular alleles cause intracellular alpha 1AT accumulation which appears to be a prerequisite for the development of hepatic injury. Liver disease is seen in only a small fraction of all patients carrying such alleles, however. The reason for this is not completely clear, but there is evidence that PI ZZ individuals 'susceptible' to liver disease carry an additional defect affecting protein degradation in the endoplasmic reticulum (ER). We characterise a newly identified defective alpha 1AT allele PI Mwürzburg (Pro369 [CCC] to Ser [TCC]) associated with a complete intracellular transport block in cell cultures in vitro. The allele PI Mheerlen, a previously described different amino acid substitution in the same position as PI Mwürzburg (Pro369 [CCC] to Leu [CTC]) is shown to cause complete retention of the mutant alpha 1AT in the ER, too, whereas in the recently described mutant allele PI Q0lisbon (Thr68 [ACC] to Ile [ATC]) a significantly reduced alpha 1AT secretion from the cells was observed. Adenovirus-mediated recombinant expression of mutant Mwürzburg and Mheerlen, and of wild-type alpha 1AT in mouse liver in vivo showed that the mutant human proteins were not secreted into the mouse plasma, in contrast with human wild-type alpha 1AT which circulated at high concentrations over several weeks. In summary, all transportation deficient alpha 1ATs analysed have the potential to cause lung disease in the homozygous state or in heterozygous carriers of another deficiency allele, and they may also cause liver disease in certain patients. The mutant PI Mwürzburg and Mheerlen alpha 1ATs are completely retained within synthesising cells, and the molecular defect of transportation in these two alleles may be similar to that in the common PI Z allele. The molecular defect in the PI Q0lisbon allele (Thr68Ile) shows similarity with the immediately neighbouring Mmineral springs mutation (Gly67Glu).

Role of the Eosinophil in Protein Oxidation in Asthma: Possible Effects on Proteinase/Antiproteinase Balance

Background: Many of the leukocytes which migrate into the tissue following allergen challenge can undergo a respiratory burst producing reactive oxygen species causing tissue damage and distorting proteinase/antiproteinase balance. The reactive oxygen species have extremely short half lives and so cannot be measured in vivo, but the protein carbonyl residues which result from protein oxidation can be measured in biological fluids. Methods: We examined protein oxidation in bronchoalveolar lavage after allergen challenge in 12 patients with atopic asthma by measuring protein carbonyl residues using a sensitive Western blotting technique. Results: We found that the median level of protein carbonyls per molecule of protein rose from 0.09 in bronchoalveolar lavage (BAL) obtained 18 h after saline challenge to 0.23 in BAL taken 10 min after segmental allergen challenge, reaching a median of 0.82 in samples obtained 18 h later (p<0.01 compared with saline control and the earlier time point). The number of protein carbonyl residues correlated strongly with the number of eosinophils recovered in the BAL (rho = 0.574, p<0.05) but not with the number of neutrophils (rho = 0.228, p = NS) or macrophages (rho = 0.178, p = NS). Western blotting showed that the majority of the modified protein comigrated with authentic α₁–antitrypsin at 53 kD. In contrast, BAL samples from patients with chronic obstructive pulmonary disease, which is characterized by an influx of neutrophils, showed that the main oxidized protein colocalized with human serum albumin, the predominant protein in the BAL. Conclusion: Our results suggest that the recruitment and activation of eosinophils accounts for much of the protein oxidation found in the BAL following allergen challenge. The main oxidized protein appears to be α₁–antitrypsin, a key antiproteinase in the airways. Inactivation of α₁–antitrypsin by oxidation may distort the proteinase/antiproteinase balance leaving the lung vunerable to proteolytic damage. Intriguingly, we have evidence to suggest that other airways diseases, characterized by recruitment of other inflammatory cells, may result in the oxidation of alternative targets.

Potential regulation of cartilage metabolism in osteoarthritis by fibronectin fragments.

There are few candidates for biochemical pathways that either initiate or amplify catabolic processes involved in osteoarthritis (OA). Perhaps, one of the most likely sources for such pathways may be within the extracellular matrix itself. This review focuses on an example of how specific degradation products of the extracellular matrix of cartilage, produced during proteolytic damage, have the potential to enhance OA-like processes. In this example, these products can induce or activate other factors, such as catabolic cytokines, that amplify the damage. The damage, in turn, enhances levels of the degradation products themselves, as in a positive feedback loop. Since these products are derived from the cartilage matrix, they could be considered barometers of the health of the cartilage that signal to the chondrocyte, through outside to inside signaling, the health or status of the surrounding matrix. The best example and most characterized system is that of fragments of the matrix protein, fibronectin (Fn), although as discussed later, other recently discovered fragment systems may also have the potential to regulate cartilage metabolism. In the case of Fn fragments (Fn-fs), the Fn-fs enhance levels of catabolic cytokines as in OA and, thus, are potentially earlier damage mediators than catabolic cytokines. The Fn-fs up-regulate matrix metalloproteinase (MMP) expression, significantly enhance degradation and loss of proteoglycan (PG) from cartilage and temporarily suppress PG synthesis, all events observed in OA. However, this Fn-f system may be involved in normal cartilage homeostasis as well. For example, low concentrations of Fn-fs enhance anabolic activities and could play a role in normal homeostasis. This system may also be involved in not only amplifying damage but also coupling damage to repair. For example, high concentrations of Fn-fs that might arise in OA temporarily offset the anabolic response of lower Fn-f concentrations and cause short-term enhanced catabolic events that are followed by slowly increasing anabolic responses. Such effects would be expected for mediators with roles in regulation of metabolism in both normal or diseased cartilage. Other products of matrix degradation have also been shown to regulate cartilage metabolism. A common mechanistic theme to these systems may be that they perturb the cartilage matrix and directly or indirectly alter function of specific receptors involved in metabolism. These concepts illustrate the potential of the cartilage matrix to regulate its composition in both health and disease.

Matrix metalloproteinases in cerebrovascular disease.

Cerebral ischemia and intracerebral hemorrhage cause extensive damage to neurons, disrupt the extracellular matrix, and increase capillary permeability. Multiple substrates participate in the cellular damage, including free radicals and proteases. Matrix metalloproteinases and serine proteases are two classes of proteases that are normally present in brain in latent forms, but once activated, contribute to the injury process. These enzymes have a unique role in the remodeling of the extracellular matrix and in the modulation of the capillary permeability. Intracerebral injection of the matrix metalloproteinase, type IV collagenase, attacks the basal lamina around the capillary and opens the blood-brain barrier. Extracellular matrix-degrading proteases are induced by immediate early genes and cytokines, and regulated by growth factors. Activity of the matrix metalloproteinases is tightly controlled by activation mechanisms and tissue inhibitors of metalloproteinases. During ischemia and hemorrhage, multiple matrix metalloproteinases and serine proteases are produced along with their inhibitors. These proteolytic enzymes are involved in the delayed injury that accompanies the neuroinflammatory response. Synthetic inhibitors to metalloproteinases reduce proteolytic tissue damage, and may limit secondary neuroinflammation.

Purification of α1 Proteinase Inhibitor from Human Plasma Fraction IV‐1 by Ion Exchange Chromatography

Alpha-proteinase inhibitor (PI) protects the lungs from proteolytic damage caused by elastase and can be used to treat congenital emphysema. We describe an improved method of purification of alpha 1 PI from redissolved fraction IV-1 paste. The process used dimethylaminoethyl anion exchange chromatography, sulfopropyl cation exchange chromatography, virus inactivation by dry heat, and tri-n-butyl-phosphate/cholate treatment, followed by a second strong cation exchange chromatography. Optimizations of loading conditions for ion exchange chromatography at small scale (20-60 ml of suspension) are described. Virus inactivation was adjusted to provide the best yield of alpha 1 PI consistent with effective inactivation. The process has been effectively scaled up. The final product was approximately 90% pure by SDS-PAGE, with a 60-70% yield from starting fraction IV-1 paste. The process has been characterized by methods including nonreduced SDS-PAGE, alpha 1 PI inhibition assay, and biuret protein assay. The method described is an effective way of preparing large quantities of alpha 1 PI from fractionated plasma.

Purification of α1 Proteinase Inhibitor from Human Plasma Fraction IV-1 by Ion Exchange Chromatography

Background and Objectives: α-proteinase inhibitor (PI) protects the lungs from proteolytic damage caused by elastase and can be used to treat congenital emphysema. We describe an improved method of purification of α₁ PI from redissolved fraction IV-1 paste. Materials and Methods: The process used dimethylaminoethyl anion exchange chromatography, sulfopropyl cation exchange chromatography, virus inactivation by dry heat, and tri-n-butyl-phosphate/cholate treatment, followed by a second strong cation exchange chromatography. Optimizations of loading conditions for ion exchange chromatography at small scale (20–60 ml of suspension) are described. Virus inactivation was adjusted to provide the best yield of α₁ PI consistent with effective inactivation. The process has been effectively scaled up. Results: The final product was approximately 90% pure by SDS-PAGE, with a 60–70% yield from starting fraction IV-1 paste. The process has been characterized by methods including nonreduced SDS-PAGE, α₁ PI inhibition assay, and biuret protein assay. Conclusion: The method described is an effective way of preparing large quantities of α₁ PI from fractionated plasma.

Local control of α1-proteinase inhibitor levels: regulation of α1-proteinase inhibitor in the human cornea by growth factors and cytokines

Alpha 1-proteinase inhibitor is a major serine proteinase inhibitor in the human cornea involved in the protection of the avascular corneal tissue against proteolytic damage. This inhibitor is upregulated systemically during infection, inflammation and injury. Cytokines that mediate the acute phase response such as IL-1 β and IL-2 increased α1-proteinase inhibitor present in corneal organ culture media. This released inhibitor represented mainly newly synthesized protein. However, IL-6, a general inducer of the acute phase response that upregulates α1-proteinase inhibitor in all other tissues and cells tested, failed to alter corneal α1-proteinase inhibitor levels over the tested period of 24 h. In addition to IL-1 β and IL-2, α1-proteinase inhibitor levels in the corneal organ culture medium increased following the addition of FGF-2 and IGF-I. The effect of the above growth factors and cytokines was relatively fast with maximal induction observed within the first 5 h. Among the tested growth factors and cytokines, IL-1 β was the most potent and increased total corneal α1-proteinase inhibitor levels approximately 2.4-fold in the cornea organ culture medium. Newly, synthesized α1-proteinase secreted into the medium increased 3.9-fold. In addition to the effect on corneal α1-proteinase inhibitor, IL-1 β also increased the amount of α1-proteinase inhibitor released by monocytes and macrophages but not by HepG2, CaCo2, and MCF-7 cells within 24 h. These results suggest that the cornea can locally control levels of α1-proteinase inhibitor in response to an inflammatory insult.

Human matrix metalloprotease activation by insults of bacterial infection involving proteases and free radicals.

We found that human matrix metalloproteases (MMPs) may be processed from their proenzyme forms (proMMP) to their active forms by two new and unique mechanisms: Firstly, by bacterial proteases such as Pseudomonas elastase and Vibrio cholerae protease, which cleave off the N-terminal autoinhibitory domain (so-called cysteine switch) from proMMPs. The second mechanism depends on free radical generation by activated polymorphonuclear leukocytes (PMNs). In this case, peroxynitrite (ONOO-) or nitrogen dioxide radical (.NO2), the reaction products of either superoxide (O2.-) or molecular oxygen (O2) and nitric oxide (.NO), are the key reactants. Both O2.- and .NO are generated by activated macrophages and PMNs as a result of immunologic responses involving various proinflammatory cytokines. .NO2 or ONOO- seems to interact with a single cysteine residue in the propeptide autoinhibitory domain, or so-called cysteine switch of proMMPs, thus transforming proMMPs into their active conformation. Furthermore, reactive oxygen species are known to inactivate the alpha1-protease inhibitor (alpha1-PI), a potent neutrophil elastase inhibitor in plasma. In addition, we found that such radicals activate MMPs which degrade and inactivate alpha1-PI by proteolysis. Thus, the activation of MMPs, accompanied by the inactivation of alpha1-PI, will bring about enhanced proteolytic damage to the matrix tissues of the infected sites by both MMPs and elastase.

A novel role for the peritrophic matrix in protecting Leishmania from the hydrolytic activities of the sand fly midgut.

The role of the peritrophic matrix (PM) in the development of Leishmania major infections in a natural vector, Phlebotomus papatasi, was investigated by addition of exogenous chitinase to the bloodmeal, which completely blocked PM formation. Surprisingly, the absence of the PM was associated with the loss of midgut infections. The chitinase was not directly toxic to the parasite, nor were midgut infections lost due to premature expulsion of the bloodmeal. Most parasites were killed in chitinase-treated flies within the first 4 h after feeding. Substantial early killing was also observed in control flies, suggesting that the lack of PM exacerbates lethal conditions which normally exist in the blood-fed midgut. Early parasite mortality was reversed by soybean trypsin inhibitor. Allosamadin, a specific inhibitor of chitinase, led to a thickening of the PM, and also prevented the early parasite mortality seen in infected flies. Susceptibility to gut proteases was extremely high in transitional-stage parasites, while amastigotes and fully transformed promastigotes were relatively resistant. A novel role for the PM in promoting parasite survival is suggested, in which the PM creates a barrier to the rapid diffusion of digestive enzymes, and limits the exposure of parasites to these enzymes during the time when they are especially vulnerable to proteolytic damage.

Cytokines and the hepatic acute phase response

The acute phase response is an orchestrated response to tissue injury, infection or inflammation. A prominent feature of this response is the induction of acute phase proteins, which are involved in the restoration of homeostasis. Cytokines are important mediators of the acute phase response. Uncontrolled and prolonged action of cytokines is potentially harmful, therefore mechanisms exist which limit the activity of cytokines; these include soluble cytokine receptors and receptor antagonists. The cytokine signal is transmitted into the cell via membrane-bound receptors. Different intracellular signalling pathways are activated by different cytokine-receptor interactions. Eventually, cytokine-inducible transcription factors interact with their response elements in the promotor region of acute phase genes and transcription is induced. Systemic inflammation results in a systemic acute phase response. However, local inflammatory or injurious processes in the liver may also induce an acute phase response, for example after partial hepatectomy and during hepatic fibrosis. The acute phase proteins induced in these conditions probably act to limit proteolytic and/or fibrogenic activity and tissue damage. The possible function of the acute phase protein alpha 2-macroglobulin in hepatic fibrosis is discussed in some detail.

Rapid Screening for α1-Antitrypsin Z and S Mutations,

α1-Antitrypsin (A1AT) is a serine protease inhibitor required for the prevention of proteolytic tissue damage, principally in the lung, by neutrophil elastase released by inflammatory cells (1). While severe A1AT deficiency is the major factor leading to emphysema and related pulmonary diseases, it is also associated with neonatal hepatitis and cirrhosis (1)(2). A1AT deficiency is an autosomal codominant disorder with a prevalence of about 1:3000 in Caucasians (3). The A1AT gene has 7 exons spanning ∼12 kb. The most common gene defect resulting in A1AT deficiency is that of a protease inhibitor (PI)-system Z mutation Glu342 to Lys, which is a single base substitution of G to A in exon 5 (4)(5). The S mutation, a Glu264 to Val change, is caused by an A to T substitution in exon 3 (6). Individuals with SS are unaffected, SZ may be symptomatic, and ZZ results in the most severe clinical symptoms. In Caucasians the prevalence of the S allele ranges from 5% to 10% and Z allele 2% to 5% depending on geographical location (7)(8). Although the frequencies are unknown in the Chinese, geographical variability of the A1AT alleles is evident by phenotypic analysis of the PI variants (9 …

Reticuloendothelial system uptake of infused 125I-trypsin in newborn and adult rats.

Previous studies have demonstrated enhanced intestinal trypsin uptake and decreased liver clearance of trypsin in newborn rats compared to adults. In order to examine the effectiveness of the reticuloendothelial system (RES) in clearing trypsin, bovine trypsin (1.25 mg/100 g body weight) plus trace 125I-trypsin were injected into the portal vein of 2-week-old (n = 57) and adult (n = 44) control rats or following RES stimulation using intraperitoneally injected lipopolysaccharide or RES suppression with intraperitoneally injected oleic acid emulsion. Plasma, liver and spleen 125I activities were assessed at 1, 5 or 15 min following infusion in control, stimulated and suppressed animals. Newborn control rats had significantly increased 125I plasma levels with decreased liver and spleen 125I activity compared to control adults. RES stimulation in the newborns did not lead to any change in liver or plasma levels although splenic values increased while adults had a decrease in liver 125I activity. RES suppression in the newborns led to increased plasma and decreased spleen 125I-trypsin values while adult rat levels were unchanged. The immature reticuloendothelial system in newborns is poorly responsive to RES stimulation although it can be made even further inefficient by RES suppression. The combination of RES immaturity and lack of response to stimulation may make newborns susceptible to proteolytic damage, especially during times of increased systemic levels of proteolytic enzymes.

Effect of protein C inhibitor (PCI) on in vitro fertilization

Protein C inhibitor (PCI) is a non-specific serpintype inhibitor (Suzuki et al., 1989) present in plasma and many other body fluids and secretions (Geiger, 1988; Laurell et al., 1992). PCI is synthesized throughout the male reproductive tract and high concentrations are found in seminal plasma (Laurell et al., 1992). In seminal plasma PCI has been shown to interact with prostate specific antigen (Christensson and Lilja, 1994). Additionally PCI inhibits the sperm protease acrosin and is immunocytochemically localized to disrupted acrosomal membranes of washed sperms, while intact sperms are negative for PCI-antigen (Zheng et al., 1994). Therefore, by inhibiting prematurely released and activated acrosin in the male reproductive tract PCI might protect spermatozoa, surrounding tissues and seminal plasma proteins from proteolytic damage. Under physiological conditions, however, acrosin is activated and released only in the female reproductive tract in the immediate vicinity of the ovum and is thought to be involved in the fertilization process by digesting a pathway for the sperm through the zona pellucida of the ovum. Inhibition of acrosin by PCI might therefore have an influence on the sperm/egg interaction. It was our aim to study PCI synthesis in the female reproductive tract and to analyze the effect of PCI on fertilization. For this purpose we used a mouse in vitro fertilization model.

Inhibition of the protease activity in tracheobronchial aspirates of horses with chronic obstructive pulmonary disease

Abstract Objectives To clarify the role of proteolytic enzymes in the pathogenesis of chronic obstructive pulmonary disease (COPD) in horses, and to investigate new possibilities for treatment of this disease by interfering in the proteolytic process. Design Effect of antiproteolytic activity of selected protease inhibitors on tracheal aspirates was studied in vitro, and the inhibition profiles were compared with those of purified proteases. Sample Population Respiratory tract secretions with antiproteolytic activity from 9 horses with COPD. Procedure Caseinolytic agar-diffusion assay. Results The protease-inhibition profile of tracheal aspirates differed from horse to horse. The profiles did not resemble that of any of the pure proteases. Acetylcysteine, pentamidine, and diminazene were most effective in inhibiting proteolytic activity in tracheal aspirates in vitro. Conclusions A mixed type of proteolytic activity is present in the respiratory tract secretions of horses with COPD. Clinical Relevance Acetylcysteine, pentamidine, and diminazene seem to have potential to be used in vivo to protect the lungs of horses with COPD from proteolytic damage. (Am J Vet Res 1996; 57:603–607)