Divergent changes in the content and activity of MMP-26 and TIMP-4 in human umbilical cord tissues associated with preeclampsia.

OBJECTIVES: Preeclampsia is the most common disorder associated with pregnancy. Our earlier findings revealed a substantial increase in the amount of matrix metalloproteinase-26 (matrilysin 2; MMP-26) in preeclamptic umbilical cord blood. The role of MMP-26 in preeclamptic umbilical cord tissue has not been fully elucidated. Some reports have indicated that the expression of matrilysin 2 and tissue inhibitor of matrix metalloproteinase 4 (TIMP-4) is coordinately regulated during progression of various diseases. STUDY DESIGN: Therefore, we decided to assess the expression and activity of MMP-26 and TIMP-4 in normal and preeclamptic umbilical cord tissues - umbilical cord arteries (UCA), vein (UCV) and Wharton's jelly (WJ). Tissues obtained from 10 normal (control material) and 10 preeclamptic umbilical cords were assessed using immunoenzymatic assay, Western immunoblotting, reverse transcriptase - polymerase chain reaction and fluorometric determination of the enzyme activity. RESULTS: All umbilical cord tissues, both control and preeclamptic, expressed MMP-26 and TIMP-4 in macromolecular complexes. Preeclampsia induced a significant increase in the content and actual activity of MMP-26 in UCV and WJ, as compared to control. The content of TIMP-4 in preeclamptic UCV and WJ was reduced. The content of MMP-26 mRNA was lower in UCA and UCV, whereas higher in WJ in preeclampsia. CONCLUSIONS: Divergent changes in MMP-26 mRNA and protein expression suggest a difference in the factors controlling the matrilysin synthesis in healthy and preeclamptic subjects. The decrease in TIMP-4 content in preeclamptic UCV might be the main reason for significantly higher actual activity of MMP-26 in that tissue. Only in preeclamptic Wharton's jelly the changes were compatible in terms of the content and activity of MMP-26 and TIMP-4. It cannot be excluded that similar alterations can be observed for the whole vascular system of newborns delivered by mothers with preeclampsia.

Trace elements as an activator of antioxidant enzymes.

Oxidative stress is a state of impaired balance between the formation of free radicals and antioxidant capacity of the body. It causes many defects of the body, e.g. lipid peroxidation, DNA and protein damage. In order to prevent the effects of oxidative stress, the organism has developed defence mechanisms. These mechanisms capture and inhibit the formation of free radicals and also chelate ion metals that catalyse free radical reactions. Trace elements are components of antioxidant enzymes involved in antioxidant mechanisms. Selenium, as a selenocysteine, is a component of the active site of glutathione peroxidase (GPx). The main function of GPx is neutralization of hydrogen peroxide (H2O2) and organic peroxide (LOOH). Furthermore, selenium is a structural part of a large group of selenoproteins that are necessary for proper functioning of the body. Manganese, copper and zinc are a part of the group of superoxide dismutase enzymes (MnSOD, Cu/ZnSOD), which catalyse the superoxide anion dismutation into hydrogen peroxide and oxygen. Formed hydrogen peroxide is decomposed into water and oxygen by catalase or glutathione peroxidase. An integral component of catalase (CAT) is iron ions. The concentration of these trace elements has a significant influence on the activity of antioxidant enzymes, and thus on defence against oxidative stress. Even a small change in the level of trace elements in the tissue causes a disturbance in their metabolism, leading to the occurrence of many diseases.

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1.

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor-UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.

[Cathepsin D and B activity in the serum of patients with urothelial bladder cancer]. / Aktywnosc katepsyn D i B w osoczu chorych na raka przejsciowokomórkowego pecherza moczowego.

UNLABELLED: The key role of cathepsin D and B is intralysosomal digestion of used cellular proteins and other proteins that enter cells through endocytosis. Under pathological conditions like cancer formation and growth, cathepsins from lysosomes are released. The aim of the study was to determin of cathepsin D and B activities in serum of patients with urothelial bladder cancer depending on disease severity and determination of its' changes after transurethral resection of tumor. MATERIAL AND METHODS: Experiment involved 50 patients. Blood samples were obtained from 18 healthy volunteers and 32 urothelial bladder cancer patients. Samples from people with suspected urothelial bladder cancer were collected three times: before the surgery, 2 weeks and 6 weeks after the surgical treatment. RESULTS: Our research showed that cathepsin D activity, measured as the level increment of acid soluble tyrosine, is the highest before the surgery in muscle invasive bladder tumor (pT2) (57,9 nmol/ml). 2 weeks and 6 weeks after the surgical treatment, cathepsin D activity is decreased. In case of cathepsin B activity, measured as the level of released p-nitroaniline, decreased, 2 weeks and 6 weeks after the surgical treatment in both cases of disease severity. CONCLUSION: Cathepsin D and B activities in the serum of patients with urothelial bladder cancer are directly proportional to disease severity and significantly higher compared with control group. Transuretral resection of the tumor leads to diminution of their activities in second and 6th week after the procedure.

Cathepsin D serum and urine concentration in superficial and invasive transitional bladder cancer as determined by surface plasmon resonance imaging.

Determination of cathepsin D (Cat D) concentration in serum and urine may be useful in the diagnosis of bladder cancer. The present study included 54 healthy patients and 68 patients with bladder cancer, confirmed by transurethral resection or cystectomy. Cat D concentration was determined using a surface plasmon resonance imaging biosensor. Cat D concentration in the serum of bladder cancer patients was within the range of 1.3-5.59 ng/ml, while for healthy donors it was within the range of 0.28-0.52 ng/ml. In urine, the Cat D concentration of bladder cancer patients was within the range of 1.35-7.14 ng/ml, while for healthy donors it was within the range of 0.32-0.68 ng/ml. Cat D concentration may represent an efficient tumor marker, as its concentration in the serum and urine of transitional cell carcinoma patients is extremely high when compared with healthy subjects.

Lysosomal exoglycosidases and cathepsin D in colon adenocarcinoma.

INTRODUCTION: Changes in the structure of membrane glycoconjugates and activity of glycosidases and proteases are important in tumor formation. OBJECTIVES: The aim of the study was to compare the specific activity of lysosomal exoglycosidases: N-acetyl-ß-D-hexosaminidase (HEX), its isoenzymes A (HEX A) and B (HEX B), ß-D-galactosidase (GAL), α-fucosidase (FUC), and α-mannosidase (MAN) with the activity of cathepsin D (CD) in serum, urine, and carcinoma tissue of patients with colon adenocarcinoma. PATIENTS AND METHODS: The specific activity of HEX, HEX A, HEX B, GAL, FUC, MAN, and CD was assayed in serum, urine, and carcinoma tissue of 12 patients with colon adenocarcinoma. RESULTS: Lysosomal exoglycosidases and CD have similar specific activity in colon adenocarcinoma tissue and urine, which is higher than their activity in serum (with the exception of the highest specific activity of CD in urine). A positive correlation was observed between the specific activity of CD and that of HEX, HEX A, FUC, and MAN in the carcinoma tissue and urine as well as between CD and GAL in the urine of patients with colon adenocarcinoma. Negative correlations were observed between protein levels and the specific activity of HEX, HEX A, FUC, MAN, and CD in the carcinoma tissue and urine, and between protein levels and GAL in urine. CONCLUSIONS: Increased degradation and remodeling of glycoconjugates in the colon adenocarcinoma tissue is reflected by increased specific activity of exoglycosidases and CD. The results suggest a strong effect of exoglycosidase action on tissue degradation and a potential role of exoglycosidases in the initiation of proteolysis.

[Cathepsin D activity in chronic rhinosinusitis with nasal polyps]. / Ocena aktywnosci katepsyny D w blonie sluzowej pacjentów z przewleklym zapaleniem zatok przynosowych z polipami.

UNLABELLED: Nasal polyposis affects about 1 to 4% of the population. Polyps develop in oedematous and inflammated mucous membrane. In spite of the intensive research the pathomechanism of their development is not fully understood. The majority of the theories concerning the development of nasal polyps emphasize the role of the inflammatory process causing the rupture of the epithelium and the basal membrane. Cathepsin D is one of important mediators of inflammatory processes, that may be involved in the pathogenesis of nasal polyposis. THE AIM OF THE STUDY: was to establish the role of the cathepsin D in the pathogenesis of nasal polyps. MATHERIAL AND METHOD: Tissues were taken from 39 patients treated with endoscopic sinus surgery due to chronic rhinosinusitis with polyps. The activity of the cathepsin D was assesed with spectrofotometric method using the specific inhibitor (pepstatin) in tissue of nasal polyps, in oedematous and the inflammated mucous membrane of the nasal conchae and the samples of mucous membrane taken from the nasal septum. RESULTS: Statistically significant difference in cathepsin D activity between polypoid tissue, inflammated mucosa and the mucous membrane of the nasal septum was detected (t-student test, p < 0.05). No difference in the activity of this enzyme was observed between the polypoid tissue and the inflammated mucosa. CONCLUSION: Increased activity of the cathepsin D in nasal polyps and inflammatory changed mucosa confirm the important role of the cathepsin D in inflammatory processes leading to damage and subsequent remodeling of mucous membrane. We believe that further research on the activity of other proteolytic enzymes is necessary to demonstrate the differences between the inflammable changed mucous membrane and nasal polyps.

Cathepsin D and carcino-embryonic antigen in serum, urine and tissues of colon adenocarcinoma patients.

BACKGROUND/AIMS: Application of neoplastic markers in early diagnosis of colorectal carcinoma has brought fresh hope to millions of sufferers. However such a marker, distinctive for this particular carcinoma and allowing its detection at a sufficiently early stage of development has not yet been found. Cathepsin D (CD) is lysosomal aspartyl proteinase. It is a component of a proteolytic cascade participating actively in neoplastic invasion as well as in metastasis formation. Carcino-embryonic antigen (CEA) is a useful marker in oncological diagnostics of colorectal cancer. CEA undergoes expression in all kinds of adenocarcinoma and is found both intercellularly and extracellularly. High concentrations of CEA in the blood serum confirm neoplastic changes in the digestive tract with high probability. The objective of this study has been to evaluate CD activity in the blood serum, urine and tumor tissues as well as in the colon biopsies which were not changed macroscopically and CEA concentration in the serum of colon adenocarcinoma, considering the extent of spread of cancer (TNM), the grade of the differentiation of cancer cell (G) as well as the tumor size. The possibility of application of CD along with CEA as markers of colon adenocarcinoma has also been examined. METHODOLOGY: The examination included the serum and urine of 21 patients as well as 12 tissues biopsies with histopathologically confirmed colon adenocarcinoma. The reference group for the blood and urine comprised of 17 healthy controls, and for the colon adenocarcinoma tissues- samples collected from 14 people from the sites most distant from the resected tumor on the boundaries which were free of cancer cells. Activity of CD in the blood serum, urine as well as tissues was determined with a modified Greczaniuk et al. method and expressed by the amount of released tyrosine as the concentration of the activity in nmolTyr/mL/6h, whereas the specific activity was expressed in nmol Tyr/mg of protein /6h. The specific activity of CD in the urine was expressed in nmol Tyr/mg of creatinine/6h. CEA concentration in the blood serum was determined by the immunoenzymatic method (MEIA) on Axym Abbot Analyzer and was expressed in ng/mL. The protein concentration was determined by the Lowry method, and the results were expressed in mg/mL. The creatinine concentration in the urine was determined by the Jaffe method (without deproteinization) and was expressed in mg/100mL. RESULTS: CD activity was increased in the blood serum (p < 0.0001) and tissues (p = 0.022) of colon adenocarcinoma patients in comparison to the reference group. CD specific activity (Tyr/mg of protein/6h) was significantly increased in serum but decreased in the urine (p < 0.0001) whereas the specific activity of CD (nmol Tyr/mg of creatinine/6h) was increased in the urine (p = 0.0001). CD specific activity has tendency to increase in colon adenocarcinoma tissues (p = 0.441) as compared to the reference group. By examining data in regard to TNM clinical-histopathological classification, G and the tumor size, it could be concluded that CD activity in serum and urine in colon adenocarcinoma patients depends on progress of cancer in which CD activity increases with TNM. A statistically significant increase in CEA concentration was found in the serum of colon adenocarcinoma patients, which was almost threefold higher than the in reference group. No significant differences in CEA concentration were found depending on TNM, G and tumor size. CONCLUSIONS: The results of this study suggest that examination of CD activity and CEA concentration in serum, as well as CD activity in the urine, might be used in oncological diagnostics of colon adenocarcinoma.

Protective effect of black tea against ethanol-induced oxidative modifications of liver proteins and lipids.

OBJECTIVE: Black tea has been recently ascertained as a source of water-soluble antioxidants that may enhance cellular antioxidant abilities. The present study was designed to investigate the efficacy of the preventive effect of black tea on oxidative modifications of liver lipids and proteins of 2-month-old rats intoxicated chronically (28 days) with ethanol. METHOD: Lipid peroxidation was estimated by measurement of lipid hydroperoxides, malondialdehyde, and 4-hydroxynonenal by high-performance liquid chromatography (HPLC) and by spectrophotometric determination of conjugated dienes. The markers of protein oxidative modification products-bistyrosine and tryptophan-were quantified by spectrofluorimetry, whereas levels of amino, sulfhydryl, and carbonyl groups were estimated spectrophotometrically. RESULTS: Ethanol intoxication caused changes in liver antioxidant abilities that led to the generation of oxidative stress and, consequently, to the significant increase in products of lipid and protein oxidative modification. Enhanced lipid peroxidation was confirmed by assessment of the concentration of lipid peroxidation products measured at all examined levels. Protein modifications were evidenced by increase in levels of bistyrosine and carbonyl groups and by decrease in concentration of tryptophan and levels of sulfhydryl and amino groups. The metabolic consequences of oxidative modifications of lipids and proteins were reduced by cathepsin B activity and translocation of this lysosomal protease into cytosol as well as markers of liver damage-alanine aminotransferase (ALT) and aspartate aminotransferase (AST)-into the blood serum. Administration of black tea to ethanol-intoxicated rats partially protected antioxidant parameters and, remarkably, prevented the significant increase in concentrations of all measured lipid peroxidation products. Moreover, the levels of markers of the protein-modification process were similar to those of the control group. Protection of biological membranes by black tea prevents changes in the permeability of these membranes and translocation of the examined enzymes. CONCLUSIONS: Our findings indicate that black tea protects proteins and lipids against oxidative modification induced by chronic ethanol intoxication, which preserves changes in redox and proteolytic homeostasis.

[Activity of cathepsin D in the blood serum and urine of patients with cancer of the stomach, pancreas and liver]. / Aktywnosc katepsyny D w surowicy krwi i moczu chorych na raka zoladka, trzustki lub watroby.

Cathepsin D is a protease involved in invasion of the cancer and metastasis formation. The purpose of the study was to evaluate a prognostic value of cathepsin D activity in blood serum and urine of patients with cancer of the stomach, pancreas and liver. The study was carried out on the samples of blood serum and urine obtained from patients with cancer of the stomach, pancreas and liver treated surgically at the First Department of General Surgery and Endocrinology of the Medical University of Bialystok. The control group consisted of healthy individuals. Activity of cathepsin D was determined in serum and urine by the Folin-Ciocaltau method with the cupric modification and was expressed in nmol Tyr/ml/6h. Specific activity of cathepsin D was determined in the urine, and was expressed in nmol Tyr/mg of protein/6h. Protein concentration in serum was assessed with Lowry et al. method and results were expressed in mg/ml. A significant increase in activity of cathepsin D in serum (p = 0.0169) and urine (p = 0.0008) and an enhanced specific activity in the urine (p = 0.0085) was found in patients with cancer of the pancreas as compared with the controls. A significantly increased activity of cathepsin was revealed in serum (p = 0.0233) of patients with cancer of the stomach. No significant differences of cathepsin D activity were found in urine of the patients with cancer of the stomach when compared to the controls. Additionally, an upward tendency (almost two-fold increase) of cathepsin D activity was shown in blood serum and an increase in the activity and in specific activity was observed in urine of both patients with cancer of the liver in comparison with the healthy individuals. There were no significant differences in the activity of cathepsin D in serum of the patients with cancer of the pancreas and stomach (p = 0.4156). A statistically significantly higher activity (p = 0.0004) and specific (0.0048) cathepsin D activity was demonstrated in urine of the patients with cancer of the pancreas in comparison with the patients with cancer of the stomach. Determination of protein level in urine proved a downward tendency in the patients with cancer of the stomach (p = 0.11109), as compared to the controls, and a statistically significant increase found in the patients with cancer of the pancreas (p = 0.0238), in comparison with the patients with cancer of the stomach. In conclusion, investigation of cathepsin D activity in the blood serum of patients with cancer of the stomach and pancreas and in the urine of the patients with cancer of the pancreas may be usefull in clinical oncological diagnostics. However, further studies of the enzyme are necessary to establish the clinical value of cathepsin D measurement.

Effect of epsilon-aminocaproylamino acids on fibrin formation.

Effect of three epsilon-aminocaproylamino acids with significant antifibrinolytic activity on polymerization of fibrin monomer, clot retraction, fibrin structure, prothrombin consumption and thrombin activity was examined. epsilon-Aminocaproyl-L-norleucine and epsilon-aminocaproyl-L-leucine were weak inhibitors of thrombin activity and epsilon-aminocaproyl-L-norleucine slightly inhibited polymerization of fibrin monomers.

Effects of epsilon-aminocaproiloaminoacids on the amidolytic activity of tissue plasminogen activator, urokinase and kallikrein.

Effect of three epsilon-aminocaproylaminoacids with a significant antifibrinolytic activity on amidolytic activity of tissue plasminogen activator (t-PA), urokinase and kallikrein was examined. epsilon-Aminocaproyl-S-benzyl)-L-cysteine and epsilon-aminocaproyl-L-norleucine were weak inhibitors of kallikrein. Weak activation of t-PA activity was observed at high concentration of the tested compounds. Only one of the examined dipeptides was a weak inhibitor of amidolytic activity of urokinase.

Green tea protection against age-dependent ethanol-induced oxidative stress.

Ethanol intoxication leads to oxidative stress, which may be additionally enhanced by aging. The aim of this study was to investigate the influence of green tea as a source of water-soluble antioxidants on the ability to prevent oxidative stress in aged rats sub-chronically intoxicated with ethanol. Two-, 12-, and 24-mo-old male Wistar rats were divided into 4 experimental groups: (1) control, (2) green tea, (3) ethanol, and (4) ethanol and green tea. Ethanol intoxication produced age-dependent decrease in the activity of serum superoxide dismutase, glutathione peroxidase, and reductase and in levels of glutathione (GSH), vitamins C, E, and A, and beta-carotene. Changes in the serum antioxidative ability were accompanied by enhanced oxidative modification of lipid (increase in lipid hydroperoxides, malondiadehyde, and 4-hydroxynonenal levels) and protein (rise in carbonyl group levels). Green tea partially protected against changes in antioxidant enzymatic as well as nonenzymatic parameters produced by ethanol and enhanced by aging. Administration of green tea significantly protects cellular components such as lipids and proteins against oxidative modification. Results indicate that green tea effectively protects blood serum against oxidative stress produced by ethanol as well as aging.

[Proteases of neutrophilic granulocytes]. / Proteazy granulocytów obojetnochlonnych.

The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B), serine proteases (granulocytic elastase, cathepsin G, protease 3), membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine), cysteine and aspartic cathepsins. The role of these proteases in the pathology and diagnostics of certain diseases was considered.