Marek Gacko

Human cathepsin D.

A literature survey was performed of human cathepsin D gene, cathepsin D biosynthesis, posttranslatory modifications, transport within the cell, substrate specificity and catalytic effect. Methods used to determine the activity and level of this proteinase as well as its role in the biochemistry and pathobiochemistry of cells, tissues and organs were considered.

Thiocyanate concentration in saliva of cystic fibrosis patients.

Thiocyanates (SCN-) are ubiquitous in nature. There are indispensable part of host defense system that act as a substrate for lactoperoxidase (LPO). In our study we present initial data on SCN- concentration in saliva of CF patients in comparison to healthy non-smokers and healthy smokers. 5 ml of saliva was collected from each subject to a sterile tube and thiocyanate concentration was measured in each sample. The results of the measurements are presented on Fig. 1. Mean concentration of SCN- in saliva of CF patients was 0.031 +/- 0.0052 g/l, in healthy non-smokers 0.039 +/- 0.0048 g/l and in healthy smokers 0.048 +/- 0.0161 g/l. The differences between each group were statistically significant. Studies on larger group of patients and probably on different material (BALF or induced sputum) should present interesting data complementing the in vitro studies.

Evaluation of Transforming Growth Factor-β Signaling Pathway in the Wall of Normal and Varicose Veins

Objective(s): Extracellular matrix remodeling in the vein wall is involved in varicose vein pathogenesis, with transforming growth factor β1 (TGF-β1) playing a potential role. The aim of the study was to assess the TGF-β signaling pathway including its receptor (TGF-β RII) and phosphorylated receptor-regulated Smads (p-Smad2/3) in varicose veins. Methods: Varicose veins from patients undergoing varicose vein surgery were the studied material, whereas normal greater saphenous veins from patients undergoing infrainguinal arterial bypass surgery were the control material. Expression of TGF-β RII mRNA was assessed with RT-PCR, whereas expression of TGF-β RII and p-Smad2/3 proteins was assessed with Western blot. Results: A significantly increased TGF-β RII mRNA level was found in varicose veins (287 ± 24%), when compared with normal veins (100 ± 26%). The receptor protein expression reflected a changed mRNA level with significantly increased TGF-β RII protein in varicose veins (290 ± 21%), when compared with controls (100 ± 16%). Enhanced TGF-β RII expression was accompanied by increased p-Smad2/3 protein expression in varicose veins (257 ± 19%) in comparison with normal veins (100 ± 9%). Conclusion(s): Increased TGF-β RII expression and activation in the wall of varicose veins may be involved in extracellular matrix remodeling related to TGF-β1 and supports its role in the disease pathogenesis.

Quantitative determination and localization of cathepsin D and its inhibitors

A literature survey was performed of the methods of quantitative assessment of the activity and concentration of cathepsin D and its inhibitors. Usefulness of non-modified and modified proteins and synthetic peptides as measurement substrates was evaluated. The survey includes also chemical and immunochemical methods used to determine the distribution of cathepsin D and its inhibitors in cells and tissues.

Evaluation of enzymes involved in proteoglycan degradation in the wall of abdominal aortic aneurysms

The abdominal aortic aneurysm (AAA) wall represents an extreme example of arterial remodeling with disturbed elastin, collagen and proteoglycan metabolism. The aim of this study was to evaluate enzymes involved in the degradation of glycosaminoglycan chains and core proteins of proteoglycans in the AAA wall. The study material consisted of wall samples from 10 AAA. Fragments of 5 normal abdominal aortas from organ donors were used as a control. The activity of endoglycosidases, exoglycosidases and sulfatases was measured using colorimetric methods. To assess matrix metalloproteinases (MMPs), Western blot and zymography were performed. The activity of endoglycosidase degrading chondroitin-4-sulfate was lower in the AAA wall. Endoglycosidase degrading heparan sulfate and dermatan sulfate, arylosulfatase B, as well as all the exoglycosidases assessed demonstrated higher activities in the AAA wall. Furthermore, increased expression of MMP1, MMP2 and MMP9 was also shown in the AAA wall. Zymography revealed decreased activity of pro-MMP2 and presence of pro-MMP9 in the AAA wall compared to the wall of normal aorta. Extensive changes in the activity of glycosaminoglycan-degrading enzymes and MMPs may influence the organization of the extracellular matrix network and lead to previously demonstrated changes in the proteoglycan and glycosaminoglycan content in the AAA wall.

Evaluation of aFGF/bFGF and FGF Signaling Pathway in the Wall of Varicose Veins

BACKGROUND Extensive extracellular matrix remodeling of the vein wall is involved in varicose veins pathogenesis. The process is controlled by numerous factors, including peptide growth factors. The aim of the study was to evaluate acidic (aFGF) and basic (bFGF) fibroblast growth factors, their receptor (FGFR) and the MAP kinase pathway (ERK 1/2) in the wall of varicose and varicose veins complicated by thrombophlebitis, when compared to normal ones. METHODS Segments of normal, varicose, and varicose veins complicated by thrombophlebitis were collected during varicose veins surgery in 17 patients. Expression and content of aFGF and bFGF were evaluated with Western blot and enzyme-linked immunosorbent assay (ELISA) methods, respectively, whereas RT-PCR was employed to assess mRNA level of growth factors. Expression of FGFR and ERK 1/2 was examined with Western blot method. RESULTS Increased aFGF expression and content were accompanied by increased aFGF mRNA level in the wall of varicose veins. Furthermore, alternatively spliced aFGF mRNA was shown in varicose veins complicated by thrombophlebitis. Expression, content, and mRNA level of bFGF were comparable in the investigated material. FGFR and ERK 1/2 expression was demonstrated in the wall of diseased veins, however, without any significant differences in comparison with the wall of normal veins. CONCLUSIONS Overexpressed aFGF in the wall of varicose veins via FGFR and the MAP kinase pathway may influence expression of enzymes involved in extracellular matrix metabolism and play a role in vein wall remodeling, as well as in the disease pathogenesis.

Activities of proteases in parietal thrombus of aortic aneurysm

Deterioration of the aortic wall resulting in formation of aneurysm may be evoked by increased activity of elastases, collagenases and lysosomal proteases. These enzymes come from macrophages and neutrophil granulocytes which are elements of the inflammatory reaction accompanying aneurysm. These cells may also come from parietal thrombus in the aneurysm lumen. The aim of this work was to determine activity of elastase, cathepsin G, collagenase-like Pz-peptidase and cathepsins A, B, C, D and E in the parietal thrombus of aortic aneurysm. The thrombus was obtained from the lumen of the aortic aneurysm of six patients during operation. Protease activities were determined using specific substrates at optimum pH. Retracted blood clot was a comparative material. The thrombus of aortic aneurysm showed two-five fold higher activity of elastases, collagenase-like Pz-peptidase and cathepsins A, D and G in comparison to the blood clot (P < 0.001). However, activity of cathepsins B, C and E in the thrombus was only slightly higher (P < 0.05). Prolonged effect of proteases coming from parietal thrombus on the aneurysm wall could evoke marked degradation of fibrillar proteins resulting in increase of aneurysm.

Renal Parenchyma Perfusion Spectrum and Resistive Index (RI) in Ultrasound Examinations With Contrast Medium in the Early Period After Kidney Transplantation

The main diagnostic method for renal graft dysfunction is color Doppler ultrasound with the use of spectrae evaluation of blood flow within the main and intrarenal arteries. Ultrasound with contrast medium (US-CM) enhances the possibilities of this tool. The aim of this study was to evaluate the efficacy of US-CM to assess renal graft perfusion among 18 kidney allograft recipients at 5 to 10 days after transplantation. Patients underwent pulse inversion sonography of the graft during intravenous injections of 2.4 mL SonoVue (Bracco-Altana, Italy). Images were quantitatively assessed using computer software to compare the time to peak contrast enhancement effect in the renal cortex and renal pyramids. The results were compared to Doppler ultrasonography of the renal arteries and estimated glomerular filtration rate (eGFR; Modification of Diet in Renal Disease [MDRD]). A correlation was observed between eGFR and blood flow parameters within the renal arteries, as well between the flow time of contrast medium from the artery within the renal hilus to the renal cortex. Increased eGFR correlated with subsequent improvement in graft function (r = -.806; P = .001), and resistive index (RI) of the renal artery was inversely related to subsequent delayed graft function (r = .544; P = .029). Negative correlations were observed between eGFR and renal artery RI, as well as between eGFR and time from renal artery contrast to maximal contrast enhancement of the renal pyramids. A negative correlation was found as well between eGFR and time difference of contrast enhancement of the cortex and pyramids. In conclusion, US-CM enhanced the efficacy of ultrasound diagnostics of the renal graft and may be used as a predictor of graft function in the early posttransplantation period.

The activity of class I, II, III and IV of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the wall of abdominal aortic aneurysms

OBJECTIVE Human blood vessels contain a huge amount of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which play a significant role in the metabolism of many biological substances and participate in various metabolic pathways. The aim of this study was the investigation of the differences between the activities of ADH and ALDH in the wall of aortic aneurysm and wall of healthy aorta, that can explain the pathological background of aneurysm development. METHODS For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity the fluorometric methods was employed. The total ADH activity and activity of class III and IV isoenzymes was measured by the photometric method. The study material consisted of vessels wall samples obtained from 45 abdominal aortic aneurysm. RESULTS The activity of the class I ADH isoenzyme was significantly lower in the wall of aortic aneurysm than in healthy aorta. The other tested classes of ADH showed the tendency to lower level of the activity in aneurysm tissue than that in wall of unchanged aorta. The activities of total ADH and ALDH were also not significantly lower in the aneurysms. CONCLUSION The decrease of the activity of class I ADH isoenzymes in the wall of aortic aneurysm may be a factor of some disorders in metabolic pathways with participation of these isoenzymes.

Cathepsin E (EC 3.4.23.34) — a review

Cathepsin E belongs to the third class of enzymes - hydrolases, a subclass of peptide bond hydrolases and a sub-subclass of endopeptidases with aspartic catalytic sites. Cathepsin E is an endopeptidase with substrate specificity similar to that of cathepsin D. In a human organism, cathepsin E occurs in: erythrocytes, thymus, dendritic cells, epithelial M cells, microglia cells, Langerhans cells, lymphocytes, epithelium of gastrointestinal tract, urinary bladder, lungs, osteoclasts, spleen and lymphatic nodes. In human cells, loci of the gene of pre-procathepsin E are located on chromosome 1 in the region 1231-32. The catalytic site of cathepsin E is two residues of aspartic acid - Asp96 and Asn281, occurring in amino acid triads with sequences DTG96-98 and DTG281-283. To date, no particular role of cathepsin E in the metabolism of proteins in normal tissues has been found. However, it is known that there are many documented pathological conditions in which overexpression of cathepsin E occurs.