Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 µM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 µM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 µM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 ± 0.8 µM and k cat = 48.4 ± 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 ± 10, 1,098 ± 91, 38.6 ± 5.2 and 37,340 ± 5,400 µM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 ± 92.8 and 310,500 ± 38,600 µM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Microencapsulation and tissue engineering as an alternative treatment of diabetes

In the 70's, pancreatic islet transplantation arose as an attractive alternative to restore normoglycemia; however, the scarcity of donors and difficulties with allotransplants, even under immunosuppressive treatment, greatly hampered the use of this alternative. Several materials and devices have been developed to circumvent the problem of islet rejection by the recipient, but, so far, none has proved to be totally effective. A major barrier to transpose is the highly organized islet architecture and its physical and chemical setting in the pancreatic parenchyma. In order to tackle this problem, we assembled a multidisciplinary team that has been working towards setting up the Human Pancreatic Islets Unit at the Chemistry Institute of the University of São Paulo, to collect and process pancreas from human donors, upon consent, in order to produce purified, viable and functional islets to be used in transplants. Collaboration with the private enterprise has allowed access to the latest developed biomaterials for islet encapsulation and immunoisolation. Reasoning that the natural islet microenvironment should be mimicked for optimum viability and function, we set out to isolate extracellular matrix components from human pancreas, not only for analytical purposes, but also to be used as supplementary components of encapsulating materials. A protocol was designed to routinely culture different pancreatic tissues (islets, parenchyma and ducts) in the presence of several pancreatic extracellular matrix components and peptide growth factors to enrich the beta cell population in vitro before transplantation into patients. In addition to representing a therapeutic promise, this initiative is an example of productive partnership between the medical and scientific sectors of the university and private enterprises.

Heterogeneous response of adipose tissue to cancer cachexia

Cancer cachexia causes disruption of lipid metabolism. Since it has been well established that the various adipose tissue depots demonstrate different responses to stimuli, we assessed the effect of cachexia on some biochemical and morphological parameters of adipocytes obtained from the mesenteric (MES), retroperitoneal (RPAT), and epididymal (EAT) adipose tissues of rats bearing Walker 256 carcinosarcoma, compared with controls. Relative weight and total fat content of tissues did not differ between tumor-bearing rats and controls, but fatty acid composition was modified by cachexia. Adipocyte dimensions were increased in MES and RPAT from tumor-bearing rats, but not in EAT, in relation to control. Ultrastructural alterations were observed in the adipocytes of tumor-bearing rat RPAT (membrane projections) and EAT (nuclear bodies).

Effect of L-arginine, dimercaptosuccinic acid (DMSA) and the association of L-arginine and DMSA on tissue lead mobilization and blood pressure level in plumbism

Lead (Pb)-induced hypertension is characterized by an increase in reactive oxygen species (ROS) and a decrease in nitric oxide (NO). In the present study we evaluated the effect of L-arginine (NO precursor), dimercaptosuccinic acid (DMSA, a chelating agent and ROS scavenger), and the association of L-arginine/DMSA on tissue Pb mobilization and blood pressure levels in plumbism. Tissue Pb levels and blood pressure evolution were evaluated in rats exposed to: 1) Pb (750 ppm, in drinking water, for 70 days), 2) Pb plus water for 30 more days, 3) Pb plus DMSA (50 mg kg-1 day-1, po), L-arginine (0.6%, in drinking water), and the combination of L-arginine/DMSA for 30 more days, and 4) their respective matching controls. Pb exposure increased Pb levels in the blood, liver, femur, kidney and aorta. Pb levels in tissues decreased after cessation of Pb administration, except in the aorta. These levels did not reach those observed in nonintoxicated rats. All treatments mobilized Pb from the kidney, femur and liver. Pb mobilization from the aorta was only effective with the L-arginine/DMSA treatment. Blood Pb concentrations in Pb-treated groups were not different from those of the Pb/water group. Pb increased blood pressure starting from the 5th week. L-arginine and DMSA treatments (4th week) and the combination of L-arginine/DMSA (3rd and 4th weeks) decreased blood pressure levels of intoxicated rats. These levels did not reach those of nonintoxicated rats. Treatment with L-arginine/DMSA was more effective than the isolated treatments in mobilizing Pb from tissues and in reducing the blood pressure of intoxicated rats.

The influence of fasting/refeeding on the lipoprotein lipase activity of adipose tissue and muscle

Lipoprotein lipase activity in adipose tissue and muscle is modulated by changes in the pattern of food intake. We have measured total lipoprotein lipase activity in adipose tissue and muscle of male Wistar rats (N = 6-10), weighing 200-250 g (~12 weeks), during the refeeding/fasting state following 24 h of fasting. Lipoprotein lipase activity in tissue homogenates was evaluated using a [³H]-triolein-containing substrate, and released [³H]-free fatty acids were extracted and quantified by liquid scintillation. Adipose tissue lipoprotein lipase activity did not completely recover within 2 h of refeeding (60% of refed ad libitum values). Cardiac lipoprotein lipase activity remained increased even 2 h after refeeding (100% of refed ad libitum values), whereas no significant changes were observed in the soleus and diaphragm muscles. Adipose tissue lipoprotein lipase activities were consistently higher than the highest skeletal muscle or heart values. It is therefore likely that adipose tissue, rather than muscle makes the major contribution to triacylglycerol clearance. There was concomitant relatively high lipoprotein lipase activity in both adipose tissue and cardiac muscle during the first few hours of refeeding, therefore cardiac muscle may contribute significantly to triacylglycerol clearance during this period. The results suggest that during fasting, increased lipoprotein lipase activity provides a complementary source of free fatty acids from circulating triacylglycerol, allowing the heart to maintain its continuous, high-energy expenditure.

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

The distribution of presumptive thoracic paraganglionic tissue in the common marmoset (Callithrix jacchus)

The aortic-pulmonary regions (APR) of seven adult marmosets (Callithrix jacchus) and the region of the right subclavian artery of a further three marmosets were diffusion-fixed with 10% buffered formol-saline solution. In both regions serial 5-µm sections were cut and stained by the Martius yellow, brilliant crystal scarlet and soluble blue method. Presumptive thoracic paraganglionic (PTP) tissue was only observed in the APR. PTP tissue was composed of small groups of cells that varied in size and number. The distribution of the groups of cells was extremely variable, so much so that it would be misleading to attempt to classify their position; they were not circumscribed by a connective tissue capsule, but were always related to the thoracic branches of the left vagus nerve. The cells lay in loose areolar tissue characteristic of this part of the mediastinum and received their blood supply from small adjacent connective tissue arterioles. Unlike the paraganglionic tissue found in the carotid body the cells in the thorax did not appear to have a profuse capillary blood supply. There was, however, a close cellular-neural relationship. The cells, 10-15 µm in diameter, were oval or rounded in appearance and possessed a central nucleus and clear cytoplasm. No evidence was found that these cells possessed a 'companion' cell reminiscent of the arrangement of type 1 and type 2 cells in the carotid body. In conclusion, we found evidence of presumed paraganglionic tissue in the APR of the marmoset which, however, did not show the characteristic histological features of the aortic body chemoreceptors that have been described in some non-primate mammals. A survey of the mediastina of other non-human primates is required to establish whether this finding is atypical for these animals.

Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin

Changes in glutathione levels were determined in tissues of 11- to 12-week-old Swiss albino mice at different stages of Dalton's lymphoma tumor growth and following cisplatin (8 mg/kg body weight, ip) treatment for 24-96 h, keeping 4-5 animals in each experimental group. Glutathione levels increased in spleen of tumor-bearing compared to normal mice (9.95 ± 0.14 vs 7.86 ± 1.64 µmol/g wet weight, P<=0.05) but decreased in blood (0.64 ± 0.10 vs 0.85 ± 0.09 mg/ml) and testes (9.28 ± 0.15 vs 10.16 ± 0.28 µmol/g wet weight, P<=0.05). Dalton's lymphoma cells showed an increase in glutathione concentration (4.43 ± 0.26 µmol/g wet weight) as compared to splenocytes, their normal counterpart (3.62 ± 0.41 µmol/g wet weight). With the progression of tumor in mice, glutathione levels decreased significantly in testes (~10%) and bone marrow cells (~13%) while they increased in Dalton's lymphoma cells (28-46%) and spleen (15-27%). Glutathione levels in kidney, Dalton's lymphoma cells and bone marrow cells (8.50 ± 1.22, 4.43 ± 0.26 and 3.28 ± 0.17 µmol/g wet weight, respectively) decreased significantly (6.04 ± 0.42, 3.51 ± 0.32 and 2.17 ± 0.14 µmol/g wet weight, P<=0.05) after in vivo cisplatin treatment for 24 h. Along with a decrease in glutathione level, the glutathione-S-transferase (GST) activity also decreased by 60% in tumor cells after cisplatin treatment. The elevated drug uptake by the tumor cells under the conditions of reduced glutathione concentration and GST activity after treatment could be an important contributory factor to cisplatin's anticancer activity leading to tumor regression. Furthermore, lower doses of cisplatin in combination with buthionine sulfoximine (an inhibitor of glutathione synthesis) may be useful in cancer chemotherapy with decreased toxicity in the host.

Endothelial cells, tissue factor and infectious diseases

Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

Building bone tissue: matrices and scaffolds in physiology and biotechnology

Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

Distribution of biglycan and decorin in rat dental tissue

Biglycan and decorin are small leucine-rich proteoglycans that play several biological and structural roles in different tissues and organs. Several reports have indicated that biglycan participates in odontoblast and ameloblast differentiation and in the calcification process. In the present study we show that the expression of biglycan changes from within the ameloblasts and odontoblasts to the extracellular space according to the stage of animal development. In predentin and in the pulp space, however, biglycan was continually expressed throughout the period of investigation. In contrast, decorin was absent in odontoblasts and in ameloblasts and was exclusively expressed in predentin throughout the period of observation. In young rats, however, decorin was expressed in the extracellular spaces of the pulp, where it was concentrated mainly in the peripheral pulp.

Female pelvic floor disorders – outcome after mesh-augmented procedures and studies on vaginal connective tissue

Pelvic floor disorders, such as urinary incontinence (UI) and pelvic organ prolapse (POP), are common disorders in women. Because of the prolonged life expectancy the prevalence of UI and POP and the probability of ending up in surgery are increasing. However, the pathophysiology behind these disorders is still unsolved. The aim of this thesis is to study possible alterations in the connective tissue in the vaginal wall in patients with and without POP. The long-term outcome and complications of mid-urethral slings (MUS) and mesh-augmented POP surgery were studied in heterogenic patient populations. More elastin and a slight increase in immunostaining of type III and V collagens in tissue samples were obtained from patients with POP compared to controls in whom type I collagen was more prominent. The studies assessing the mesh-augmented procedures revealed good efficacy and high patient satisfaction after a long-term follow-up. Patients operated on because of mixed incontinence and with BMI >30 kg/m² reported significantly more urinary symptoms and a lower quality of life than the patients operated on because of stress urinary incontinence and the ones with BMI ≤30 kg/m². The objective outcome was equal between the groups. Mesh exposure through vaginal mucosa occurred in 23 % of the patients after POP surgery, most of these being asymptomatic. There are alterations in connective tissues in patients with POP. Mid-urethral sling procedures produced good long-term cure rates and patient satisfaction. As to the prolapse surgery, in spite of relatively high exposure rate, mesh-augmented procedure proved to be safe and effective method for the correction of POP.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

Hepatitis C virus seroprevalence and genotypes in patients with diffuse connective tissue diseases and spondyloarthropathies

Many extrahepatic manifestations, including rheumatic diseases, have been reported to be associated with hepatitis C virus (HCV) infection. In order to investigate the prevalence of HCV infection among patients with rheumatic diseases, in the present study we interviewed 367 patients and tested their blood samples for HCV antibodies (anti-HCV) by an enzyme-linked immunosorbent assay. Anti-HCV-reactive samples were retested for confirmation by a line immunoassay and also for HCV RNA detection by the polymerase chain reaction. HCV RNA-positive samples were genotyped by INNO-LIPA. An overall HCV infection prevalence of 1.9% (7/367) was found. Of the 7 HCV-infected patients, 4 had systemic lupus erythematosus and 3 rheumatoid arthritis, resulting in positivity rates of 2.3 and 3.4%, respectively. HCV RNA genotyping revealed the presence of subtypes 1a (57.1%), 1b (28.6%) and 3a (14.3%). The clinical course was favorable for all HCV-infected patients, except one, who died due to renal insufficiency related to lupus nephritis. These results demonstrate a low HCV infection prevalence among the population studied. In the few positive cases, we observed no adverse influence of this infection on the clinical evolution of the rheumatic disease.