Nuclear exclusion of transcription factors associated with apoptosis in developing nervous tissue

Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues.

The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus

We have shown that tissue-type plasminogen activator (tPA) and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP) response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment). For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 ± 0.2 ml/min, N = 7) decreases significantly (P = 0.016) when compared to normal rats (3.2 ± 0.3 ml/min, N = 6). The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80%) cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 ± 1.3 µg/min, N = 4) was slower (P = 0.003) than the clearance rate by the liver of normal rats (22.5 ± 0.7 µg/min, N = 10). After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment), tPA was cleared by the perfused liver at 16.2 ± 2.4 µg/min (N = 5), suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

Role of gelatinases MMP-2 and MMP-9 in tissue remodeling following acute lung injury

Acute lung injury is characterized by a severe disruption of alveolo-capillary structures and includes a variety of changes in lung cell populations. Evidence suggests the occurrence of rupture of the basement membranes and interstitial matrix remodeling during acute lung injury. The dynamic equilibrium of the extracellular matrix (ECM) under physiological conditions is a consequence of the balance between the regulation of synthesis and degradation of ECM components. Matrix metalloproteinases (MMPs) represent a group of enzymes involved in the degradation of most of the components of the ECM and therefore participate in tissue remodeling associated with pathological situations such as acute lung injury. MMP activity is regulated by proteolytic activation of the latent secreted proenzyme and by interaction with specific tissue inhibitors of metalloproteinases. This review details our knowledge of the involvement of MMPs, namely MMP-2 and MMP-9, in acute lung injury and acute respiratory distress syndrome.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

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.