TRPM2 channel deficiency prevents delayed cytosolic Zn2+ accumulation and CA1 pyramidal neuronal death after transient global ischemia.

Transient ischemia is a leading cause of cognitive dysfunction. Postischemic ROS generation and an increase in the cytosolic Zn(2+) level ([Zn(2+)]c) are critical in delayed CA1 pyramidal neuronal death, but the underlying mechanisms are not fully understood. Here we investigated the role of ROS-sensitive TRPM2 (transient receptor potential melastatin-related 2) channel. Using in vivo and in vitro models of ischemia-reperfusion, we showed that genetic knockout of TRPM2 strongly prohibited the delayed increase in the [Zn(2+)]c, ROS generation, CA1 pyramidal neuronal death and postischemic memory impairment. Time-lapse imaging revealed that TRPM2 deficiency had no effect on the ischemia-induced increase in the [Zn(2+)]c but abolished the cytosolic Zn(2+) accumulation during reperfusion as well as ROS-elicited increases in the [Zn(2+)]c. These results provide the first evidence to show a critical role for TRPM2 channel activation during reperfusion in the delayed increase in the [Zn(2+)]c and CA1 pyramidal neuronal death and identify TRPM2 as a key molecule signaling ROS generation to postischemic brain injury.

Limitations of macroscopic fluorescence imaging for the estimation of tumour growth in an orthotopic glioma mouse model.

Imaging methods based on light detection are being increasingly used for the non-invasive assessment of tumour growth in animal models. In contrast with bioluminescence imaging, there are no studies assessing the use of macroscopic fluorescence imaging for the longitudinal monitoring of tumour growth in an orthotopic glioma mouse model. Glioma cells expressing the red-shifted fluorescent protein mKate2 were orthotopically implanted to NOD-rag mice and the tumour size estimated by macroscopic fluorescence imaging was compared to the tumour volume determined morphometrically. There was no significant correlation between the data obtained by non-invasive macroscopic fluorescence imaging and post mortem morphometry. In addition, the fluorescence imaging failed to detect a morphometrically verified difference in tumour volume between animals with tumours expressing a potential tumour suppressor gene and controls. The fluorescence signal was affected by the spatial pattern of tumour growth and substantially attenuated by the interfering brain tissue. Our results indicate that the fluorescence signal emitted by glioma cells reflected not only the tumour mass, but also its spatial distribution. Macroscopic planar FLI in an epi-illumination mode and a conventional source of excitation light therefore appears to be more suitable for semi-quantitative assessment of the tumour growth especially in the case of superficially located tumours rather than for precise volume estimation of the xenografts located deep within the brain tissue.

Compartment- and malignance-dependent up-regulation of γ-glutamyltranspeptidase and dipetidylpeptidase-IV activity in human brain gliomas.

γ-Glutamyltranspeptidase (GGT, syn. γ-Glutamyltransferase) and dipeptidylpeptidase-IV (DPP-IV) activity participates in metabolic and growth control of normal and tumor cells by processing biologically active peptides. Here, we report on up-regulation of these enzymes in human brain gliomas determined by catalytic enzyme histochemistry and immunocytochemistry. Higher activity of GGT was found in 50%, 68% and 81% of WHO grade II, III and IV tumors, respectively. The process started at/near the microvasculature, from where it spread to the parenchyma. On average, the enzyme activity in grade II, III and IV gliomas exceeded controls 2.0, 3.0 and 3.5-fold, respectively. Up-regulation of DPP-IV-like activity also started at the microvasculature, but mainly in pericytes and mononuclear-like cells around the vessels and dispersed in the parenchyma. Marked elevation of this enzyme activity, comprising also tumor parenchyma, occurred only in grade IV glioblastomas (65% patients; 3.6 times above controls) which can, therefore, help in their differentiation from grade III gliomas. The increase of total DPP-IV-like activity also included its two enzymatic homologs, the canonical DPP-IV/CD26 and FAP-1α. The increase in GGT is supposed to be a tumor grade dependent response of microvasculature and tumor astrocytes to stress induced by tissue hypoxia and/or the metabolic aberrancies. The increase in DPP-IV-like activity in high-grade tumors can be attributed to inflammatory/scavenging processes performed by the mononuclear-like cells and, in glioblastomas, also to regressive changes in the structure and function of the microvasculature and tumor parenchyma, including astrocyte stress response. The inverse relationship between DPP-IV-like activity and Ki67 in most glioblastomas and shorter survival time of patients with low activity of this enzyme also suggest its anti-oncogenic effects.

Expression pattern of dipeptidyl peptidase IV activity and/or structure homologues in cancer.

Proline at the second position of the N-terminus of biologically active peptides involved in cell growth regulation is an evolutionarily conserved motif protecting them against cleavage by non-specific proteases. Just a small number of proline-specific hydrolases including dipeptidyl peptidase IV (DPP-IV) and related molecules is capable of cleaving such post-prolyl bond. DPP-IV, originally described on the basis of its enzymatic activity, is a ubiquitous, multifunctional homodimeric plasma membrane glycoprotein of type II. Subsequently, several other molecules related to DPP-IV by their enzymatic activity and/or sequence were discovered and classified as "dipeptidyl peptidase IV activity and/or structure homologues" (DASH). Along with canonical DPP-IV this group comprises DPP-IVbeta, DPP-II, DPP6, DPP8, DPP9, DPP10 and fibroblast activation protein alpha (FAP-alpha). Recent observations of deregulated expression of several DASH molecules in multiple human cancers led to the assumptions of their pathogenetic relevance in cancerogenesis. Here we review recent information about selected DASH molecules in human malignancies.

Methods of RNA purification. All ways (should) lead to Rome.

Ribonucleic acid (RNA) represents an important target of a wide array of laboratory anal yses. Thus, RNA purification is a critical first preceding step of a number of preparative and analytical methods, important particularly in diagnostics of dozens of viral, bacterial, and parasitic diseases, dia gnosis of inherited disorders, and tumours, as well as in basic research. To provide relevant and reliable results, techniques of molecular biology used for such purposes require pure and intact molecules of purified RNA. Moreover, RNA has to be purified effectively and reproducibly from various heterogeneous materials such as fresh or frozen tissues, cell lines, PCR products or long-term chemically preserved samples. Principally, methods of RNA purification can be divided into three groups. The first group of methods is based on organic phenol:chloroform extraction. The second group encompasses methods of RNA purification by means of its ability to bind specific surfaces in the presence of chaotropic salt, and the third group includes methods exploiting RNA isolation on isopycnic gradients. Although RNA can be isolated from either prokaryotic or eukaryotic organisms, this review is to give out a basic outline of methods available for eukaryotic, with emphasis on mammalian, tissues.

Modulation of substance P signaling by dipeptidyl peptidase-IV enzymatic activity in human glioma cell lines.

Dipeptidyl peptidase-IV (DPP-IV, CD26) is a serine protease almost ubiquitously expressed on cell surface and present in body fluids. DPP-IV has been suggested to proteolytically modify a number of biologically active peptides including substance P (SP) and the chemokine stromal cell derived factor-1alpha (SDF-1alpha, CXCL12). SP and SDF-1alpha have been implicated in the regulation of multiple biological processes and also induce responses that may be relevant for glioma progression. Both SP and SDF-1alpha are signaling through cell surface receptors and use intracellular calcium as a second messenger. The effect of DPP-IV on intracellular calcium mobilization mediated by SP and SDF-1alpha was monitored in suspension of wild type U373 and DPP-IV transfected U373DPPIV glioma cells using indicator FURA-2. Nanomolar concentrations of SP triggered a transient dose dependent increase in intracellular calcium rendering the cells refractory to repeated stimulation, while SDF-1 had no measurable effect. SP signaling in DPP-IV overexpressing U373DPPIV cells was not substantially different from that in wild type cells. However, preincubation of SP with the DPP-IV overexpressing cells lead to the loss of its signaling potential, which could be prevented with DPP-IV inhibitors. Taken together, DPP-IV may proteolytically inactivate local mediators involved in gliomagenesis.

TP53 and progression from Barrett's metaplasia to oesophageal adenocarcinoma in a UK population cohort.

BACKGROUND AND AIMS: Oesophageal adenocarcinoma frequently develops on a background of metaplastic Barrett's epithelium. The development of malignancy is accompanied by genetic alterations, which may be promising biomarkers of disease progression. METHODS: A case control study was conducted nested within a large unselected population based cohort of Barrett's patients. Incident oesophageal malignancies and high grade dysplasias were identified. For each case up to five controls were matched on age, sex, and year of diagnosis. Biopsies from the time of diagnosis of Barrett's epithelium were stained immunohistochemically for TP53, cyclin D1, cyclooxygenase 2 (COX-2), and beta-catenin proteins. RESULTS: Twenty nine incident oesophageal malignancies and six cases of high grade dysplasia were identified. The odds of diffuse or intense TP53 staining were substantially elevated in biopsies from patients who developed oesophageal adenocarcinoma compared with controls (odds ratio (OR) 11.7 (95% confidence interval (CI) 1.93, 71.4)). This difference was also present when all cases were considered (OR 8.42 (95% CI 2.37, 30.0). Despite the association with TP53 staining, only 32.4% of cases had an initial biopsy showing diffuse/intense TP53 staining. There were no significant associations between cyclin D1, COX-2, or beta-catenin staining and case control status. The OR for positive staining for both TP53 and COX-2 was markedly increased in cases compared with controls (OR 27.3 (95% CI 2.89, 257.0)) although only 15% of cases had positive staining for both markers. CONCLUSIONS: Immunohistochemical detection of TP53 expression is a biomarker of malignant progression in Barrett's oesophagus but sensitivity is too low to act as a criterion to inform endoscopic surveillance strategies. Additional biomarkers are required which when combined with TP53 will identify, with adequate sensitivity and specificity, Barrett's patients who are at risk of developing cancer.

Up-regulation of gamma-glutamyl transpeptidase (GGT) activity in growth perturbed C6 astrocytes.

Activity of gamma-glutamyl transpeptidase (GGT) was studied in astrocyte-like C6 glial cells modulated in growth and maturation by different concentration of serum and dibutyryl cyclic AMP (Db-cAMP) supplement in culture medium. After reduction of serum concentration from 10% to 0.1%, the number of GGT positive cells determined histochemically increased 3.1 times and the GGT activity/mg protein in whole cell lysates was 5.1 times higher. In cultures with 0.1% serum + Db-cAMP, the histochemically and biochemically assayed GGT activity exceeded 5.1 and 7.9 times the values measured in control 10% serum cultures, respectively. The up-regulation of GGT was accompanied by an inhibition of proliferation, enhanced differentiation and hypertrophy of cells. In addition, the process of metabolic perturbation and/or cellular stress was revealed in these cultures by the (i) growth-support release followed by shrinkage and death of a small number of cells and (ii) higher oxidation of 2'7'dichlorofluorescein diacetate to its fluorescent form in the adherent/viable cells. The observed up-regulation of GGT is considered to primarily reflect increased metabolism of glutathione and/or the maintenance of the redox potential in cells stressed by sub-optimal concentration of serum and Db-cAMP supplement. The concomitant cellular hypertrophy and differentiation and their relationship to increased activity of GGT await further investigation. The study suggests that up-regulation of GGT can contribute to adaptation of astrocytic cells to metabolic and/or oxidative perturbances occurring under various pathological conditions, including radiation- and drug-induced toxicity.

Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.

Quaternary benzo[c]phenanthridine alkaloids as inhibitors of dipeptidyl peptidase IV-like activity baring enzymes in human blood plasma and glioma cell lines.

Quaternary benzo[c]phenanthridine alkaloids (QBA), fagaronine (FA), sanguinarine (SA), chelerythrine (CHE) and the QBA extract from Macleya cordata (EX) exerted differential inhibitory effect on the hydrolytic activity of particular dipeptidyl peptidase (DPP)-like enzyme isolated from human blood plasma and from human and rat glioma cell lines. The low-MW form of DPP-IV-like enzyme activity, corresponding most probably to DPP-8, observed only in glioma cells but not in human plasma, was inhibited preferentially by SA, CHE and EX, and only slightly by FA. The alkaloid inhibitory effect was concentration-dependent in the range 25-150 mM and directly pH-related. In addition, a subtle but consistent inhibition of the intermediate-MW form of DPP-IV-like enzyme activity, ascribed to DPP-IV/CD26, observed only in human plasma and of the attractin (high-MW form of DPP-IV-like enzyme activity, expressed in U87 glioma cells) by the studied alkaloids was observed. We conclude that some of the QBA biological effects could be determined by tissue and cell type specific dipeptidyl peptidase IV-like molecules expression pattern.

Cisplatin induced gamma-glutamyltransferase up-regulation, hypertrophy and differentiation in astrocytic glioma cells in culture.

Gamma-glutamyltransferase (GGT) hydrolyses gamma-glutamylated peptides, including glutathione and transports amino acids into the cells. The enzyme is up-regulated in some tumors, especially those with a higher degree of malignancy and resistance to cytostatics. In this study we examined the effects of Cisplatin (1.6 x 10(-5)M) on the activity of GGT in astrocytic C6 glioma cells in cultures monitored for growth, morphology and differentiation. Initially (24 h), the drug inhibited cell division and later (96 h), it caused apoptotic death of about half of the population. The more resistant and surviving cells became hypertrophic and more differentiated, as indicated by their larger size and higher protein content, including the maturation- specific GFAP. In addition, the activity of GGT was significantly elevated in these cells at 48 h and onwards. At 96 h, the biochemically determined enzyme activity was between 230% and 330% above the controls. Compared to the protein content, the GGT activity started to increase later (48 h) but it grew steeper towards 72-96 h. Similarly, histochemical analysis revealed a manifold increase in the number of GGT+ cells in the population and higher intensity of staining per cell from at 48 h and onwards. The study showed that the transformed astrocytic cells can up-regulate GGT activity as part of an adaptation and/or, survival-enhancing reaction triggered by Cisplatin.

Functional cross-talk of Ca2+-mobilizing endothelin receptors in C6 glioma cells.

There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells. In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells. Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content. Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2. The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration. However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone. Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells. Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts. In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor. The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation.

Cytotoxicity of natural compounds in hepatocyte cell culture models. The case of quaternary benzo[c]phenanthridine alkaloids.

The quaternary benzo[c]phenanthridine alkaloids (QBA) produce a plethora of species- and tissue-specific effects but the molecular basis of their biological activities remain mysterious. The objective of the present study was to investigate the cytotoxicity of QBA alkaloids, sanguinarine (SA), chelerythrine (CHE), fagaronine (FA), and the extract from Macleaya cordata in primary cultures of human and porcine hepatocytes. The cellular damage was assessed by the MTT assay, lactate dehydrogenase (LDH) leakage and the determination of intracellular glutathione (GSH) levels. The results are summarised as follows: (i) The alkaloids tested in doses 0.1 and 10 microM did not display statistically significant cytotoxicity for 0-3 h incubation; (ii) SA and CHE showed the dose- and time-dependent toxicity within the range 25-100 microM whereas FA was not toxic; (iii) the LDH leakage into the medium was higher for SA than for CHE, thus revealing a potent potential of SA to disturb cell-membrane integrity; (iv) after 3 h incubation with 100 microM SA/CHE, mitochondrial dehydrogenase activity (MTT assay) and the cellular GSH levels decreased to residual values of about 40% suggesting that mitochondria are unlikely to be a primary target for SA/CHE in the cell; (v) no differences were found in the response to QBA application in human vs porcine hepatocyte.

Expression of attractin and its differential enzyme activity in glioma cells.

Attractin/mahogany protein was previously shown to be involved in a number of physiological and pathological events, including immune system regulation, body weight control, pigmentation, myelinization, and tumor susceptibility. Human attractin has an enzymatic activity resembling dipeptidyl peptidase IV (DPP-IV). In the central nervous system, attractin has been detected in neurons but not in glial cells up to now. We show the expression of attractin mRNA and protein in glioma cell lines at different degree of transformation. In human U373 and U87 glioma cells (Grades III and IV), membrane-bound attractin displays hydrolytic activity amounting to 5 and 25% of total cellular DPP-IV-like enzyme activity, respectively. Such activity has not been observed in the rat C6 glioma cells (Grade I). Attractin presence in glioma, but not in normal glial cells, together with its differential enzymatic activity, suggests its role in growth properties of tumors of glial cell origin.

Dipeptidyl peptidase IV in two human glioma cell lines.

There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III) and U87 glioblastoma multiforme (Grade IV) lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further studies of function of this enzyme in human glioma cells.

Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities?

Membrane-bound proteases are widely distributed among various cell systems. Their expression in a particular cell type is finely regulated, reflecting the specific functional cell implications and engagement in defined physiological pathways. Protein turnover, ontogeny, inflammation, tissue remodeling, cell migration and tumor invasion are among the many physiological and pathological events in which membrane proteases play a crucial role, both as effector as well as regulatory molecules. The presence of proline residues gives unique structural features to peptide chains, substantially influencing the susceptibility of proximal peptide bond to protease cleavage. Among the rare group of proline-specific proteases, dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) was originally believed to be the only membrane-bound enzyme specific for proline as the penultimate residue at the amino-terminus of the polypeptide chain. However, other molecules, even structurally non-homologous with the DPP-IV but bearing corresponding enzyme activity, have been identified recently. This review summarizes the present knowledge of "DPP-IV activity- and/or structure-homologues" (DASH) and provides some insight into their multifunctional roles.

Heterogeneity of dipeptidyl peptidase IV from C6 rat glioma cells.

Dipeptidyl peptidase IV is known to be involved, due to both hydrolytic and non-hydrolytic mechanisms, in various cell functions of normal and cancer cells as well. In this report dipeptidyl peptidase IV substrate and pH preferences, some inhibition parameters, freezing/thawing sensitivity and stability against hydrolysis by trypsin were studied in C6 rat glioma cells. Our results confirmed substantial heterogeneity of dipeptidyl peptidase IV population. Such observation is important to avoid methodological artifacts and to decrease risk of misinterpretations in biological studies.

[Hydraulic prosthesis for regulating the sling tension in the treatment of urinary incontinence in women]. / Prótesis hidráulica para regular la tensión del "sling" en el tratamiento de la incontinencia urinaria en la mujer.

OBJECTIVE: To improve the results of corrective surgery for severe urinary incontinence in the female (sphincteric lesion) utilizing a new prosthesis that allows adjusting the degree of tension of the band or urethrocervical sling. METHODS: We have added the use of a subcutaneous hydraulic device to the classical pubovaginal sling procedure. The device can be gradually filled by percutaneous punction to adjust the urethral closure pressure. RESULTS: All patients had previously undergone different procedures and had grade III stress urinary incontinence. With the hydraulic device, the success rate (complete continence) was 95.5% (21/22 patients) and the complication rate was 9% (2 cases; one developed infection and the other chronic urinary retention). CONCLUSIONS: The hydraulic device described herein is easy to place, low-cost and carries a minimum morbidity. Its use is indicated in patients who have previously undergone surgery, with a sphincteric lesion and who require precise adjustment of the urethrocervical sling. The degree of tautness can be adjusted repeatedly according to the clinical course of a patient without requiring reoperation.

Calcium-mediated endothelin signaling in C6 rat glioma cells.

Endothelin induced intracellular Ca(2+)signaling was studied in C6 rat glial cells. Endothelins 1 and 3 increased transiently intracellular Ca(2+)concentration, endothelin 3 being less potent inducer. Dibutyryl-cAMP treated cells responded with less sensitivity. While BQ123, a specific endothelin A receptor antagonist, inhibited both endothelins induced response in proliferating cells, it failed to inhibit in dibutyryl-cAMP treated ones. IRL1620, a specific endothelin B receptor agonist, was devoid of any significant effect. Although re-stimulation by both endothelins after endothelin-1 did not cause any Ca(2+)oscillation, both endothelins evoked new Ca(2+)transient after endothelin-3 stimulation. Our findings suggest that endothelin induced Ca(2+)signaling is mediated probably through the receptor A in proliferating C6 cells. The lack of both BQ123 and IRL 1620 effect in dibutyryl-cAMP treated cells could be caused by an alteration of endothelin A receptor alone, by a change of receptor expression pattern, or by more complex postreceptor mechanism.