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F Radvanyi

Publications and source records attributed to F Radvanyi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Moderate intergenerational and somatic instability of a 55-CTG repeat in transgenic mice.

Myotonic dystrophy (DM) is associated with the expansion of a (CTG)n trinucleotide repeat in the 3' untranslated region (UTR) of the DM protein kinase gene (DMPK). The (CTG)n repeat is polymorphic and varies in size between 5 and 37 repeats in unaffected individuals whereas in affected patients there are between 50 and 4,000 CTGs. The size of the (CTG)n repeat, which increases through generations, generally correlates with clinical severity and age of onset. The instability of the CTG repeat appears to depend on its size as well as on the sex of the transmitting parent. Moreover, mitotic instability analysis of different human DM tissues shows length mosaicism between different cell lineages. The molecular mechanisms of triplet instability remain elusive. To investigate the role of genomic sequences in instability, we produced transgenic mice containing a 45-kb genomic segment with a 55-CTG repeat cloned from a mildly affected patient. In contrast to other mouse models containing CAG repeats within cDNAs, these mice showed both intergenerational and somatic repeat instability.

Animals↗
PubMed1997

[The PSA gene: value in detection of circulating prostate cancer cells].

There has been a renewed interest in detection of circulating cancer cells due to the simplicity, specificity and sensitivity of a molecular biology technique: RT-PCR. This detection is based on the concept of specificity of an RNA sequence expressed by a cancer cell. PSA appears to be the ideal marker for circulating prostate cancer cells due to its specificity. The authors review the genes of the kallikrein family and discuss the advantages and difficulties involved in the use of PSA messenger RNA as a marker for circulating prostate cancer cells.

Blotting, Northern↗
PubMed1996

Functions of fibroblast and transforming growth factors in primary organoid-like cultures of normal human urothelium.

Previous studies have indicated that growth factors such as epidermal growth factor, transforming growth factor alpha, and fibroblast growth factor 1 (FGF-1) have important regulatory functions in murine urothelial wound healing and tumorigenesis. Immunocytochemical analyses suggest that these factors are also involved in human urothelium. Yet, little is known about the functional effects of these growth factors on human urothelial cells. We established organoid-like primary cultures of normal human urothelium on porous membranes. Direct functional effects of growth factors were examined on confluent cultures reflecting intact urothelium. Immunocytochemistry was performed with a panel of specific antibodies against growth factors and their receptors on both cultures and the corresponding tissue sections. Lacking the appropriate antibodies, we performed reverse transcriptase PCR to detect FGF receptor mRNA in cultures and dissected tissue. The proliferation was stimulated by transforming growth factor alpha, FGF-1, and weakly by FGF-7, but not by FGF-2. TGF beta 1 inhibited proliferation. In contrast to mouse urothelium, none of the growth factors showed an effect on differentiation. The functional data correlate with the urothelial expression of epidermal growth factor receptors, TGF beta receptor types I and II, the (low) protein expression of FGF receptor 1, and the presence of FGF-7 receptor (FGF receptor 2 (IIIb)) mRNA. The organotypic nature of the cultures permits the study of growth factor interactions between urothelial cells. The data indicate that FGF-1, transforming growth factor alpha, and TGF beta 1 contribute differently to the maintenance of human urothelium.

Base Sequence↗
PubMed1995

Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas.

Increased serum levels of human chorionic gonadotropin beta subunit (hCG beta) were described previously in patients with bladder cancer. To obtain insight into such production of hCG beta, the expression of hCG beta 7, 8, 5, and 3 genes in bladder carcinomas and normal urothelia was investigated by reverse transcription PCR. Surprisingly, hCG beta mRNAs were detected in both normal urothelial and carcinomatous cells. However, tumor progression was characterized by different patterns of transcription of the hCG beta genes; the beta 7 gene was the only gene transcribed in normal urothelia and Ta tumors included in this study, whereas in addition to beta 7, genes beta 5, 8, and 3 were transcribed in T1 to T4 tumors. Moreover, transcription levels of the latter three genes increased with the stage of the disease. These observations showed that dramatic modifications in the expression of hCG beta genes accompany progression of bladder carcinomas.

Base Sequence↗
PubMed1995

Androgen suppressed apoptosis is modified in p53 deficient mice.

Several in vitro studies have provided evidence that the tumor suppressor protein, p53, is involved in the cell death process referred to as apoptosis. The recent development of p53 knock-out mice has enabled further investigation into the function of p53 for apoptosis, in vivo. Radiation-induced apoptosis is suppressed in such mice, yet other forms of apoptosis do not seem to be significantly affected. In this report, we present evidence that such male p53 nullizygous mice have less apoptosis in the prostate glands associated with the first 4 days following castration. Ventral prostate glands were obtained from normal, heterozygous p53-null and p53 nullizygous mice at daily intervals after castration. These tissues were stained for apoptosis with the use of the in situ and labeling method and apoptotic bodies were quantified by microscopy. Although labeled apoptotic bodies were observed in post-castrated tissues from all of these genetic variant mice, the onset of apoptosis was delayed and the occurrence of apoptosis was significantly reduced in the p53 nullizygous mice when compared to normal controls. Heterozygous p53-null mice were intermediate for these criteria. Examination of the internucleosomal DNA fragmentation pattern at 2 days of castration supports a significant diminution of prostate cell apoptosis in nullizygous p53 mice. Additionally, large nucleated and multinucleated cells were detected in the prostate epithelium of noncastrated p53 nullizygous mice and these abnormal cells were increased after castration. Flow cytometric analysis of these tissues confirmed a high number of 4C and 8C DNA content cells in the p53 nullizygous prostates and their frequency was increased by castration. In concordance with an earlier study, we conclude that functional p53 protein is not essential for prostate epithelial cells to undergo castration-induced apoptosis. However, wild-type p53 does appear to enhance this process, especially in the early period following castration, and this protein may regulate an aberrant prostate cell cycling activity that follows castration.

Androgens↗
PubMed1995

Expression of MAGE genes in transitional-cell carcinomas of the urinary bladder.

Human genes MAGE-1 and MAGE-3 code for distinct antigens, which are recognized on melanoma cells by autologous cytolytic T lymphocytes (CTL). These antigens may constitute useful targets for anti-cancer immunotherapy, since no expression of MAGE genes has been observed in normal tissues other than testis. Out of 57 samples of primary transitional-cell carcinomas of the bladder, 12 (21%) expressed MAGE-1 and 20 (35%) expressed MAGE-3. All but one of the tumors expressing MAGE-1 also expressed MAGE-3. Genes MAGE-2 and MAGE-4, which are closely related to MAGE-1 and MAGE-3, were expressed by 30% and 33% of the tumors respectively. MAGE expression was more frequent in advanced tumor stages: 61% of the invasive tumors (stage > or = T2) were positive for expression of at least one of the four genes, whereas only 28% of the superficial tumors (stages Ta and T1) expressed these genes.

Antigens, Neoplasm↗
PubMed1994

Purification of an acidic phospholipase A2 from Bothrops lanceolatus (fer de lance) venom: molecular and enzymatic properties.

The acidic phospholipase A2 from Bothrops lanceolatus venom has been purified by gel filtration on Sephadex G-50 and ion exchange chromatography on CM-cellulose. Analysis by FPLC on Mono-Q column of the purified phospholipase A2 indicated that it is a mixture of several isoenzymes. The two major isoforms consist of a single polypeptide chain with mol. wts of 14,500 and 15,000, which slightly differ in their isoelectric point (4.9 and 5.3) and amino acid composition. However, enzymatic and pharmacological properties of the various isoenzymes are identical. The phospholipase from B. lanceolatus venom is characterized by a progressive increase in the rate of hydrolysis when enzymatic activity is determined with crude egg yolk as substrate in the absence of detergent. This phenomenon, which is not observed with mixed micelles of lecithin-detergent, is not due to the presence of a phospholipase A2 inhibitor in the venom, as previously suggested by several investigators in the case of other Bothrops and Cobra venoms. It is rather a catalytic characteristics of B. lanceolatus venom phospholipase, the enzymatic activity of which depends on the physical state of phospholipids. Bothrops lanceolatus acidic phospholipase A2 is non-toxic.

Amino Acids↗
PubMed1994

Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells.

After differential screening of a cDNA library constructed from quail neuroretina cells infected with the v-myc containing avian retrovirus MC29, we have isolated a cDNA clone Pax-QNR, homologous to the murine Pax6 which is mutated in the autosomal dominant mutation small eye (Sey) of the mouse and aniridia in man. Here we report the characterization of Pax-QNR/Pax-6 expression in the chicken, quail, and mouse pancreas. In situ hybridization performed with E3 chick embryos demonstrated that, in addition to the documented expression of Pax-QNR/Pax-6 in the neural tube, this gene is also expressed in the pancreatic bud. This expression is later restricted to discrete parts of the organ. From bacterially expressed Pax-QNR peptides we obtained rabbit antisera (paired domain, serum 11; domain between paired and homeo, serum 12; homeodomain, serum 13; and carboxyl-terminal part, serum 14) capable of specifically recognizing Pax-QNR/Pax-6 proteins (48, 46 kilodaltons) in cell lines derived from alpha- and beta-pancreatic cells, but not from exocrine derived cell lines. We conclude that Pax-QNR/Pax-6 represents another gene expressed both in the endocrine pancreas and neuro-ectodermic tissues.

Animals↗
PubMed1993

Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action.

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase-A2 subunit (CB) and a non-enzymic, non-toxic subunit (CA). Crotoxin complex (CACB) dissociates upon interaction with membranes: CB binds while CA does not. Moreover, CA enhances the toxicity of CB by preventing its non-specific adsorption. Several crotoxin isoforms have been identified. Multiple variants of each subunit give different crotoxin complexes that can be subdivided into two classes: those of high toxicity and low enzymic activity and those of moderate toxicity and a high phospholipase-A2 activity. In this study, we demonstrate that the more-toxic isoforms block neuromuscular transmission of chick biventer cervicis preparations more efficiently than weakly toxic isoforms. The less-toxic crotoxin complexes have the same Km and Vmax as CB alone. In contrast, the more-toxic isoforms are enzymically less active than CB. These differences correlate with the stability of the complexes: less-toxic isoforms are less stable (Kd = 25 nM) and dissociate rapidly (half-life about 1 min), whereas the more-toxic isoforms are more stable (Kd = 4.5 nM) and dissociate more slowly (half-life 10-20 min). The rate of interaction of crotoxin complexes with vesicles of negatively charged phospholipids paralleled the rate of dissociation of the complexes in the absence of vesicles. The differences of pharmacological and biochemical properties of crotoxin isoforms indicate that the stability of crotoxin complexes plays a major role in the synergistic action of crotoxin subunits: a stronger association between the two crotoxin subunits would account for their slower dissociation rate, a weaker enzymic activity, a slower interaction with phosphatidylglycerol vesicles, a faster blockade of neuromuscular transmission and a higher lethal potency.

Animals↗
PubMed1993

Pancreatic beta cells cultured from individual preneoplastic foci in a multistage tumorigenesis pathway: a potentially general technique for isolating physiologically representative cell lines.

Culturing and comparing the discrete stages of tumorigenesis provide a route to defining important components of the cancer phenotype and, in addition, present the opportunity to establish cell cultures more representative of normal cells than the ultimate malignant cancer cells. Herein we report that preneoplastic foci in one multistep tumorigenesis pathway can be cultured in vitro and show that they preserve distinctive characteristics of the normal cells from which they arose, pancreatic beta cells. In the RIP1-Tag2 line of transgenic mice, which express the simian virus 40 T antigen in insulin-producing beta cells, pancreatic islets develop into vascularized tumors in a multistage pathway. We established conditions for reproducible derivation of beta-cell lines from individual hyperplastic islets that have not yet developed into solid tumors. Most of these cell lines, designated beta HC, release insulin at physiological concentrations of glucose. In contrast to tumor-derived lines (beta TC), which are not properly regulated, the ability of the beta HC lines to respond correctly to glucose correlated with maintenance of normally depressed levels of low-Km hexokinases. Glutamic acid decarboxylase (GAD), an early autoantigen in type I diabetes, was detected in most of the beta HC lines. The relative levels of the two forms of this enzyme (GAD65 and GAD67) varied significantly between the different cell lines, suggesting independent regulation. Class I major histocompatibility complex antigens were detected on the beta HC cells, and the levels of surface major histocompatibility complex expression correlated with their capacity to serve as targets in a cytotoxic T-cell killing assay. The beta HC lines will be of value for studies of beta-cell physiology, autoantigenicity, and tumor development. This work suggests the possibility of culturing preneoplastic stages of other cancers, both to address the mechanisms of transformation and to provide a source of cells that maintain important qualities of their normal progenitors.

Adenoma, Islet Cell↗
PubMed1991

Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma.

In a transgenic mouse model, dermal fibrosarcomas develop in a pathway comprised of at least three stages: mild fibromatosis, aggressive fibromatosis, and fibrosarcoma. The latter two stages are highly vascularized when compared with both the normal dermis and the initial mild lesion. Analysis of cell cultures derived from biopsies of these lesions has revealed that basic fibroblast growth factor (bFGF) is synthesized in all three stages and in normal dermal fibroblasts derived from the same mice. Unexpectedly, there is a change in the localization of bFGF from its normal cell-associated state to extracellular release in the latter two stages, which is concomitant both with the neovascularization seen in vivo and with the tumorigenicity of these cell lines. Thus, in this multistep tumorigenesis pathway there appears to be a discrete switch to the angiogenic phenotype that correlates with the export of bFGF, a known angiogenic factor.

Animals↗
PubMed1991

Crotoxin, a phospholipase A2 neurotoxin from snake venom, interacts with epithelial mammary cells, is internalized and induces secretion.

Prolactin (PRL) induces liberation of arachidonic acid (AA) from phospholipids of lactating mammary epithelial cells and stimulates casein secretion. In order to investigate the possible involvement of phospholipase A2 (PLA2) activity in the hormonal control of casein secretion by PRL, we examined the effects of crotoxin, a PLA2 neurotoxin from snake venom, on mammary epithelial cells. Crotoxin is made of two subunits: a basic PLA2 with low toxicity (component B, CB) and an acidic, non-toxic and enzymatically inactive component A (CA) which enhances the pharmacological action of CB. While CA is inactive, the PLA2 subunit (CB) induces an accumulation of secretory products in the lumen of mammary acini, an extensive development of the Golgi apparatus. The secretion of newly synthesized casein is increased in the presence of CB and this effect is inhibited by nordihydroguaiaretic acid (NDGA) and caffeic acid, two inhibitors of the lipoxygenase pathway which also prevent stimulation of secretion by PRL. Further, CB transiently induces the release of radiolabelled AA from mammary tissues previously labelled with [14C]AA, the highest release being observed between 15 s and 5 min of contact with CB and CA. Immunofluorescence labelling by anti-CB antibodies of epithelial mammary tissues previously incubated with CA, CB or a combination of CA and CB indicates that CB binds to epithelial cells and is internalized, at least in part, and that CA enhances both CB binding and its internalization. These observations emphasize the involvement of PLA2 in the control of casein secretion and suggest that PLA2 acts intracellularly.

Animals↗
PubMed1989

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids.

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.

Cations, Divalent↗
PubMed1989

A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin.

Phospholipase A2 activity can be determined fluorometrically in the presence of serum albumin using phospholipids labeled at the sn-2-acyl position with 10-pyrenyldecanoic acid. In the water reaction medium 10-pyrene phospholipids form vesicles and the monomer fluorescence of the pyrene is negligible due to pyrene-pyrene interaction. Upon phospholipid hydrolysis 10-pyrenyldecanoic acids are produced and tightly bind to albumin so that a monomer pyrene fluorescence is observed. We obtained an excellent parallelism between hydrolysis determined by a classical extraction method and that followed by direct and continuous spectrofluorometric recording of the monomer emission of pyrene. This assay can measure picomole amounts of phospholipids hydrolyzed per minute so that picogram quantities of phospholipases A2 from pancreas or from venoms can be measured. Phospholipase activity remains proportional to enzyme concentration over three orders of magnitude. The method can be used to quantify the phospholipase A2 activity of crude extracts of low specific activity.

Hydrolysis↗
PubMed1989

Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab).

Rabbit antibodies were prepared against both purified catalytic (component-B) and purified non-catalytic (component-A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross-react with crotoxin-like toxins from the venom of several Crotalus species as well as with single-chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross-reactions of anti-component-A and anti-component-B sera with crotoxin and with its isolated components A and B showed that component-A exposes determinants of low immunogenicity which are present on component-B, whereas the major antigenic determinants of component-B are not present on component-A. Anti-component-B antibodies, but not anti-component-A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non-precipitating anti-component-B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immunoprecipitate.

Antibodies↗
PubMed1989

Binding of crotoxin, a presynaptic phospholipase A2 neurotoxin, to negatively charged phospholipid vesicles.

Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of less than 1 microM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to less than 1 microM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.

Chemical Phenomena↗
PubMed1989

Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin.

Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.

Crotoxin↗