Photoaffinity labeling and expression cloning of extracellular ATP receptors of cardiac myocytes.
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Adenosine Triphosphate↗
Biomedical subjects
A Scarpa
Publications and source records attributed to A Scarpa.
p53 gene mutation spectrum in hepatocellular carcinoma.
In order to clarify the significance of mutation of the p53 tumor suppressor gene in the genesis and development of human hepatocellular carcinoma (HCC) in an aflatoxin B1 low-exposure area, the spectrum, i.e., incidence, type, and site, of p53 gene mutations was examined in 169 tissue samples resected mainly from Japanese patients using single-strand conformation polymorphism analysis and direct sequencing. Forty-nine tumors (29%) showed a p53 mutation (39 point mutations and 10 frameshifts). The point mutations comprised 18 transitions, only 4 of which occurred at CpG sites, and 21 transversions. Two evolutionarily conserved domains, IV and V, contained 65% of all mutations and codon 249 was the most frequent mutation site (7/49). The spectrum of p53 mutation did not differ among HCCs in relation to the type of hepatitis virus infection, sex, age, and background liver disease of patients, tumor size, or presence of metastasis, but incidence and site were significantly associated with the degree of differentiation of cancer cells. In poorly differentiated HCC, p53 mutation was frequent (54%) and clustered on domains IV and V, whereas in well or moderately differentiated HCC, the mutation was less frequent (21%) and equally distributed on domains II to V. Restriction fragment length polymorphism analysis revealed loss of heterozygosity on chromosome 17p in 55 (69%) of 80 informative cases and in 34 (95%) of 36 cases with p53 mutation. Therefore, p53 gene mutation is suggested to occur independently of the type of viral infection or status of preexisting liver disease and to occur preferentially in moderately and poorly differentiated HCCs in association with or after loss of another p53 allele as a late event of HCC progression.
Adult↗
Characterization of myocardial extracellular ATP receptors by photoaffinity labelling and functional assays.
Extracellular ATP receptors in rat ventricular myocytes were investigated through intact cell photolabelling followed by protein isolation. 8-Azido-ATP (8Az-ATP) was used for labelling under specific conditions determined by parallel functional studies. In those studies ATP-induced cytosolic Ca2+ transients were irreversibly and specifically inhibited by UV-photolyzed 8Az-ATP, but not by 2-azido-ATP (2Az-ATP), even in the presence of high concentrations of phosphonucleotides not affecting myocardial ATP receptors. Under those conditions background labelling is minimized and radioactive 8Az-ATP specifically labels a band of 45-48 kDa on a SDS gel. Labelling under the above conditions in the presence of ATP gamma S or 2-methylthio-ATP (2-meSATP), which are distinct for two functionally different cardiac ATP receptors, shows two different proteins within the same band consistent with the possible labelling of these two receptors.
Adenosine Diphosphate↗
Mutation pattern of the p53 gene as a diagnostic marker for multiple hepatocellular carcinoma.
Hepatocellular carcinoma, sometimes shows multiple tumor nodules, therefore poses a problem of differential diagnosis between cancers of multifocal and those of metastatic origin. Conventionally, pathological criteria have been used for this purpose, but these are largely subjective. In order to facilitate more objective differential diagnosis of multiple hepatocellular carcinoma, we used the pattern of mutation of the p53 gene as a marker for each tumor nodule. We studied 58 nodules from 26 cases of multiple hepatocellular carcinoma using polymerase chain reaction-single strand conformation polymorphism analysis, a simple method for detecting mutations. p53 gene mutations were detected in 65% (17 of 26) of cases. The internodule mutation patterns were heterogeneous in 11 cases and homogeneous in 6, enabling a multifocal origin to be diagnosed in the former and a metastatic origin in the latter at the genetic level. Moreover, the origin of recurrent tumors was determined from the mutation pattern. It is concluded that analysis of p53 mutations seems to be useful for differentiating the origin of multiple cancers, since the information it yields is essentially objective.
Base Sequence↗
Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C.
A large Mg2+ cell uptake against concentration gradients is stimulated in collagenase-dispersed rat myocytes by carbachol and in hepatocytes by carbachol or vasopressin. The signalling pathway(s) responsible for this stimulation of Mg2+ uptake was investigated by using various activators or inhibitors of protein kinase C (PKC) and by correlating Mg2+ uptake with cell PKC activity and cAMP content. In both cell preparations, the direct stimulation of PKC by diacylglycerol analogs or phorbol esters reproduce the same pattern of Mg2+ uptake as that induced by carbachol or vasopressin. These data indicate that the activation of PKC is responsible for a stimulation of Mg2+ uptake by myocytes or hepatocytes, whereas increase in cAMP in these cells stimulates Mg2+ release.
Animals↗
Association of Epstein-Barr virus genome with mixed cellularity and cellular phase nodular sclerosis Hodgkin's disease subtypes.
Association of Epstein-Barr virus (EBV) genome with Hodgkin's disease (HD) histological subtypes was investigated using the polymerase chain reaction (PCR). A highly significant association of EBV genome with the mixed cellularity (MC) subtype (10 positive out of 15 cases; 66%) was observed, suggesting a possible etiopathogenetic role of EBV in the induction of this subset. By contrast, a markedly lower frequency of association with EBV genome was found in nodular sclerosis (NS) (12 positive out of 46 cases; 26%) and in nodular lymphocytic predominance (NLP) (0 positive out of 5 cases) HD subtypes. In addition, in the NS series, the presence of EBV genome was mainly restricted to the 'cellular phase' NS subset. This finding strengthens the possibility, suggested by clinico-pathological features and survival rates, that 'cellular phase NS' is a disease more akin to MC than to typical NS HD.
Genome, Viral↗
Neoplastic epithelial cells in a subset of human thymomas express the B cell-associated CD20 antigen.
A series of 36 human thymomas have been immunohistochemically analyzed using a panel of antibodies recognizing B-cell markers including CD20. Most thymomas exhibiting the cortical pattern, according to the criteria of Marino and Muller-Hermelink, were characterized by areas of medullary differentiation containing variable numbers of CD20+ B lymphocytes, thus mimicking the medulla of normal thymus. On the other hand, B cells were absent or rare in thymomas recognized as mixed using the same morphological criteria. Surprisingly, we observed in most mixed thymomas variable numbers of CD20+ spindle cells, characterized by long slender processes. Using double-marker analysis we could demonstrate the epithelial nature of these cells (expression of keratin and lack of lymphoid and B-cell-related markers). The immunoreactivity of thymoma epithelial cells with L26, an antibody widely used in the characterization of B-cell lymphomas, can represent a drawback of practical relevance in the differential diagnosis of mediastinal tumors.
Antibodies, Monoclonal↗
Immunodetection of proliferating cell nuclear antigen assesses the growth fraction and predicts malignancy in endocrine tumors of the pancreas.
Thirty-five endocrine tumors of the pancreas, 17 functioning and 18 nonfunctioning, were immunohistochemically studied for the expression of proliferating cell nuclear antigen (PCNA) using 19A2 and PC10 monoclonal antibodies. The proportion of PCNA-reactive cells (PCNA index) ranged from 0.2 to 27% in functioning tumors and from 0.1% to 55% in nonfunctioning tumors. PCNA index showed a statistically significant correlation with mitotic and Ki67 indexes. The median values of PCNA index identified three groups of patients: group A (PCNA < or = 2%), including 13 functioning and six nonfunctioning tumors; group B (PCNA between 2 and 5%), including three functioning and three nonfunctioning tumors; group C (PCNA > 5%), including one functioning and nine nonfunctioning tumors. All group A tumors were confined to the pancreas. In group B, the functioning tumors were limited to the pancreas, and the nonfunctioning tumors extended to extrapancreatic tissues. All group C patients had extrapancreatic extension of the disease. At follow-up, a PCNA index higher than 5% correlated to a decreased mean survival. Our data suggest that PCNA index is a reliable tool to assess the growth fraction, discern local from advanced diseases, and predict malignancy in pancreatic endocrine tumors.
Adolescent↗
Synergism between cAMP and ATP in signal transduction in cardiac myocytes.
ATP transiently increases the intracellular Ca2+ concentration in cardiac myocyte suspensions. Pretreatment with norepinephrine (NE) greatly potentiates the ATP response. We performed experiments on adult rat myocyte suspensions loaded with fura-2 to investigate the mechanism of NE potentiation. We found that forskolin (an activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase), and permeative adenosine 3',5'-cyclic monophosphate (cAMP) analogues potentiate the increase in cytosolic Ca2+ concentration induced by ATP. NE, forskolin, and 8-(4-chlorophenylthio)-cAMP all increase Vmax of the Ca2+ response curve of ATP. Measurement of cAMP by radioimmunoassay confirmed that the changes in the ATP response were accompanied by an increase in cAMP. These results suggest that the noradrenergic potentiation of the ATP-induced Ca2+ mobilization involves cAMP as a second messenger. Patch-clamp studies of isolated myocytes showed that neither NE nor forskolin alters the inward current elicited by ATP, but rather they increase the duration of secondary slow action potentials elicited by ATP. NE also increases the Ca2+ current through L-type Ca2+ channels in the myocytes. We conclude that NE potentiates the ATP-induced Ca2+ transient by increasing cAMP levels and that one of the early events is the increase of the inward Ca2+ current during the action potential.
Adenosine Triphosphate↗
Ca2+ mobilization by extracellular ATP in rat cardiac myocytes: regulation by protein kinase C and A.
Activation of protein kinase C (PKC) modulates the mobilization of intracellular Ca2+ induced by extracellular ATP in rat ventricular myocytes. Pretreatment of myocytes with PKC activators attenuated both the ATP-induced Ca2+ transient and the noradrenergic potentiation of the Ca2+ response. Various PKC activators decreased both the basal cAMP level and the cAMP levels that had been elevated by norepinephrine, forskolin, or 3-isobutyl-1-methylxanthine. The inhibitory effects of PKC activators were reversed by the PKC inhibitor staurosporine. The ATP-induced Ca2+ response is an integrated response resulting from ATP eliciting an inward cation current (IATP), cellular depolarization, Ca2+ influx through Ca2+ channels, and Ca2+ release from the sarcoplasmic reticulum. We used the whole cell voltage-clamp technique to investigate which steps of this integrated response are affected by PKC. PKC activators did not significantly affect the IATP. In contrast, PKC activators decreased the basal Ca2+ current (ICa) or Ba2+ current and the beta-adrenergic-stimulated ICa. These results suggest that PKC-induced suppression of the ATP-induced Ca2+ response and the beta-adrenergic-potentiated Ca2+ response is achieved at least partially by decreasing the intracellular cAMP level and ICa.
Adenosine Triphosphate↗
cAMP control of Mg2+ homeostasis in heart and liver cells.
The increase of intracellular cAMP level in intact myocytes and hepatocytes induced by beta-adrenergic stimulation or by forskolin or permeant cyclic AMP analogues induces a sizeable Mg2+ efflux from the cells. Approximately 10-15% of total cell Mg2+ is mobilized over 5-8 min stimulation. The addition of cAMP to digitonin-permeabilized hepatocytes also mobilizes Mg2+ from intracellular compartment(s). Furthermore, cAMP stimulates a major Mg2+ extrusion from isolated rat liver mitochondria, indicating this compartment as the main source of Mg2+ mobilization in liver cells.
Animals↗
Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.
The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.
Adenosine Triphosphate↗
A splice site mutation of the beta-spectrin gene causing exon skipping in hereditary elliptocytosis associated with a truncated beta-spectrin chain.
We studied a French kindred with hereditary elliptocytosis associated with a spectrin variant (spectrin LePuy) containing a beta-spectrin chain that is truncated at its C terminus (Dhermy, D., Lecomte, M., Garbarz, M., Bournier, O., Galand, C., Gautero, H., Feo, C., Alloisio, N., Delaunay, J., and Boivin, P. (1982) J. Clin. Invest. 70, 707-715). The structure of the 3' end of the beta-spectrin gene, the region encoding the C terminus of beta-spectrin, was determined. Nucleotide sequencing of amplified genomic DNA revealed a mutation at position +4 (A----G) of the 5' donor consensus splice site of the intron following the third-to-last exon (exon X) in one beta-spectrin allele of a heterozygous patient. Agarose gel electrophoresis of polymerase chain reaction-amplified cDNA revealed an extra band of lower molecular weight, suggesting that the shortened beta-spectrin chain of spectrin LePuy arises from aberrant mRNA splicing. Nucleotide sequencing of the shorter cDNA amplification product revealed that the sequences encoding exon X were absent. Southern blotting of cDNA amplification products confirmed this result. The skipping of exon X causes a shift in the normal reading frame resulting in the encoding of a new amino acid sequence at the C terminus of the mutant beta-spectrin chain. A new in-frame stop codon is encountered following a single residue of this novel sequence.
Amino Acid Sequence↗
Evidence of c-myc gene abnormalities in mediastinal large B-cell lymphoma of young adult age.
Six cases of mediastinal large B-cell lymphoma (MLCL) with sclerosis were analyzed for the presence and patterns of c-myc and bcl-2 loci rearrangements, and for the presence of Epstein-Barr virus DNA sequences by Southern blot hybridization, c-myc gene alterations were found in three of six cases. Two cases showed the presence of mutations or small rearrangements at the 3' end of the first exon. The c-myc gene abnormalities found in these two cases are similar to those observed in the translocation 8;14 of the endemic Burkitt's lymphomas or in its variants t(2;8) and t(8;22). A third case showed a major rearrangement of c-myc gene, with truncation within its first intron, similar to those observed in sporadic Burkitt's and in acquired immunodeficiency-associated lymphomas. None of the cases displayed bcl-2 gene rearrangements or contained viral sequences. Our data suggest a possible role for a translocation-mediated c-myc activation in the pathogenesis of MLCL. Conversely, bcl-2 gene and Epstein-Barr virus do not appear to be involved in the pathogenesis of these peculiar lymphomas. The association between c-myc structural modifications and MLCL also seems to be of relevance in light of the peculiar tendency of this tumor to involve unusual extranodal site (eg, kidney), reminiscent of the spreading attitude of Burkitt's limphomas.
Adult↗
An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216).
Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C-terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.
Adolescent↗
Improved renal transplant preservation using a modified intracellular flush solution (PB-2). Characterization of mechanisms by renal clearance, high performance liquid chromatography, phosphorus-31 magnetic resonance spectroscopy, and electron microscopy studies.
A number of new intracellular renal flush solutions have been found to be more efficacious than Collins-2 (C-2) solution in extending organ viability during simple cold storage. However, the mechanism of action of these solutions remains poorly understood. To delineate better underlying intracellular mechanisms, we studied a modified, simple, hypothermic, intracellular (340 mOsm/kg) flush solution (PB-2). The development of PB-2 solution is based on the ability of some of its individual components to minimize ischemic adenine nucleotide (AN) catabolism and endothelial post "reperfusion injury." Preliminary results in 10 canine autorenal transplants show a significant (P less than 0.02) improvement in renal recovery and viability (recipient posttransplant inulin clearance and survival) after 50 h of cold storage compared with 10 canine kidneys similarly preserved using conventional C-2 flush solution. High performance liquid chromotography (HPLC) studies show a significant (P less than 0.01) loss of AN using C-2, while PB-2 was associated with regeneration of AN within 45 min of reperfusion. Magnetic resonance spectroscopy using phosphorus 31 (31P-MRS) showed more high energy phosphorus metabolites (phosphomonoester and nicotinamide-adenine-dinucleotide phosphate: P less than 0.001) at 50 h cold storage using PB-2 compared with C-2. Electron micrographs (EM) revealed normal microcapillary morphology for the PB-2 group; however, moderate vascular red and white blood cell clumping was observed in the C-2 group. Characterization of the basic preservation mechanisms by HPLC, 31P-MRS, and EM studies indicates that PB-2 solution enhances renal preservation by diminution of both reperfusion injury and the loss of intracellular high energy metabolites that are necessary for viability.
Animals↗
Modulation of the B cell response to T-independent antigens by prostaglandins: evidence for effector system cross-talk.
The extracellular environment is a major factor in determining the responsiveness of a cell to particular stimuli. For example, E series prostaglandins suppress B cell responses to T-independent antigens, mitogen stimulation of DNA synthesis and proliferation, and the primary immune response. We investigated the effects of prostaglandins on the intracellular signals generated by receptor-coupled effector systems in B lymphocytes. Pretreating splenocytes from athymic nude mice with forskolin, PGE1, or PGE2 decreased the magnitude of anti-IgM-induced changes in cytosolic free [Ca2+]. Addition of 8-Br-cAMP, forskolin, PGE1, or PGE2 following stimulation with anti-IgM resulted in a decrease in the intracellular calcium signal measured by fluorescence-activated cell sorting using Indo-1 as a Ca2+ indicator. This decrease was not a result of an inhibition of influx across the plasma membrane. Thus activation of adenylate cyclase by prostaglandins modifies the generation of signals by phosphoinositidase C. This effector system cross-talk between adenylate cyclase and phosphoinositidase C is consistent with and may account for the inhibitory effects of prostaglandins in B cell responses.
8-Bromo Cyclic Adenosine Monophosphate↗