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Biomedical subjects

A Scarpa

Publications and source records attributed to A Scarpa.

At least 145 records · Page 8Linked to original sources
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

Calcium currents in the A7r5 smooth muscle-derived cell line. Calcium-dependent and voltage-dependent inactivation.

Inactivation of a dihydropyridine-sensitive calcium current was studied in a cell line (A7r5) derived from smooth muscle of the rat thoracic aorta. Inactivation is faster with extracellular Ca2+ than with Ba2+. In Ba2+, inactivation increases monotonically with depolarization. In Ca2+, inactivation is related to the amount of inward current, so that little inactivation is seen in Ca2+ for brief depolarizations approaching the reversal potential. Longer depolarizations in Ca2+ reveal two components of inactivation, the slower component behaving like that observed in Ba2+. Furthermore, lowering extracellular Ca2+ slows inactivation. These results are consistent with the coexistence of two inactivation processes, a slow voltage-dependent inactivation, and a more rapid current-dependent inactivation which is observable only with Ca2+. Ca(2+)-dependent inactivation is decreased but not eliminated when intracellular Ca2+ is buffered by 10 mM BAPTA, suggesting that Ca2+ acts at a site on or near the channel. We also studied recovery from inactivation after either a short pulse (able to produce significant inactivation only in Ca2+) or a long pulse (giving similar inactivation with either cation). Surprisingly, recovery from Ca(2+)-dependent inactivation was voltage dependent. This suggests that the pathways for recovery from inactivation are similar regardless of how inactivation is generated. We propose a model where Ca(2+)- and voltage-dependent inactivation occur independently.

Animals↗
PubMed1991

Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin.

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.

Animals↗
PubMed1991

False-positive immunostaining of normal epithelia and carcinomas with ascites fluid preparations of antimelanoma monoclonal antibody HMB45.

HMB45 is a melanoma-specific monoclonal antibody that has found widespread use in diagnostic pathology. Recent reports, however, have suggested that this antibody may cross-react with a small number of carcinomas and other epithelial cells. The authors tested the hypothesis that these latter reports represent examples of false-positive immunostaining by comparing the immunostaining on breast, salivary gland, and lung tumors with the following: (1) a commercial ascites preparation of this monoclonal antibody; (2) a protein A-purified antibody preparation derived from ascites fluid; and (3) supernatant fluid obtained from the hybridoma cell line. The authors found that all examples of nonmelanoma immunostaining in the carcinomas tested were eliminated with the nonascites fluid preparations, whereas strong immunostaining of melanomas was retained. The authors conclude that contaminated commercial ascites fluid preparations of HMB45 may account for most, if not all, of the reports of nonmelanoma immunostaining with HMB45.

Antibodies, Monoclonal↗
PubMed1991

Intracellular ionized calcium changes in squid giant axons monitored by Fura-2 and aequorin.

Squid giant axons were injected simultaneously with Ca indicators Fura-2 and aequorin. Fura-2 was calibrated in situ by measuring fluorescence at 510 nm upon UV excitation at 340 nm, 360 nm, and 380 nm with a time-sharing multiple wavelength spectrofluorimeter. Limiting values for dye fluorescence were obtained by allowing a massive load of Ca to enter the axon with the aid of procedures such as prolonged depolarization in the presence of CN (for saturation) and by sequestration of all Ca present in the axoplasm accomplished with injection of EGTA into the axon (for a zero-Ca signal). The average intracellular Ca concentration obtained with Fura-2 was 184 nM. The sensitivity of Fura-2 to intracellular Ca is at least as great as that of aequorin, thus permitting its use in the characterization of Ca homeostasis mechanisms such as Na-Ca exchange. It was found, however, that for voltage-clamp experiments requiring an internal current electrode, Fura-2 is not a convenient Ca probe because electrode reactions in the axoplasm denature the dye, thereby restricting its use in characterization of Ca movements associated with electrically induced changes in membrane potential. A comparison of aequorin luminescence with Fura-2 fluorescence demonstrated that light output by aequorin is linear with intracellular Ca concentrations up to values of 750 nM, changing to a square law relationship from 750 nM up to 10 microM Ca.

Aequorin↗
PubMed1991

Regulation of cytosolic pH of cultured mesangial cells by prostaglandin F2 alpha and thromboxane A2.

Prostaglandins (PG) and thromboxane A2 (TxA2) have marked vasoactive effects on the renal glomerular microcirculation. Exposure of cultured mesangial cells to PGF2 alpha and TxA2 mimetics results in a rapid elevation of free cytosolic Ca2+ ([Ca2+]i) followed by contraction and cell proliferation. We studied whether other ionic changes mediate these effects of eicosanoids on cells of rat and human origin. Cytoplasmic pH (pHi) was monitored in cells loaded with the fluorescent, intracellularly trapped pH-sensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. PGF2 alpha in rat cells and the TxA2 mimetic U-46619 in human cells induced rapid, dose-dependent cytosolic acidification followed by recovery and net alkalinization mediated by enhanced Na(+)-H+ exchange. The early acidification was also stimulated by ionomycin and Ca2(+)-mobilizing peptides, implicating a Ca2(+)-dependent mechanism. Alkalinization was abolished by removal of extracellular Na+ and by amiloride. Both components of the responses were inhibited by phorbol myristate acetate, which could mimic alkalinization, suggesting a regulatory role of protein kinase C in activation of the Na(+)-H+ exchanger by eicosanoids. Vasoconstrictor arachidonate metabolites may control glomerular cell function by a signaling mechanism centered on concurrent changes of pHi and [Ca2+]i.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Extracellular ATP activates coordinated Na+, Pi, and Ca2+ transport in cardiac myocytes.

Activation of an ATP receptor has previously been shown to induce cytosolic [Ca2+] transients in rat ventricular myocytes. A slower but larger [Ca2+] increase which can cause cell hypercontraction follows the transient when extracellular Pi is increased. This second phase of the [Ca2+] response is stimulated by ATP or adenosine 5'-(gamma-thio)triphosphate in a medium containing 11.2 mM Pi, but not by high concentrations of 2-methylthio-ATP, which stimulate only the initial [Ca2+] transient. Replacing medium Na+ with N-methyl-D-glucamine suppresses this Pi-dependent [Ca2+] increase following ATP addition, suggesting a causal relationship between Na+ transport and Ca2+ influx. Blocking voltage-sensitive Na+ channels, Na(+)-H+ exchange, or Na(+)-K(+)-Cl- cotransport did not reduce ATP-induced cell hypercontraction in 11.2 mM Pi medium, suggesting that these transporters are not involved. ATP stimulation of Na(+)-Pi cotransport was investigated with isotopic methods. The results were consistent with the hypothesis that extracellular ATP stimulates Na(+)-Pi cotransport, which activates Na(+)-Ca2+ exchange. A novel Pi-dependent ATP receptor-effector system has been demonstrated in cardiac cells, and it may have significant effects on cellular transport, contractility, and bioenergetics.

Adenosine Triphosphate↗
PubMed1991

Melanocyte-marker-HMB-45 is regularly expressed in angiomyolipoma of the kidney.

HMB-45 (melanocytic cell-specific monoclonal antibody) immunoreactivity was investigated in 10 cases of angiomyolipoma (AML) (1 with massive regional lymph node involvement) of the kidney and detected in all of them. No HMB-45 immunoreactivity was found in other tumors of the region which can occasionally be confused with AML, such as renal cell carcinoma, Wilms' tumor, and retroperitoneal sarcoma (leiomyosarcoma and liposarcoma). These findings indicate that HMB-45 is not a melanocyte-restricted marker and suggest that its expression might be useful in distinguishing AML from other tumors of the kidney and retroperitoneum.

Antibodies, Monoclonal↗
PubMed1991

Characterization of calcium channel forming activity of Entamoeba histolytica in model biological membranes.

Entamoeba histolytica is an invasive enteric protozoan parasite whose cytolytic activity is associated with irreversible increases in target cell intracellular calcium. We studied the effect of homogenates of virulent E. histolytica on calcium permeability of bovine chromaffin granules and rat liver mitochondria, model membrane systems whose mechanisms of ion transport and permeability are well characterized. Treatment of chromaffin granules, with an extract of E. histolytica resulted in a dose-dependent increase in calcium uptake. These effects were similar to that seen with the calcium ionophore A23187 and indicate that the homogenate acts as a divalent cation ionophore. However, unlike what is observed with A23187, the Ca2+ uptake was greater in the presence of the permeable anion Cl- than in its absence. Also, in contrast to the calcium ionophore A23187, a homogenate of E. histolytica caused no calcium release from rat liver mitochondria but resulted in a dramatic increase in the rate of calcium accumulation. The amebic homogenate did not bind calcium in the absence of mitochondria. The increase in mitochondria calcium uptake occurred only in the presence of respiration and with normal state 3 and 4 oxygen consumption. Calcium accumulation occurred both in the presence and absence of 5 mM Pi. Substitution of NaCl or KCl for sucrose in the medium did not alter the enhanced mitochondria calcium uptake. Most data are consistent with the hypothesis that the E. histolytica homogenate is acting as a calcium ionophore transporting charged calcium electrophoretically across the chromaffin granules and the mitochondria inner membrane. This activity may contribute to the cytopathogenicity by E. histolytica.

Animals↗
PubMed1991

Immunohistochemical evidence of abnormal expression of the antioncogene-encoded p53 phosphoprotein in Hodgkin's disease and CD30+ anaplastic lymphomas.

The gene encoding p53 phosphoprotein, originally believed to be an oncogene, recently has been proposed as a candidate antioncogene (tumor-suppressor gene). Abnormalities of the p53 gene expression have been demonstrated in different human malignancies including carcinomas and sarcomas, but little information concerning p53 immunoreactivity in human lymphomas is so far available. In this study immunohistochemical staining for p53-protein was performed on frozen- and paraffin-embedded samples from patients with Hodgkin's (HD) and non-Hodgkin's lymphomas (NHL). No p53 immunoreactivity could be demonstrated in any cell type in nonneoplastic lymphoid samples, including germinal center cells in reactive lymph nodes and cortical thymocytes. On the other hand, a significant proportion of p53+ neoplastic cells was observed in 23 of 31 cases of HD and 17 of 68 cases of NHL. All positive lymphoma cases were diagnosed as high-grade or CD30+ anaplastic NHL. The demonstration of abnormal expression of p53 protein in these diseases can contribute to addressing unresolved issues regarding the origin and pathogenesis of HD and CD30+ anaplastic lymphomas.

Antibodies, Monoclonal↗
PubMed1990

Hormonal control of Mg2+ transport in the heart.

Magnesium is abundant in the mammalian body and the second most abundant cation in cells. Because the concentration of intracellular free Mg2+ is relatively high (0.2-1 mM), Mg2+ is unlikely to act as a second messenger, like Ca2+, by rapidly changing its cytosolic concentration. But changes in Mg2+ do have profound effects on cellular metabolism, structure and bioenergetics. Key enzymes or metabolic pathways, mitochondrial ion transport, Ca2+ channel activities in the plasma membrane and intracellular organelles, ATP-requiring reactions, and structural properties of cells and nucleic acids are modified by changes in Mg2+ concentration. Yet, although some information is available from giant cells and bacteria, little is known about the regulation of intracellular Mg2+ in mammalian cells. Here we report a new transport mechanism for Mg2+ across the sarcolemma of cardiac cells in both intact hearts and dissociated myocytes. We show that noradrenaline, through beta-adrenergic stimulation and increase of cyclic AMP, stimulates a large efflux of Mg2+ from cardiac cells. This transport is of major dimensions and can move up to 20% of total cellular Mg2+ within a few minutes.

Animals↗
PubMed1990

Norepinephrine evokes a marked Mg2+ efflux from liver cells.

The addition of norepinephrine to perfused rat livers and to collagenase isolated hepatocytes induced a marked and dose-dependent magnesium efflux. The addition of beta-adrenergic receptor antagonists, but not alpha-antagonists, completely blocked the Mg2+ efflux. The Mg2+ efflux could also be induced by forskolin and by permeable cAMP analogues. By contrast, the addition of carbachol or vasopressin induced a Mg2+ influx into isolated hepatocytes. These results indicate that a significant Mg2+ efflux from liver cells can be induced through the beta-adrenergic receptors and that it is mediated through the cytosolic cAMP levels.

Animals↗
PubMed1990

Expression and gene rearrangement of the T-cell receptor in human thymomas.

Human thymomas are epithelial neoplasms frequently associated with an exuberant lymphoid component. This mixture of epithelial cells and lymphocytes closely mimicks the organization of normal thymic cortex. However, it is not known whether thymocytes in thymoma express the T-cell receptor (TCR) for the antigen. We have analyzed the molecular configuration of TCR genes and their phenotypic expression in eight thymomas. In all we detected polyclonal rearrangements of TCR genes and cytoplasmic expression of TCR molecules in most thymocytes, thus indicating that rearranged TCR genes in thymomas are functioning genes. In addition, these findings suggest that the epithelial component of thymomas, even if neoplastic, is still capable of directing thymocyte differentiation.

Adolescent↗
PubMed1990

Peripheral giant cell granuloma: evidence for osteoclastic differentiation.

Nine cases of peripheral giant cell granuloma of the oral cavity have been immunohistochemically analyzed to assess the nature of the giant cells. Giant cells were unreactive when tested with antibodies recognizing myelomonocytic and macrophage markers (lysozyme, MAC 387, HAM 56) but showed strong immunoreactivity with MB1, an antibody reactive with osteoclasts. It is concluded that giant cells characterizing giant cell granuloma exhibit a phenotype distinct from other giant cells found in sites of chronic inflammation and may be true osteoclasts.

Antibodies, Monoclonal↗