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Biomedical subjects

A Scarpa

Publications and source records attributed to A Scarpa.

At least 19 recordsLinked to original sources

The FHIT gene is expressed in pancreatic ductular cells and is altered in pancreatic cancers.

We examined 2 normal pancreata, 21 primary pancreatic ductal cancers, and 19 pancreatic cancer cell lines for Fhit expression and FHIT gene status. The normal pancreas expressed Fhit protein in the cytoplasm of ductular cells, whereas interlobular and larger ducts, acini, and insulae of Langerhans were negative. Fhit protein was detected by immunoblot assay in 11 pancreatic cancer cell lines; of the 8 cell lines lacking Fhit protein, 7 lacked FHIT mRNA and 1 showed an abnormally sized transcript. DNA from five of these eight cell lines showed homozygous loss of FHIT exon 5. In 8 of the 21 primary cancers, Fhit expression was detected by immunohistochemistry. Reverse transcription-PCR analysis of 6 of the 13 cases lacking Fhit showed normal-sized FHIT product in 3 cases and a mixture of normal and abnormal products in the other 3. Sequencing showed that abnormal bands were missing variable numbers of exons. Loss of microsatellite DNA markers internal to the FHIT gene was observed in 10 of 13 primary cancers lacking Fhit protein (homozygous in two cases) and in only 1 of the 8 cancers expressing Fhit protein. In nine primary cancers, four expressing and five lacking Fhit protein, it was possible to obtain pure cancer DNA by microdissection. Three of the five microdissected cases lacking Fhit protein exhibited homozygous deletion of FHIT exon 5. In conclusion, the lack of Fhit protein in pancreatic cancers correlated with absence or alteration of FHIT mRNA and was often associated with FHIT gene anomalies.

Acid Anhydride Hydrolases

Mucinous cystic tumors of the pancreas: clinicopathological features, prognosis, and relationship to other mucinous cystic tumors.

The clinicopathological features of 56 patients with mucinous cystic tumors (MCTs) of the pancreas were studied. Particular attention was paid to the prognosis of MCTs and the relationship to their ovarian, hepatic, and retroperitoneal counterparts. To distinguish MCTs from pancreatic intraductal papillary-mucinous tumors, MCTs were defined as tumors lacking communication with the duct system and containing mucin-producing epithelium, usually supported by ovarian-like stroma. All 56 tumors occurred in women (mean age 48.2 years) and were preferentially (93%) located in the body and tail of the pancreas. In accordance with the WHO classification, MCTs were divided into adenomas (n = 22), borderline tumors (n= 12), and noninvasive and invasive carcinomas (n = 22). Survival analysis revealed the extent of invasion to be the most significant prognostic factor (p<0.0001). Malignancy correlated with multilocularity and presence of papillary projections or mural nodules, loss of ovarian-like stroma, and p53 immunoreactivity. Stromal luteinization with expression of tyrosine hydroxylase, calretinin, or alpha inhibin was found in 66% of the cases. We conclude that the biologic behavior of MCTs is predictable on the basis of the extent of invasion. The similarities (i.e. gender, morphology, stromal luteinization) between pancreatic MCT and its ovarian, hepatobiliary, and retroperitoneal counterparts suggest a common pathway for their development.

Adenocarcinoma, Mucinous

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion.

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.

Amino Acid Sequence

Pancreatic carcinoma: correlation between E-cadherin and alpha-catenin expression status and liver metastasis.

BACKGROUND: Dysfunction of the E-cadherin/catenin-mediated cell-cell adhesion system has been associated with invasiveness and poor differentiation of human carcinomas. However, its importance in the genesis of liver metastasis has not been examined sufficiently. METHODS: A series of 26 primary pancreatic carcinomas and the concomitant liver metastases from 15 of them, obtained at autopsy, were analyzed for E-cadherin and alpha-catenin protein expression by immunohistochemistry. RESULTS: Both E-cadherin and alpha-catenin expression were preserved in 15 (58%) and reduced in 11 (32%) of the 26 primary pancreatic carcinomas. In the former 15 primaries, carcinoma cells were attached to each other tightly, whereas the latter 11 primaries showed isolated or loosely connected attachments. The metastatic ratio was higher in tumors exhibiting tight adhesion than in those with loose adhesion: 73% and 36%, respectively (P = 0.059). E-cadherin and alpha-catenin expression patterns in liver metastases basically followed those in the corresponding primaries (P < 0.01). CONCLUSIONS: Reduced E-cadherin and alpha-catenin expression in primary pancreatic carcinoma has no significant predictive value regarding the presence of liver metastasis. Rather, there is a greater tendency for liver metastasis in cases in which the integrity of the E-cadherin/catenin-mediated cell-cell adhesion system is intact.

Adult

Clonality of B-cells in portal lymphoid infiltrates of HCV-infected livers.

Evidence has been accumulating in favour of a role for hepatitis C virus (HCV) in the pathogenesis of human lymphoproliferative disorders. HCV infection has been documented in the majority of patients with essential mixed cryoglobulinaemia type II (MC-II); in patients with HCV infection, B-cell clonal expansion have been detected in peripheral blood and bone marrow, and a high prevalence of B-cell non-Hodgkin's lymphomas has been documented. Liver biopsies in chronic hepatitis C frequently show portal lymphoid infiltrates with features of B follicles, whose clonality has not yet been investigated. This study has analysed the B-cell clonality of portal lymphoid infiltrates from 16 patients with chronic HCV hepatitis. Portal tracts showing obvious lymphoid infiltrates were microdissected from the paraffin-embedded liver tissue sections and the clonality of lymphoid B-cells was tested using a polymerase chain reaction (PCR) approach designed to identify immunoglobulin heavy chain gene (IgH) rearrangements. A successful IgH-PCR analysis was achieved in 35 lymphoid infiltrates from 11 patients (seven with the four without MC-II) and yielded a single band in 21 cases, two bands in ten cases, and three bands in four cases. Comparison of the IgH-PCR amplification bands obtained from the different lymphoid aggregates of the same biopsy revealed that they differed in size. This finding indicates that each aggregate derives from the proliferation of one or a few founder B-cells, which are not related to each other. The results obtained in patients with and without MC-II were similar, suggesting that the presence of B-cell clonal proliferations in liver biopsies is independent of the occurrence of B-cells producing monoclonal IgMk cryoglobulins.

B-Lymphocytes

Pancreatic endocrine tumours: evidence for a tumour suppressor pathogenesis and for a tumour suppressor gene on chromosome 17p.

Two molecular pathways leading to cancer are known. Common-type cancers arise from the 'tumour suppressor' pathway, characterized by gross chromosomal changes and allelic losses (LOH) in an average of 25 per cent or more of randomly chosen chromosomal loci. The 'mutator pathway' has been recognized in a subset of cancers, characterized by widespread microsatellite DNA instability and rarity of chromosomal losses. The present study has investigated 20 pancreatic endocrine tumours (PETs) for loss of heterozygosity (LOH) at seven chromosomal loci (3p14, 7q31-32, 11q13, 13q14, 18q21, 17p13, and 17q21); microsatellite instability; and Ki-ras, N-ras, and p53 gene mutations. LOH was found in an average of 24 per cent of the chromosomal loci analysed. No tumour showed microsatellite instability. Ki-ras and p53 mutations were each found in one case. The frequency of losses was higher in malignant (40 per cent) than in benign (17 per cent) tumours (p = 0.009), and the specific chromosome 17p13 LOH was associated with extrapancreatic extension of disease (p = 0.007), high proliferative activity (p = 0.001), and absence of progesterone receptors (p = 0.01). A common deleted region on chromosome 17p13 and the rarity of p53 gene mutations suggest the existence of a novel tumour suppressor gene involved in the pathogenesis of PETs in this chromosomal area.

Adolescent

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.

Animals

Molecular pathogenesis of sporadic duodenal cancer.

Whether duodenal adenocarcinoma should be considered as a gastrointestinal or as a peripancreatic cancer is a matter of debate, as is the opportunity and type of treatment. We investigated 12 such cancers for the genetic anomalies involved in the pathogenesis of gastrointestinal malignancies, including (a) those occurring in common-type cancers - allelic losses at chromosomes 3p, 5q, 17p and 18q, and Ki-ras and p53 alterations; and (b) those characteristic of mutator-phenotype cancers - microsatellite instability and TGF-betaRII gene mutations. We found Ki-ras and p53 mutations in five (42%) and eight cancers (67%), respectively; chromosome 3p, 5q, 17p and 18q allelic losses in two of nine (22%), six of ten (60%), six of nine (67%) and three of ten (30%) informative cancers, respectively. Finally, three cancers (25%) showed widespread microsatellite instability and two of them had a TGF-betaRII gene mutation. Our data suggest that duodenal cancers may arise from either of the two known pathogenetic molecular pathways of gastric and colorectal cancers. The majority of our cases were highly aggressive cancers with frequent chromosomal changes and p53 mutations as observed in the common-type gastrointestinal malignancies, while widespread subtle alterations characteristic of mutator-phenotype cancers occurred in a minority, which also showed a favourable long-term outcome.

Adult

Chromosome 5 allelic losses are early events in tumours of the papilla of Vater and occur at sites similar to those of gastric cancer.

During our studies of DNA fingerprinting of tumours of the pancreas and papilla (ampulla) of Vater, using arbitrarily primed polymerase chain reaction (AP-PCR), we noticed two bands showing a decreased intensity in six of ten ampullary tumours with respect to matched normal tissues. Those bands were both assigned to chromosome 5. Such a finding was somewhat in contrast with the reportedly low frequency of APC gene mutations in ampullary cancers, located at chromosome 5q21, and suggested that loci different from that of APC might be the target of chromosome 5 allelic losses (LOH) in these tumours. Therefore, we analysed chromosome 5 LOH in a panel of 27 ampullary tumours, including eight adenomas, four early- and 15 advanced-stage cancers, using 16 PCR-amplified CA microsatellite polymorphic markers spanning the entire chromosome. Nineteen cases (70%) showed LOH, and the interstitial deletions found in these tumours described two smallest common deleted regions, in which putative suppressor genes might reside. They were at 5q13.3-q14 and at 5q23-q31 respectively, which correspond to those found in gastric tumours. In addition, the presence of 5q LOH in six of eight adenomas and in three of four early-stage cancers suggests that such phenomena occur at early stages of neoplastic progression of the ampullary epithelium.

Adenoma

A high copy suppressor screen reveals genetic interactions between BET3 and a new gene. Evidence for a novel complex in ER-to-Golgi transport.

The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34 degrees. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone alpha-factor at 37 degrees. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

Amino Acid Sequence

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells.

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.

Alkaloids

Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver.

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+ with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30-50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ . micrometer -2 . min-1 after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+ but not Ca2+/Mg2+ exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

Animals

Molecular approach in human tumor investigation: oncogenes, tumor suppressor genes and DNA tumor polyomaviruses (review).

Molecular analysis are useful for diagnosis, prognosis and follow-up of the patients, as well as for addressing therapeutic choices. Most of the molecular methods are based on the analysis of nucleic acids. The DNA and RNA methodologies of routine applicability include Southern and Northern hybridizations and polymerase chain reaction (PCR) techniques. Southern blot hybridization recognizes major DNA rearrangements, and detection of oncogenic viral sequences present in high copy number, whereas PCR-based methods allow the detection of gross chromosomal modifications, fine gene alterations and low amount of tumor virus footprints. PCR techniques also allow the analysis of the partially degraded nucleic acids from formalin-fixed paraffin-embedded tissues. We present an overview of the use of molecular techniques for the analysis, diagnosis, prognosis and follow-up of neoplastic diseases, using examples from our experience in both leukemias and solid tumors.

Genes, Tumor Suppressor

Prompt hepatitis C virus suppression following hepatitis B virus superinfection in chronic untreated hepatitis C.

The natural course of chronic hepatitis C virus infection after hepatitis B virus superinfection is not clear since it is difficult to determine the chronology of the double infections. We report on a case of de novo hepatitis B virus infection in the course of chronic untreated hepatitis C, in which the time of hepatitis B virus infection is actually known. The patient eliminated HCV-RNA, both from serum and from liver tissue, soon after the clinical onset of the acute hepatitis B. Liver histology featured hepatitis with severe portal inflammation and high-grade periportal and intralobular necro-inflammatory lesions. This observation demonstrates that hepatitis C virus replication can be promptly and spontaneously suppressed by acute hepatitis B virus superinfection.

Acute Disease

The human ALL-1/MLL/HRX antigen is predominantly localized in the nucleus of resting and proliferating peripheral blood mononuclear cells.

The ALL-1 gene is an important regulator of embryonal and hematopoietic development, and structural variants of the human gene generated by chromosomal translocations and other genomic alterations presumably act as oncogenes in the pathogenesis of acute leukemias and other hematological malignancies. Antisera against two different epitopes of the human ALL-1 protein (anti-ALL1-N and anti-ALL1-C) were produced. Both sera revealed indistinguishable patterns of antigen localization in human peripheral blood mononuclear cells (PBMCs). In resting PBMCs, the antigen was distributed in a speckled pattern across the nuclei, with an increased density at the nuclear envelope and the nuclear indentation. In mitotically stimulated PBMCs, the antigen surrounded the condensing chromosomes but did not colocalize with chromatin or the nuclear scaffold. The antigen is considered a marker for a novel nuclear subcompartment, a perichromosomal area termed the "chromosomal envelope." In Western blot experiments, the anti-ALL1-N serum reacted with a polypeptide corresponding to the expected full-length 430-kDa ALL-1 protein. Recombinant proteins representing the AT-hook and zinc binding subdomains of the ALL-1 protein interacted in vitro with a degenerate mixture of double-stranded oligodeoxynucleotides. Thus, the ALL-1 protein probably is a DNA-binding protein with both a sequence-unspecific (AT-hook) and a sequence-specific (zinc binding subdomains) double-stranded DNA binding mode.

Animals

Psychophysiology of anger and violent behavior.

Antisocial behavior is a complex phenomenon that arises out of multiple causes involving biologic, psychological, and social forces. Moreover, different forms of violent antisocial behavior may each result from different biopsychosocial pathways. The overview of human psychophysiologic findings presented in this article provides some support for this notion. In particular, the finding of psychophysiologic underarousal (e.g., reduced resting HR and SC levels, increased slow-wave EEG, poor classical conditioning) is one of the most robust and best replicated findings in antisocial populations. The majority of these studies consist of populations exhibiting nonviolent antisocial behavior or milder forms of aggression. Findings of underarousal in institutionalized criminal samples are very few in number and are not well-replicated. The relationship of psychophysiologic underarousal to antisocial behavior, therefore, may be specific to covert forms of antisocial behavior and perhaps to some less severe forms of violent behavior. On the other hand, violence associated with anger and emotional aggression (which is often more impulsive, less controlled, and reactive to some perceived provocation) may have very different psychophysiologic underpinnings. It was suggested that risk factors for emotional aggression include a predisposition to negative affect/arousal and an inability to regulate that affect/arousal. It also was suggested that this effect will be most pronounced in individuals experiencing stressors or adverse social environments, where negative affect and arousal would be increased. Laboratory studies have suggested that overarousal may facilitate aggression in situations in which someone has been provoked. Clinical studies also have indicated a relationship between increased physiologic arousal, negative emotionality, and aggression/antisocial behavior in some populations, with increases in aggression in those also exposed to adverse home environments. Thus, the relationship of increased psychophysiologic arousal to antisocial behavior may be more specific to angry or emotional violence. It is important to note that these psychophysiologic distinctions are speculative for few studies actually have differentiated type of violence in their design. Pitts did group children according to proactive or reactive aggression and found reduced HR levels in both groups, but a substantial increase in HR only in the reactive aggressive group. Lakosina and Trunova found increased SC responsivity in psychopathic individuals characterized by affective violence. These studies provide some initial support for underarousal in proactive/instrumental aggression and overarousal in emotional aggression. It is important, however, that more studies be done with subtypes of violence to test the notion of such differential psychophysiologic patterns. Lastly, some definitional and methodologic considerations need to be mentioned. First, a distinction exists between physiologic arousal and reactivity. Typically, arousal refers to psychophysiologic activity that occurs during a resting state, whereas reactivity refers to activity that occurs in response to some stimulus. Although some studies did assess measures during a resting state, the majority of findings on over-arousal in relation to emotional aggression more accurately reflect psychophysiologic and emotional reactivity to a situation or stimulus. Second, arousal or reactivity are nonspecific terms that can refer to any psychophysiologic response system (e.g., electrodermal, cardiovascular, cortical, and so on). Responses from these systems typically do not correlate in the general population. Thus, it would be worthwhile for future studies to include more than one response system (as done by Raine et al) and see if the arousal/reactivity measures converge. If the measures converge, a general model of arousal or reactivity is supported. (ABSTRACT TRUNCATED)

Affect

Heart rate and skin conductance in behaviorally inhibited Mauritian children.

This study tested predictions that inhibited versus uninhibited children exhibit higher heart rate (HR) and skin conductance (SC) arousal. Mauritian children (N = 1,795) were tested at age 3 and classified as inhibited, middle, or uninhibited on the basis of social behavior. HR level and several SC measures were obtained immediately before or during a tone task. Inhibited children displayed significantly higher HR and SC levels and longer SC latency relative to uninhibited children. Results remained regardless of ethnicity, gender, height, weight, respiratory complaints, or crying behavior. Findings suggest that HR and SC levels may be early indicators of inhibited or uninhibited behavior at age 3 and support the notion of heightened sympathetic reactivity due to limbic arousal in inhibited children.

Arousal