Biomedical subjects

M Falconi

Publications and source records attributed to M Falconi.

At least 109 records · Page 6Linked to original sources

PubMed1998

A spectroscopic and molecular dynamics study of native and of a mutant of Xenopus laevis Cu,Zn superoxide dismutase: mechanistic consequences of replacing four charged amino acids on the 'electrostatic' loop.

Neutralisation by site-directed mutagenesis of four charged and highly conserved residues of the electrostatic loop of Cu,Zn superoxide dismutase from Xenopus laevis, involved in the electrostatic attraction of the substrate: Lys120-->Leu, Asp130-->Gln, Glu131-->Gln and Lys134-->Thr, gives rise to a mutant enzyme which displays an affinity for monovalent inhibitor anions, such as N3-, higher than that of the wild type. Analysis of 300 ps of molecular dynamics simulation carried out on the wild type and on the Xenopus laevis Cu,Zn superoxide dismutase mutant indicates that the two proteins display a distinct dynamical behaviour. In particular the root mean square deviation from the starting structure, the number of residues in random coil conformations, the number of residues in unfavourable regions of the Ramachandran plot indicate that the mutant displays a rigidity higher than the native enzyme. This is also evidenced by the loss of dynamical cross correlations in the simulation of the mutant, which on the other hand are present in the wild type. Moreover the mutant protein shows a different organisation of the backbone-to-backbone hydrogen bonds network that generates a rigid structure leading to an increase of the active site accessibility when compared to the native enzyme. It is suggested that the rigid state in which the mutant is confined, accompanied by the increase of the solvent accessible surface of the active site may explain the difference in reactivity toward the inhibitor anion.

Amino Acid Sequence↗

PubMed1998

Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS.

The expression of plasmid-borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H-NS, one of the main nucleoid-associated proteins, controls the temperature-dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (approximately 32 degrees C). This temperature-dependent transcriptional regulation has been reproduced in vitro and the targets of H-NS on the virF promoter were identified as two sites centred around -250 and -1 separated by an intrinsic DNA curvature. H-NS bound cooperatively to these two sites below 32 degrees C, but not at 37 degrees C. DNA supercoiling within the virF promoter region did not influence H-NS binding but was necessary for the H-NS-mediated transcriptional repression. Electrophoretic analysis between 4 and 60 degrees C showed that the virF promoter fragment, comprising the two H-NS sites, undergoes a specific and temperature-dependent conformational transition at approximately 32 degrees C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H-NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H-NS-dependent thermoregulation of virulence gene expression.

Bacterial Proteins↗

PubMed1998

Conserved enzyme-substrate electrostatic attraction in prokaryotic Cu,Zn superoxide dismutases.

The catalytic activity of wild type Escherichia coli Cu,Zn superoxide dismutases and of two mutants in which two lysine residues conserved in most bacterial Cu,Zn superoxide dismutases have been replaced by serine was investigated by pulse radiolysis and Brownian dynamics simulations. Experimental and computational data show that neutralization of Lys60 strongly reduces the catalytic activity of the enzyme (approximately 50%), indicating that this residue has a primary role in the electrostatic attraction of the substrate towards the catalytic copper. Neutralization of Lys63 does not significantly influence the catalytic rate constant. The results suggest that prokaryotic Cu,Zn superoxide dismutases have evolved an electrostatic mechanism to facilitate the enzyme-substrate encounter that is functionally equivalent to that already found in the eukaryotic enzymes.

Amino Acid Sequence↗

PubMed1998

Nuclear morphology during the S phase.

In order to evaluate at the ultrastructural level the chromatin arrangement during the S phase of the cell cycle, the detection of Bromodeoxyuridine (BrdU) by immunogold has been performed in synchronized 3T3 fibroblasts, regenerating liver, and Friend Leukemia Cells (FLC). After a 5-minute BrdU pulse, this label is detected in 10-nm-wide fibers, organized as lacework and assumed to be replication units. In the early part of the S phase, DNA replication units are localized exclusively in the dispersed chromatin domains far from the nuclear envelope. In the middle S, replication occurs at the border between condensed and dispersed chromatin and, finally, in late S, it mainly occurs in perinuclear heterochromatin regions. After replication, the 10-nm fibers can condense in heterochromatin without translocation. Chromatin is highly dispersed in early S and computer image analysis shows an increase in condensed chromatin areas ranging from 13 to 18% at the end of the S phase with a temporal and morphological pattern of distribution characteristic for each cell type. Scanning transmission electron microscopy demonstrates a regular and repetitive structure of dispersed chromatin, represented by a ring-like arrangement of the 10-nm fibers; assuming the same spatial distribution, gold particles that identify incorporated BrdU confirm this organization. By evaluating the organization and the distribution of DNA replication units during S phase, the results suggest that DNA replication occurs at a nucleosomal-like fiber level and that replicating enzymes machinery moves over a fixed template.

3T3 Cells↗

PubMed1998

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↗

PubMed1998

Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.

Animals↗

PubMed1998

Controlled clinical trial of pefloxacin versus imipenem in severe acute pancreatitis.

BACKGROUND & AIMS: Antibiotic prophylaxis in severe pancreatitis has recently yielded promising clinical results, with imipenem significantly reducing the incidence of infected necrosis compared with an untreated control group. On the bases of pefloxacin's spectrum of action and pancreatic penetration, we investigated whether such drugs represent a valid alternative to imipenem. METHODS: In a multicenter study, 60 patients with severe acute pancreatitis with necrosis affecting at least 50% of the pancreas were randomly allocated to receive intravenous treatment for 2 weeks with pefloxacin, 400 mg twice daily (30 patients), or imipenem, 500 mg three times daily (30 patients), within 120 hours of onset of symptoms. Age, sex, body weight, Ranson and Apache II scores, C-reactive protein, etiology, and time from onset of symptoms to treatment were well matched in the two groups. RESULTS: The incidences of infected necrosis and extrapancreatic infections were 34% and 44%, respectively, in the pefloxacin group and 10% and 20% in the imipenem group. Imipenem proved significantly more effective in prevention of pancreatic infections (P </= 0.05). Mortality was not significantly different in the two groups. CONCLUSIONS: Despite its theoretical potential, pefloxacin is inferior to imipenem in the prevention of infections associated with severe pancreatitis.

Adult↗

PubMed1998

A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli.

We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the expression of the virF gene present on the large virulence plasmid of Shigella and enteroinvasive Escherichia coli in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the VirG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of H-NS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30 degrees C. The expression of a virF-lacZ translational fusion in E.coli wild type and in an hns-defective derivative grown at 30 degrees or 37 degrees C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virF expression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controls virF expression by binding to the virF promoter and by repressing its expression at low temperature and at low pH.

Bacterial Proteins↗

PubMed1998

Long-term follow-up of patients with chronic pancreatitis in Italy.

BACKGROUND: We investigated the epidemiologic, clinical, and radiologic aspects of a mixed medicosurgical series of chronic pancreatitis patients observed at the University of Verona Centre for the Study of Pancreatic Diseases over the period 1971-1995. METHODS: Even though the pathogenesis of chronic pancreatitis has yet to be clarified and the classification is still debatable, the patients were subdivided in accordance with the Marseilles-Rome classification into those with alcoholic, obstructive, familial, and idiopathic forms of the disease. A total of 715 patients were analysed with a median follow-up period of 10 years (range, 1-25 years). RESULTS AND CONCLUSIONS: At the end of follow-up the dropout rate amounted to 7.1% (51 patients), and 176 patients (24.6%) had died. Data are reported about the type of chronic pancreatitis, sex, and age distribution at the onset of the disease, drinking and smoking habits before onset and during follow-up, and incidence of calcifications, pain, diabetes, steatorrhoea, and pseudocysts. Surgical aspects, survival curves, and causes of death are also analysed, and the most frequent concomitant diseases in chronic pancreatitis sufferers are discussed.

Adolescent↗

PubMed1998

Surgical treatment of pancreatic metastases from renal cell carcinomas.

Pancreatic metastases from a renal cell carcinoma are rare and may occur long after manifestation of the primary disease. Resection of the metastases should be regarded as the best treatment. In our center, owing to the slow evolution of these secondaries, we perform resections capable of limiting the destruction of the pancreatic parenchyma as far as possible. The use of 'atypical' resections of the pancreas is characterized by a higher incidence of postoperative complications, particularly fistulas. Despite this, we believe that adjusted resection is to be advocated because of the possibility of additional remote secondaries, the shorter duration of surgery, the preservation of the glandular parenchyma and intact adjacent organs, such as duodenum, stomach, and spleen, and the fact that there have been no reports on local recurrences.

Aged↗

PubMed1998

Ultrastructural aspects of the DNA polymerase alpha distribution during the cell cycle.

We studied the nuclear topography of the replicating enzyme DNA polymerase alpha in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase alpha antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase alpha observed in G1- and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase alpha complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase alpha. The evidence of DNA polymerase alpha circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.

Bromodeoxyuridine↗

PubMed1998

Immunocytochemical discrimination between early and late S phase: a new approach to the study of gingival epithelium proliferation in rats.

The renewal of the free gingival margin epithelium in rats was studied evaluating 5-bromodeoxyuridine (BrdU) incorporation in proliferating cells by means of an immunocytochemical method. We found a close correspondence between light and electron microscopy patterns of BrdU incorporation at a nuclear level. BrdU was localized in the inner interchromatin regions in cells starting DNA synthesis, while it was localized in the peripheral heterochromatin domains in cells terminating the S phase. This possibility of discriminating cells in early S phase from cells in late S is able to provide far more information as to the time at which a labeled cell starts proliferation than that obtainable with 3H-thymidine autoradiography. This, in turn, permits detection of cells that start proliferation in a wide period of time by means of a single BrdU administration. Rats treated at 7 a.m. demonstrated higher proliferation than rats treated at 7 p.m., supporting the existence of circadian variations in the epithelial renewal. Proliferative events take place by consecutive activation of at least three replication waves, producing clusters of labeled cells which could be observed in rats sacrificed at 10 a.m. In rats treated once with BrdU at 7 a.m., the clusters were localized in both the basal and suprabasal layer of the epithelium; in rats further injected with BrdU at the same time, the clusters increased in size, progressively extending throughout the epithelium. In this way, the renewal of the free gingival margin epithelium does not proceed randomly, but by consecutive activation of discrete units or clusters of basal cells, which then extend to the upper layers. This can be followed at a morphological level as a progression of labeled cells, which move from the basal layer to the epithelium surface in approximately 82-85 hours.

Animals↗

PubMed1998

To what extent is surgery superior to endoscopic therapy in the management of chronic pancreatitis?

The surgeon was the only figure involved in the management of chronic pancreatitis patients unresponsive to medical treatment, until a few years ago. Nowadays, because of less invasive, endoscopy offers a seductive alternative to surgery. Up to now no clinical prospective and randomized data comparing the results of the two different approaches are available. Surgery seems to be the only solution for chronic pancreatitis with duodenal stenosis and the last chance of eliminating diagnostic uncertainty. Also in the case of biliary tract involvement surgery should be regarded as the procedure of choice, inasmuch as the stenosis is benign and generally long-lasting, and endoscopic treatment would have to be repeated several times; endoscopy, in this indication, should be reserved only for patients who present contraindicating surgery conditions (such as severe jaundice, colangitis etc.); the endoscopist should assess whether to insert a stent or a naso-biliary drainage tube referring the patient back to the surgeon once good clinical conditions have been restored. Endoscopy and surgery should be regarded not as adversaries in the management of chronic pancreatitis and its complications, but as complementary procedures in an integrated approach. The maximum degree of complementarity should be achieved in the management of pseudocysts and in cases presenting severe, incapacitating pain. In selected cases endoscopy can play a definitive role. The generally good surgical outcomes, moreover, should convince endoscopists not to insist with repeated, hazardous manoeuvres in cases of failure. Particularly interesting is the possibility of performing endoscopic sphincterotomy combined with extracorporeal shock-wave lithotripsy prior to surgical treatment in cases of chronic calcifying calcific pancreatitis. The crushing of the calculi and partial clearance of the duct have simplified surgery and complete clearance of the duct in those patients receiving such treatment in our experience.

Chronic Disease↗

PubMed1997

The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending.

Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase-mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS demonstrated here for the first time. DNase I foot-printing and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H-NS of the oligomerization domain (C-domain) of bacteriophage lambda cI repressor.

Amino Acid Sequence↗

PubMed1997

Pancreatic cystic manifestations in von Hippel-Lindau disease.

CONCLUSION: In view of the frequent absence of symptoms related to pancreatic lesions, screening tests for VHL should always include assessment of the pancreas and, considering the frequency of polycystic manifestations, VHL should always be borne in mind in the differential diagnosis of multiple pancreatic cysts, especially when occurring in young patients and in the absence of a positive history of pancreatic disease. BACKGROUND: Von Hippel-Lindau disease (VHL) is a hereditary disease transmitted with an autosomal dominant character and characterized by hemangioblastomas of the central nervous system and retina, renal tumors and cysts, and pheochromocytoma. Pancreatic manifestations of VHL are reported in the literature with incidences ranging from 16 to 29% of cases and consist mainly in cystadenomas of the serous type and in multiple cystic lesions, often with complete replacement of the gland. METHODS AND RESULTS: We report five cases of VHL with a polycystic pancreas as the main or only manifestation, all devoid of symptoms related to involvement of the pancreas, who were referred to our Pancreatic Surgery center with diagnoses of multiple pancreatic pseudocysts of undefined origin.

Adult↗

PubMed1997

Evaluation of UICC TNM classification for pancreatic cancer. A study of 228 patients.

CONCLUSION: A different stage grouping of TNM factors can improve the predictivity of the UICC TNM classification for pancreatic cancer. Nevertheless, the Japanese Pancreas Society (JPS) classification maintains a higher prognostic value. BACKGROUND: The use of a reliable staging classification facilitates the evaluation of anticancer treatments and the correct management of patients. The aim of the present study was to evaluate the prognostic value of three modified UICC TNM classifications, obtained by different stage grouping of the UICC TNM factors, comparing their predictivity to the standard UICC and the JPS classifications. METHODS: Clinical material consisted of 228 patients who underwent resection for pancreatic cancer. The reliability of the classifications was analyzed by the following methods: univariate analysis of stage survival curves; multivariate analysis of each classification after adjusting for grading and radicality; and correlation between the patients' distribution in the stages of each classification and in survival classes. RESULTS: The following modified UICC classification allowed a better differentiation of stage II and III survival (P = 0.08) than standard UICC (P = 0.74): stage I: T1N0M0; stage II: T1N1M0/T2N0M0: stage III: T2N1M0/T3 any NM0; stage IV: M1. The JPS classification better discriminated between the different stages (P < 0.001). All classifications had an independent prognostic value by multivariate analysis. The correlation between stages and survival classes was higher for the JPS classification than either UICC TNM classification or the modified UICC classifications.

Adenocarcinoma↗

PubMed1997

The essential dynamics of Cu, Zn superoxide dismutase: suggestion of intersubunit communication.

A 300-ps molecular dynamics simulation of the whole Cu, Zn superoxide dismutase dimer has been carried out in water, and the trajectory has been analyzed by the essential dynamics method. The results indicate that the motion is defined by few preferred directions identified by the first four to six eigenvectors and that the motion of the two monomers at each instant is not symmetrical. The vectors symmetrical to the eigenvectors are significantly sampled, suggesting that, on average, the motions of the two subunits will exchange. Large intra- and intersubunit motions involving different subdomains of the protein are observed. A mechanical coupling between the two subunits is also suggested, because displacements of the loops surrounding the active site in one monomer are correlated with the motion of parts of the second toward the intersubunit interface.

Computer Simulation↗

PubMed1997

The role of surgery in the major early complications of severe acute pancreatitis.

The early complications of severe acute pancreatitis may constitute a dramatic clinical dilemma in the first 2 weeks of the disease, when the surgical approach is made even more difficult by failure to define the precise extent of the necrotic component of the disease. Moreover, the surgical indication itself is not always based on clear guidelines to which the clinician can refer, and this is due to factors of two types: (i) the intrinsic complexity of the pancreatitis syndrome in its early toxic stages and (ii) the difficulty in understanding the relevant information reported in the literature in this connection, which is often incomplete and based on confused terminology. While the surgical indication is universally accepted in the case of infection of the necrotic tissue (an event, however, which is by no means frequent in the early stages of severe pancreatitis), the development of multi-organ failure despite adequate intensive care is a potential indication which not all specialists go along with, at least not as regards the ideal timing of the intervention. Other surgical indications which have emerged are evidence of complete rupture of the main pancreatic duct and the presence of very extensive sterile necrosis. As things stand at present, however, we are witnessing a general tendency to postpone surgery, since delayed surgery is associated with a lower incidence of complications than is the case with early surgery. If, as is known, the role of surgery is aimed mainly at the treatment of superinfections and severe multi-organ failures, targeted antibiotic prophylaxis and earlier, more complete anti-enzymatic therapy may, as suggested by a number of pilot studies, offer a promising alternative to invasive procedures which are sometimes risky, though indispensable, in an attempt to save patients who would otherwise have no chance of survival.

Acute Disease↗