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D Neri

Publications and source records attributed to D Neri.

89 records · Page 5Linked to original sources
PubMed1992

Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.

Amides↗
PubMed1992

Support of 1H NMR assignments in proteins by biosynthetically directed fractional 13C-labeling.

Biosynthetically directed fractional incorporation of 13C into proteins results in nonrandom 13C-labeling patterns that can be investigated by analysis of the 13C-13C scalar coupling fine structures in heteronuclear 13C-1H or homonuclear 13C-13C correlation experiments. Previously this approach was used for obtaining stereospecific 1H and 13C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the 1H spin-system identifications. Studies of the hydrolysates of fractionally 13C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of 13C-13C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear 1H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the gamma- and delta-methyl groups of Ile. In addition to the methyl resonances, the gamma CH2 groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the delta-carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.

Carbon Isotopes↗
PubMed1992

Precise vicinal coupling constants 3JHN alpha in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments.

Improved experimental schemes for the recently introduced J-modulated [15N,1H]-correlation experiment for measurements of the homonuclear amide proton-C alpha proton vicinal coupling constants, 3JHN alpha, in uniformly 15N-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of 3JHN alpha. The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M = 7.3 kDa), where at 13 degrees C precise values of 3JHN alpha in the range 2.0-9.5 Hz were obtained for all residues with resolved 15N-1H cross peaks. It was then applied to the Antennapedia homeodomain complexed to a synthetic 14-base pair DNA fragment (molecular weight of the complex approximately 18 kDa). The 3JHN alpha values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.

Amino Acid Sequence↗
PubMed1990

Selective variceal decompression and its role relative to other therapies.

Seventy patients, selected from 265 patients with proved variceal bleeding, underwent a distal splenorenal shunt (DSRS) procedure with or without splenopancreatic disconnection (SPD). Alcoholic cirrhosis was the cause of portal hypertension in 57% of the patients. The operative mortality was 13% (Child's classes A and B 2%, class C 66%). Despite fewer varices in all of the patients, variceal rebleeding and death occurred in one patient (2%). Late portal perfusion was observed in 91% of the patients, with worsening in 23%, compared with the preoperative study. Persistent hepatocyte necrosis and incomplete SPD were the most significant prognostic factors for decreased perfusion (presence and absence of necrosis, 38% and 12%, respectively; DSRS and DSRS with SPD, 43% and 12%, respectively). SPD also decreased ongoing hepatocyte damage. Post-shunt encephalopathy was clinically evident in 7% of the patients, but after electroencephalographic evaluation, it increased to 24.6%. Significant factors in its development included decreased portal perfusion (62% versus 14%), active hepatitis (48% versus 17%), and incomplete SPD (43% versus 14%). The higher late liver-related mortality was associated with a lack of or decreased portal perfusion and the absence of SPD.

Esophageal and Gastric Varices↗
PubMed1989

Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling.

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.

Carbon Isotopes↗
PubMed1989

The role of the leukocyte adherence inhibition (LAI), CA 19-9, and tissue polypeptide antigen (TPA) tests in the diagnosis of pancreatic cancer.

The leukocyte adherence inhibition (LAI) assay measures host cell-mediated tumor immunity. The original test used by Halliday and Miller was modified by substituting tissue extracts with purified tumor antigens (gastrointestinal cancer antigen [GICA]) with the aim of reducing false-positive results in the diagnosis of patients with early pancreatic cancer. By our modified technique, the LAI assay identified four of five pancreatic cancers at an early stage and gave only one false-positive result in 16, showing a sensitivity of 80% and a specificity of 94%. Later stages of the disease responded poorly on the test. These results were significantly better than those by the CA 19-9 and tissue polypeptide antigen (TPA) tests. Should our findings be confirmed in larger material over a longer follow-up period, the LAI test could be proposed as a useful tool in the early diagnosis of pancreatic cancer.

Antigens, Neoplasm↗
PubMed1985

Double-blind preference and compliance multicentre study in osteoarthritis: once-a-day treatment.

Two-hundred-and-three female patients (mean age: 58 yrs; SD: 8.2 yrs) suffering from osteoarthritis entered this late phase IV multicentre, stratified according to previous therapy (e.g. ketoprofen, naproxen, aspirin, indomethacin or indoprofen), randomized, double-blind, between within-patient trial of 2-week duration. Each patient received either diclofenac SR 100 mg/day (D), piroxicam 20 mg/day (P), or placebo (P1 by oral route. Clinical evaluation (functional class; pain assessment; osteoarthritic condition; joint motility and stiffness) was performed at entry, as well as after the first and the second week. Patient compliance and reported signs and symptoms were recorded after the first week and at the end of the trial. Patient preference, as regards previous therapy, and global evaluation (both by the physicians and the patients) were checked at the end of the trial. The clinical evaluation showed a superiority of D and P over P1. No difference was seen between the two active drugs. Placebo effect was very strong. Global evaluation was significantly in favour of D and P. Patient compliance was extremely good (greater than or equal to 95%). Diclofenac was preferred to naproxen, aspirin and indomethacin, while piroxicam and placebo were preferred only to aspirin. The tolerability of the two active drugs was good and comparable. A significantly lower number of patients complaining of unwanted effects (u.e.) was detected in the placebo group. The number of patients withdrawn for u.e. was similar in the three trial groups.

Adult↗
PubMedDate not supplied

The use of biosensor technology for the engineering of antibodies and enzymes.

Recently developed scientific instrumentation featuring surface plasmon resonance detection allows the detection of biomolecular interactions in real time and without chemical modification of the binding partners. These biosensors are proving invaluable tools in protein engineering, particularly in research aimed at the isolation and improvement of protein binders and catalysts from macromolecular repertoires containing billions of individual members. This article reviews the use of biosensor technology for the isolation and characterization of engineered antibodies and enzymes.

Animals↗
PubMedDate not supplied

[Care at the patient's bedside. Non-invasive bioethics].

This paper aims at describing the circumstances of the birth of Bioethics. In the first section, the paper points out some historical data supporting the widely diffused opinion according to which Bioethics started in the USA at the beginning of the '70s. In the second section, one of the most important factors in the birth of Bioethics is discussed: namely, the changes in medical enterprise and health care ethics. In the third section the impact of Bioethics on clinical medicine at the bedside is discusses, and, in the last section, one of the most important practical application of bioethical reflection is presented: the creation of Hospital Ethics Committees.

Bioethics↗
PubMedDate not supplied

Liver transplantation with vena cava in situ and selective use of temporary portacaval shunt or portal clamping.

BACKGROUND/AIMS: The recipient hepatectomy with vena cava in situ in liver transplantation has overcome the need of venous-venous bypass thanks to temporary porta caval shunt or portal clamping. METHODOLOGY: 150 orthotopic liver transplants in 137 patients were performed and the vena cava in situ technique was used in 142 (venous bypass in 7, temporary porta caval shunt in 49, portal clamping in 87). The suprahepatic cava veins anastomosis was performed with Belghiti in 97 and piggyback techniques in 45. RESULTS: There were no differences in operative and warm ischemia times nor in blood requirements, while a greater stability of body temperature was documented in the vena cava In Situ group: in the latter temporary porta caval shunt preserved the temperature better than portal clamping (P < 0.01). In anhepatic phase mean artery pressure decreased in veno-venous bypass and increased in the vena cava In situ groups (P < 0.01). The venous return and the cardiac performances (anhepatic phase) were better preserved in the vena cava In Situ group. (P < 0.0001). CONCLUSIONS: Temporary portal caval shunt or portal clamping and piggyback or Belgiti Techniques allow a better hemodynamic stability through out the procedure, obviating the need for veno-venous bypass or fluid overload, if selectively used.

Adult↗
PubMedDate not supplied

[Endoscopic approach to biliary stones: experience acquired in a general surgery unit. Comparison of 2 periods].

The outcome of the laparoscopic technique, that in the first years needed to be applied in non complicated situations, imposed a more frequent use of ERCP preoperatively; this procedure was originally confined to a handful of European and American centers, but later spread to almost all large hospitals. Improvements in the techniques and materials have gone side by side with more specific indications and the assessment of complications. The purpose of the present study was to analyze the experience of a General Surgery Unit in terms of acquiring and developing skills in treating biliary stones by ERCP.

Cholangiopancreatography, Endoscopic Retrograde↗
PubMedDate not supplied

[Transplantation of isolated hepatocytes into the spleen of rats].

The effects of splenic transplantation of hepatocytes on survival time and hepatic regeneration were studied in adult rats subjected to: 1) 90% hepatectomy plus portocaval shunt (graft of 4 x 10(6) hepatocytes) and 2) 90% hepatectomy (graft of 4 x 10(6) and 8 x 10(6) hepatocytes); data were compared with those obtained in hepatectomized rats. Hepatic regeneration was significantly higher in transplanted rats and, within animals with grafted hepatocytes more pronounced in rats without porto-caval shunt. This support the hypothesis that a hepatotrophic factor is produced by grafted hepatocytes: its activity is more pronounced in the absence of porto-caval shunt since all splenic blood reach the liver. The activity of the putative hepatocyte growth factor does not depend on the number of grafted hepatocytes since no significant differences in survival time and hepatic regeneration was observed between rats grafted with 4 x 10(6) and 8 x 10(6) hepatocytes.

Animals↗
PubMedDate not supplied

[Diffusion of smoking among high school students at U.S.L. No. 18 of the Tuscan region].

Between young people: 51% of male have never smoked, and only 38% of the females. If increase the age, increase also the percentage of whom that have had first meeting with cigarette smoke. Begin previously boys and they smoke more strongly. Today young people begin to smoke always more precociously, even if they are more informed.

Adolescent↗