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P Neri

Publications and source records attributed to P Neri.

At least 55 records · Page 3Linked to original sources
PubMed1992

NMR studies of an FK-506 analog, [U-13C]ascomycin, bound to FK-506-binding protein.

Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for [U-13C]ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons. The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the [U-13C]ascomycin/FKBP. This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506. However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A. The methods used for resonance assignment and structure calculation are presented in detail. Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket. This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.

Carbon Isotopes↗
PubMed1992

Molluscan sperm proteins: Ensis minor.

The three major proteins, EM1, EM5 and EM6, from the mature sperm of the bivalve mollusc Ensis minor have been partially sequenced in order to establish which category they belong to and their potential for phosphorylation. Protein EM1 is protamine-like with about 50% basic amino acids, some of which are included in SK(R) repeats. Three SPXX potential phosphorylation sites were observed in the N-terminal domain. EM1 does not fold (Giancotti et al. (1983) Eur. J. Biochem. 136, 509-516). Protein EM6 (approx. 270 residues) is histone H1-like, having a globular domain homologous to other H1 family proteins. The N-domain of EM6 contains SK(R) repeats like EM1, but there are few, if any, SPXX sites in the chain. Proteins EM1 and EM6 are the two proteins specific for mature sperm. Protein EM5, of about 150 residues and present at lower levels than EM1 and EM6, is also an H1-family molecule. A sequence from its globular domain shows close homology to chicken H5 and to sea urchin somatic H1. Its presence may relate to the existence of a low level of nucleosomal structure.

Amino Acid Sequence↗
PubMed1992

Identification of solvent-exposed regions of an FK-506 analog, ascomycin, bound to FKBP using a paramagnetic probe.

The solvent-exposed regions of (U-13C)ascomycin when bound to its putative target protein, FKBP, have been identified based on the different proton longitudinal relaxation rates (R1 = 1/T1) measured in the absence and presence of the paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethyl-piperidinyl-1-oxy (HyTEMPO). The proton T1S of bound ascomycin were determined using a pulse sequence (T1-HMQC) which consists of a 180 degree proton pulse and a variable delay (tau) followed by a heteronuclear multiple quantum correlation (HMQC) experiment. The solvent-exposed regions of ascomycin determined by these experiments are compared to NOE data in which ascomycin/FKBP contacts were identified and to the X-ray structure of the FK-506/FKBP complex.

Binding Sites↗
PubMed1991

1H, 13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site.

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two- and three-dimensional NMR experiments involving selectively 15N- and uniformly 15N- and 15N,13C-labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.

Amino Acid Isomerases↗
PubMed1991

NMR studies of [U-13C]cyclosporin A bound to human cyclophilin B.

NMR data (1H and 13C chemical shifts, NOEs) on [U-13C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-13C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574-6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.

Amino Acid Isomerases↗
PubMed1991

NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding.

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.

Amino Acid Isomerases↗
PubMed1991

Iron carrier proteins facilitate engraftment of allogeneic bone marrow and enduring hemopoietic chimerism in the lethally irradiated host.

Cell-free supernatants of rabbit bone marrow were fractionated, separated, and purified by Ultrogel and Superose chromatography. A single fraction promoted engraftment of allogeneic bone marrow and enduring hemopoietic chimerism across the H-2 barrier in lethally irradiated mice. This "bio-active" fraction, analyzed by reducing SDS-PAGE electrophoresis, and transblotted on PVDF membrane, and purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main prealbumin band that when examined for primary structure by Edman degradation, proved to be rabbit transferrin. This was also attested by highly specific precipitation of the prealbumin band with polyclonal antibodies to rabbit transferrin. Iron-saturated human transferrin, lactotransferrin, and egg transferrin (conalbumin) were assayed in irradiated C57BL/6 mice infused with bone marrow from histoincompatible BALB/c donors. Mice treated with iron-loaded transferrins survive and develop enduring allogeneic chimerism with no discernible signs of graft-versus-host disease. Iron carrier proteins thus provide an unique means of achieving successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations.

Amino Acid Sequence↗
PubMed1991

Tyrosine 65 is photolabeled by 8-azidoadenine and 8-azidoadenosine at the NAD binding site of diphtheria toxin.

8-Azidoadenine and 8-azidoadenosine, two photoactivatable derivatives of adenine and adenosine, are competitive inhibitors of diphtheria toxin of similar potency with respect to their parent compounds. On irradiation, the two tritium-labeled photoactivatable azidoadenines bind covalently and specifically to an enzymic fragment of diphtheria toxin that is known to bind to NAD. This photolabeling is protected by the enzyme substrate NAD. The radiolabeled protein was fragmented, and the radioactive fragments were sequenced. Tyr-65 is labeled specifically by both photoreagents, and its labeling was reduced strongly when NAD was present during irradiation. Labeling is also reduced strongly by adenine, adenosine, and nicotinamide. These results suggest that Tyr-65 is at the NAD binding site of diphtheria toxin and that the competitive inhibitors adenine, adenosine, and nicotinamide bind to the same portion of the catalytic center of the toxin.

Adenine↗
PubMed1991

Structure and antigenic activity of rubella E1 glycoprotein synthetic peptides.

Minimal sequences of rubella E1 glycoprotein epitopes were previously identified as the tripeptide 250PER252 for the EP2 epitope, the tetrapeptide 260ADDP263 for the EP3 epitope, and the tripeptide 273EVW275 plus the octapeptide 278PVIGSQAR285 for the EP1 epitope. In order to establish for each epitope the shortest sequence that was able to give the maximum binding with human antirubella immunoglobulins, synthetic peptides with increasing number of residues flanking these essential parts of rubella E1 glycoprotein epitopes were synthesized and examined for their antigenic activity. Usually higher activity was observed with progressively longer homologues, whereas the additions of Pro-271, Pro-278 to 272GEVWVT277 peptide, and additions of Ala-248 to 249TPERP253 and 249TPERPR254, led to an abrupt decrease in binding. Taken together, our results indicated that the antigenic activity of the whole antigen could be dissected and reproduced using synthetic peptides of appropriate structure for each epitope.

Amino Acid Sequence↗
PubMed1991

1H- and natural abundance 15N-NMR studies of a derivative of a rabies glycoprotein fragment.

Using a combination of one- and two-dimensional methods, 1H- and 15N-nmr spectroscopy has been employed to perform the complete assignment and the structural determination of the immunogenic undecapeptide CTTTNSRGTTT in DMSO solution. Nuclear Overhauser enhancement spectroscopy experiments indicated the presence of secondary structures, mainly turn-like structures, which only represent a family, albeit a dominant one, of an ensemble of conformations available to the peptide. Since reverse turns may play an important role as intermediates in protein folding, the experimental observations described here may link the immunological and theoretical approaches to protein folding.

Amino Acid Sequence↗
PubMed1991

An antigenic determinant common to human chorionic somatomammotropin and human growth hormone revealed by limited proteolysis of immune complexes.

An epitope of human chorionic somatomammotropin for one of the monoclonal antibodies raised against the whole antigen has been identified. We compared the release of peptides from limited proteolysis of the antigen in the presence and absence of the related antibody. Using enzymes of different specificity, we could determine the amino acid sequence that can be considered at least inclusive of the epitope. The monoclonal antibody selected is 100% cross-reactive with human growth hormone, so the antigenic determinant identified is shared by the two protein hormones.

Amino Acid Sequence↗
PubMed1991

Role of manganese in MG-63 osteosarcoma cell attachment to fibrinogen and von Willebrand factor.

Some integrin receptors have been reported to be functionally distinct in different cell types. In endothelial and melanoma cells, the vitronectin receptor, alpha v beta 3 binds fibrinogen (fg) and von Willebrand factor (vWf) in addition to vitronectin itself, whereas it fails to do so in MG-63 osteosarcoma cells. In this report, it is shown that, in the presence of Mn2+, MG-63 cells attach more efficiently to vitronectin and acquire the de novo capacity to adhere to fg- and vWf-coated surfaces. The latter phenomenon occurs with full cell spreading, F-actin microfilament organization, and alpha v and beta 3 clustering at focal contacts. In contrast, beta 1 and beta 5 do not localize to adhesion plaques under the same experimental conditions. An antiserum to the beta 3 chain and a synthetic peptide containing the sequence Gly-Arg-Gly-Asp-Ser-Pro block MG-63 attachment to fg and vWf in the presence of Mn2+. The minimal active concentration of Mn2+ is in the range of 0.1 to 1 microns. These data suggest that the acquired capacity of MG-63 to attach to fg and vWf in the presence of Mn2+ is mediated by alpha v beta 3 and that differences in alpha v beta 3 receptor specificity may be modulated by exogenous factors.

Amino Acid Sequence↗
PubMed1990

HPLC immunoaffinity purification of rabies virus glycoprotein using immobilized antipeptide antibodies.

It has been reported that the acetylcholine receptor may be used by the rabies virus to concentrate at sites in proximal to peripheral nerves. It has also been reported that the binding site for the receptor is located within the 190-203 region of the virus glycoprotein on the basis of its structural homology with the toxic center of snake neurotoxins, which are well known cholinergic ligands. We prepared monoclonal antibodies against the synthetic tetradecapeptide having the same sequence as the putative binding site of the rabies virus. One of three antibodies (clone 2PV 36-74) was able to recognize both the whole virus and its peplomeric glycoprotein and could bind acetylcholine. It was also able to inhibit the binding both of alpha-bungarotoxin and rabies virus glycoprotein to the acetylcholine receptor. We have covalently bound 2PV 36-74 to an HPLC affinity column and utilized it for specific purification of rabies virus glycoprotein. The immunoaffinity chromatographic method we describe is very sensitive and highly specific. Moreover this procedure does not denature the sample and is vary rapid and efficient.

Animals↗
PubMed1990

[The effect of iron carrier proteins on the transplantation of H-2 locus-incompatible bone marrow in irradiated mice].

Rabbit bone marrow supernatants were fractionated and purified by Ultrogel and Superose chromatography. A unique fraction promoted engraftment of allogenic bone marrow and enduring hemopoietic chimerism across the histocompatibility (H-2) barrier in lethally irradiated mice. This fraction analysed by reducing SDS-PAGE electrophoresis and transblotted on PVDF membrane or purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main pre-albumin band that was examined for primary structure by Edman degradation. It appeared to be rabbit transferrin. Iron saturated human transferrin, lactotransferrin and egg transferrin (conalbumin) were then tested in irradiated C57B1/6 mice transplanted with bone marrow from histoincompatible BALB/CJ donors. Most mice treated with iron-loaded transferrins survived and developed enduring allogeneic chimerism with no discernible signs of graft-versus-host disease at 10 months posttransplant. Observation of these animals is still carried on. Iron carrier proteins seem to provide a novel unexpected means for achieving a successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations and may open a new approach in the clinical area.

Animals↗