Biomedical subjects

P Ferrara

Publications and source records attributed to P Ferrara.

At least 163 records · Page 9Linked to original sources

PubMed1991

Expression and pharmacological characterization of the human peripheral-type benzodiazepine receptor in yeast.

Recently we cloned the cDNA coding for the putative human peripheral-type benzodiazepine receptor (hPBR). This report describes the expression of this cDNA in Saccharomyces cerevisiae and the characterization of the recombinant protein. The expression was achieved by placing the receptor cDNA under the control of a galactose-regulated artificial promoter. After galactose induction, the transformed cells expressed a functional hPBR which displayed a Kd for the specific peripheral-type ligand [3H]PK11195 of 9.9 +/- 1.3 nM and a maximal binding capacity of 249,300 +/- 50,400 sites/cell. The pharmacological characterization of the recombinant receptor, determined in competitive ligand binding experiments, agrees closely with that described for the natural receptor expressed by human cells. Furthermore, the binding was stereospecific as shown by the displacement of the [3H]PK11195 binding by PK14067 (-Q1) and not by PK14068 (+Q1). Photolabeling experiments showed that transformed cells expressed a 18 kDa protein which was specifically labeled with [3H]PK14105. Altogether these results show that the cDNA transfected in yeast encodes a 18 kDa protein with the expected characteristics of the hPBR.

Affinity Labels↗

PubMed1991

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.

Amino Acid Sequence↗

PubMed1991

Cloning and sequencing of PYBP, a pyrimidine-rich specific single strand DNA-binding protein.

In the human transferrin gene promoter, PRI and DRI are positive cis-acting elements interacting respectively with two families of proteins, Tf-LF1 and Tf-LF2. In this paper, we report the purification from rat liver nuclei, of one of these factors, PYBP, as well as the cloning and the sequencing of its cDNA. PYBP is a DNA-binding protein, purified as a 58 kDa doublet which binds only to single strand pyrimidine-rich DNA present for example in PRI and DRI. The protein binds also to a similar polypyrimidine tract present in one of the two strands of a DNA regulatory element of the rat tyrosine aminotransferase gene enhancer. PYBP gene is transcribed ubiquitously as a roughly 2.8 kb RNA which is likely to be subject to an alternative splicing. PYBP is highly homologous to a mouse nuclear protein, as well as to PTB, its human version, which interacts specifically with the pyrimidine tracts of introns. Primary structure information and predicted secondary structure elements of the protein indicate that PYBP contains four sequence repeats. Each of these repeats appears to exhibit the typical RNA recognition motif found in several proteins interacting with RNA or single strand DNA. Finally several hypotheses concerning the biological function of PYBP are presented.

Amino Acid Sequence↗

PubMed1991

Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor.

The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.

Amino Acid Sequence↗

PubMed1991

Human interleukin 1 beta fused to the human growth hormone signal peptide is N-glycosylated and secreted by Chinese hamster ovary cells.

A hybrid gene consisting of the sequences coding for the signal peptide of human growth hormone and the mature form of interleukin-1 beta (IL-1 beta) was chemically synthesized. This sequence was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of recombinant IL-1 beta, which was secreted into the culture medium mainly as a 22-kDa form. Expression in the presence of tunicamycin, an inhibitor of N-glycosylation, led to the complete disappearance of the 22-kDa form and the appearance of a new form of 17.5 kDa, indicating that the hybrid protein had been both processed and N-glycosylated. However, transformed cells producing mature IL-1 beta without a signal peptide produced the predicted 17.5-kDa nonglycosylated form. These results suggest that fusion to a heterologous leader sequence allowed IL-1 beta to be translocated across the membrane of the endoplasmic reticulum and to be transported and secreted by the exocytotic pathway.

Amino Acid Sequence↗

PubMed1991

Role of interleukin 6 in the inflammatory response after cataract surgery. An experimental and clinical study.

The mechanism of uveitis following cataract surgery, which is mediated in part by the cyclooxygenase pathway, is complex, the complexity of which is likely to be due to several factors. We investigated the possible local involvement of interleukin 6 (IL-6) in this response. Using a specific bioassay, we showed a dramatic increase of IL-6 levels (> 4 x 10(3) times) in all of the aqueous humors of 12 patients following cataract surgery. The IL-6 levels in serum samples were below detection limits, indicating a local production of this cytokine. In addition, the injection of highly purified recombinant interleukin 6 in the anterior chamber of the eye in rabbits resulted in an inflammatory response. These observations suggest that IL-6 may play a crucial role in the occurrence of inflammation after cataract surgery, and it may lead to new therapeutic approaches to this problem.

Animals↗

PubMed1991

Topological maps of protein sequences.

A new method based on neural networks to cluster proteins into families is described. The network is trained with the Kohonen unsupervised learning algorithm, using matrix pattern representations of the protein sequences as inputs. The components (x, y) of these 20 x 20 matrix patterns are the normalized frequencies of all pairs xy of amino acids in each sequence. We investigate the influence of different learning parameters in the final topological maps obtained with a learning set of ten proteins belonging to three established families. In all cases, except in those where the synaptic vectors remains nearly unchanged during learning, the ten proteins are correctly classified into the expected families. The classification by the trained network of mutated or incomplete sequences of the learned proteins is also analysed. The neural network gives a correct classification for a sequence mutated in 21.5% +/- 7% of its amino acids and for fragments representing 7.5% +/- 3% of the original sequence. Similar results were obtained with a learning set of 32 proteins belonging to 15 families. These results show that a neural network can be trained following the Kohonen algorithm to obtain topological maps of protein sequences, where related proteins are finally associated to the same winner neuron or to neighboring ones, and that the trained network can be applied to rapidly classify new sequences. This approach opens new possibilities to find rapid and efficient algorithms to organize and search for homologies in the whole protein database.

Amino Acid Sequence↗

PubMed1991

Isolation, purification and primary structure of insulin from the turtle Chrysemys dorbigni.

Insulin A and B chains from pancreas of the turtle Chrysemys dorbigni have been purified to homogeneity, and their primary structures have been determined. The sequence of the A chain is G-I-V-E-Q-C-C-H-N-T-C-S-L-Y-Q-L-E-N-Y-C-N, and that of the B chain is A-A-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-S-P-K-A. The amino acid sequence of Chrysemys insulin is identical to that of another turtle (Pseudemys scripta), the chicken, and turkey. When compared with alligator insulin, it has three conservative substitutions in the B chain. However, there are seven substitutions when compared with the insulin of the rattlesnake.

Amino Acid Sequence↗

PubMed1991

Human recombinant dimeric IL-6 binds to its receptor as detected by anti-IL-6 monoclonal antibodies.

Three different epitopes of the human IL-6 (IL-6) molecule were recognized by the mAb B-E4 (IgG2b), B-E8 (IgG1) and B-F6 (IgG1). The affinities of these three mAb for IL-6 differ little in several assays but if ranked by affinity they fall into the following order B-E8 greater than B-E4 greater than B-F6. B-E4 and B-E8 mAb, recognizing two different epitopes, are inhibiting mAb in the bioassay with the IL-6 depending cell line B9, however B-E8 has an inhibiting activity higher than B-E4. Both human natural IL-6 (HnIL-6) and human recombinant IL-6 (HrIL-6) were inhibited but not the murine natural IL-6 (MnIL-6). Surprisingly, not only the non-inhibiting mAb (B-F6) recognizes the HrIL-6 fixed to the receptor but also the inhibiting mAb B-E4 and B-E8. This together with the results obtained in a sandwich ELISA where the same mAb was used as both catcher and tracer to detect HrIL-6, it was concluded that dimeric HrIL-6 is able to fix the IL-6 receptor. Competition studies between monomeric HnIL-6 and dimeric HrIL-6 showed that the affinity of the dimeric HrIL-6 for the receptor was higher than that of HnIL-6.

Animals↗

PubMed1991

Low dose aprotinin as blood saver in open heart surgery.

Bleeding after open heart surgery is still a great concern for the surgeon, especially when the surgical field has been revised accurately and hemostatic stitches and electrical cauterization have been used extensively. Among non-surgical adjuncts, aprotinin has been reported as very effective in reducing complications. At the time we started using this drug, we intended to test two different dosages lower than those reported in the literature. We evaluated three groups of 18 patients: the first (A) received about 350 mg of aprotinin from the start of anesthesia up to the end of operation (140 mg in the priming of cardio-pulmonary bypass and 70 mg/h i.v. during the procedure; the second (A/2) received half that dose (i.e. 70 mg and 35 mg, respectively), and the third (C) did not receive aprotinin. We compared in these groups: postoperative bleeding, blood transfusions, red blood cells, hemoglobin, hematocrit, platelets. The results were good only in the A group: bleeding was reduced and few transfusions were required. The patients in the A/2 and C groups did not show significant differences. From our observations we conclude that aprotinin is a useful adjunct, but has to be given in the proper dose.

Aprotinin↗

PubMed1991

Primary structure of alpaca growth hormone.

Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptides in the mixture were purified by reverse phase HPLC and N-terminal determination and an amino acid analysis of each was performed. Data obtained and the already known primary structure of the equine growth hormone allowed the assembly-by homology-of a definite sequence of amino acids for the polypeptide chain of the protein. Present data provide further information about the relationship between growth factors.

Amino Acid Sequence↗

PubMed1990

Separation by cation-exchange high-performance liquid chromatography of three forms of Chinese hamster ovary cell-derived recombinant human interleukin-2.

Purified recombinant (r) interleukin 2 (IL-2) produced by a transformed Chinese hamster ovary cell line shows a single peak when analysed by reversed-phase high-performance liquid chromatography, but it can be resolved into three forms by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These three forms were successfully isolated by narrow-bore ion-exchange chromatography through optimization of the elution conditions. The addition of n-propanol as an organic modifier to the mobile phase proved to be essential for the recovery of the protein from the column in a yield of 90% or better based on protein quantification and biological activity determination. This chromatographic method was used for the purification of these three rIL-2 forms which represent variable glycosylation of a single polypeptide chain. A comparison of the biological activities using the murine CTLL-2 cell proliferation assay showed that the specific activities of the three forms are similar.

Animals↗

PubMed1990

Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock.

A synthetic gene coding for the C-terminal 153 amino acids of the human interleukin-1 beta (IL-1 beta) was used to produce large quantities of recombinant IL-1 beta in Escherichia coli. The expression of the synthetic gene was under the control of an inducible promoter. The recombinant protein was released from the cells by an osmotic shock. This procedure did not lyse the cells. The IL-1 beta that represented 90% of the total extracted protein was purified to homogeneity by a single chromatographic step. Sequence analysis revealed a heterogeneous N-terminal sequence resulting from the cleavage of the N-terminal methionine in 50% of the molecules and of both the N-terminal methionine and alanine in the other 50%. This recombinant IL-1 beta had a specific activity of 1.3 x 10(8) international units per mg.

Base Sequence↗

PubMed1990

A new extra sequence at the amino terminal of a mu heavy chain disease protein (DAG).

The primary structure of a human mu heavy chain (DAG) protein is described. The native protein is a circular decamer with a molecular weight (Mr) of 500 kDa, each decamer being constituted of the constant domains C mu 2, C mu 3 and C mu4 and interlinked by 15 disulfide bridges. At its NH2-terminal each monomeric chain starts with an "extra sequence". The amino acid sequence of this segment is Arg-Gln-Ser-Asp-Asp-Pro-Val-Leu-Arg-Gly-Thr-Thr-Val-Pro-Val-Thr-Glu and its reinitiation point is located at Val223 (Gal numbering), at the beginning of C mu 2. This sequence has no homology with any other protein included in the present databases.

Amino Acid Sequence↗

PubMed1990

Relationship between the antigenic topography and the structure of human growth hormone.

Monoclonal antibodies (MAb) to human GH (hGH) were used to correlate the antigenic topography of the hormone with its structure. Competition experiments performed in a solid phase RIA system allowed us to measure the reactivity toward the MAb of the following hGH derivatives: hGH 20K (a natural variant lacking residues 32-46), hGH selectively modified in His or Met residues, hGH with the C and/or N-terminal disulfide bond reduced and carbamidomethylated, and hGH cleaved between residues 142-143. Results indicated that fragment 32-46 participates in the structure of epitopes EB1/EB3 and that the C-terminal bridge is located in epitope 10D6, whereas opening of both disulfide bridges alters the entire hGH antigenic surface. His-151 and Met-170 were placed in epitopes NA71 and AC8, respectively, whereas His-18 and Met-14 would be involved in the hGH antigenic domain formed by overlapping epitopes 3C11, 10C1, and HG3. MAb AE5, AE12, and AC3 define a flexible hGH region related to sequence 134-150; the respective epitopes show high conformational mobility induced by modifications in other regions of the molecule. Binding of the different hGH derivatives to lactogenic receptors from female rat liver gave some insights on the localization of the hormone-binding site. Epitopes EB1/EB3 and 10D6 were discarded because there was not a direct correlation between their drastic immunological alterations and the binding properties of the respective hGH derivatives. In the same way, epitopes AE5, AE12, and AC3 were excluded from the hGH-binding domain because a disruption in those sites did not affect the hGH interaction with receptors. We conclude that the hGH structure defined by epitopes 3C11, 10C1, and HG3 is probably related to the binding properties of the hormone.

Animals↗

PubMed1990

Lymphocyte populations of non-scleronodular Hodgkin's disease subtypes in different stages of lymphocyte depletion. An immunophenotypic and quantitative study.

Twenty-one cases of non-scleronodular Hodgkin's disease with variable lymphocyte contents were studied immunophenotypically and quantitatively to analyse the distribution of different lymphocyte populations and to determine whether selective loss of lymphocyte subpopulations accompanies overall lymphocyte depletion. In Hodgkin's tissue B-cells were scanty and unevenly distributed in samples with both many and few lymphocytes. Several large B, LN1-positive (possibly activated) cells were observed in a few cases. CD3-positive T-lymphocytes predominated in all cases; the same cells were also UCHL1-positive, thus expressing characteristics of mature T-memory cells. CD4-positive lymphocytes were usually more numerous than CD8-positive lymphocytes, but quantitative evaluation of the latter showed that they did not decrease in proportion to any diminution of the whole lymphocyte population. This finding suggests that in the process of lymphocyte depletion more CD4-positive lymphocytes than CD8-positive lymphocytes are lost, and this might account for the impairment of cell-mediated immunity in Hodgkin's disease.

Antibodies, Monoclonal↗

PubMed1990

Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2.

We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2.

Acetylgalactosamine↗

PubMed1990

[Tha data management of a breast radiology section via a computerized archive].

In the Institute of Radiology of the University of Trieste, from January to December 1988, 7123 patients underwent breast diagnostic procedures following a screening program for breast cancer or in order to evaluate specific symptoms. The data relative to this series of cases were collected and processed using the system described in a colleague's paper. Several sets of data became available concerning the percentage distribution of the techniques employed, their main indications, the distribution of pathological features in relation to different indications, the diagnostic value of combining different techniques, and the suggestions based on the findings collected. Both mammography and US proved to be able to solve the great majority of diagnostic problems, sometimes also as a tool to guide bioptic procedures. Other techniques (galactography, pneumocystography, CT) were devoted to specific diagnostic problems. The results are interesting in view of their clinical, epidemiological and management implications.

Breast Neoplasms↗