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P Ferrara

Publications and source records attributed to P Ferrara.

At least 199 records · Page 11Linked to original sources
PubMed1987

Characterization of recombinant glycosylated human interleukin 2 produced by a recombinant plasmid transformed CHO cell line.

A recombinant plasmid containing expression units for human pre-interleukin 2 (pre-IL-2) and the selectable marker mouse DHFR, was constructed and used to transform DHFR- CHO cells to the DHFR+ phenotype. Selected colonies were isolated and tested for IL-2 production. Twelve highly IL-2-producing clones were amplified in stepwise increasing concentrations of methotrexate. The IL-2 secreted into the culture medium by one of these clones was purified to homogeneity and partially characterized. N-terminal sequence analysis showed that pre-IL-2 was correctly processed during secretion. SDS gel electrophoresis and chromatofocusing experiments in conjunction with neuraminidase treatment indicated a posttranslational glycosylation of the secreted mature protein similar to that described for the tetrasaccharide structure of the N2 form of natural IL-2. This recombinant IL-2 has a specific activity of 2.5 x 10(7) U/mg.

Amino Acid Sequence↗
PubMed1987

Rat pheochromocytoma tyrosine hydroxylase is phosphorylated on serine 40 by an associated protein kinase.

Tyrosine hydroxylase, a key enzyme in the biosynthesis of catecholamines, was previously shown to be phosphorylated on four distinct serine residues in PC12 cell cultures, each one being specific for the kinase system involved (McTigue, M., Cremins, J., and Halegoua, S. (1985) J. Biol. Chem. 260, 9047-9056). A cAMP- and Ca2+-independent protein kinase was found to be associated with tyrosine hydroxylase purified from rat pheochromocytoma tumor. The use of this activity and the availability of a large amount of purified tyrosine hydroxylase allowed identification of the site phosphorylated by this kinase activity. A peptide of 1.5 kDa (about 12 residues long), carrying the phosphorylation site, was released from 32P-labeled tyrosine hydroxylase by limited proteolysis with trypsin. This peptide was isolated from trypsinized tyrosine hydroxylase by sequential gel filtration and ion exchange chromatographies. Analysis by thin layer chromatography of an acid hydrolysate of the peptide revealed that it contained phosphoserine. The sequence determination of the peptide showed that it corresponded to the residues 38-45 in the tyrosine hydroxylase primary structure (Arg-Gln-Ser(P)-Leu-Ile-Glu-Asp-Ala). Thus, the associated kinase phosphorylated Ser-40, one of the phosphorylation sites for the cAMP-dependent protein kinase also found in rat pheochromocytoma tumors. These results are compared to those recently appearing in a report by Campbell et al. (Campbell, D. G., Hardie, D. G., and Vulliet, P. R. (1986) J. Biol. Chem. 261, 10489-10492).

Amino Acid Sequence↗
PubMed1987

Role of radiation therapy in glomus tumor.

Since 1964, we have reported twenty-five cases of glomus tumors. Given the evolution of modern surgical technique, we have divided our cases into two groups. The first group includes patients seen from 1964 to 1975: sixteen cases, of whom eleven underwent successful radiotherapy, with a follow-up after ten years. Since 1975 we have adhered to the following therapeutic principles: We have given radiotherapy (5000 rads in twenty-eight days) to patients older than 65 with glomus tumors that had invaded posterior and medial cranial fossa and carotid canal (type C-D, according to Fisch). We have operated on patients with glomus tumors type A-B. Patients treated since 1975 (N = 9) are in the second group.

Glomus Jugulare Tumor↗
PubMed1987

[Clinical dosimetry in xeromammography using the low dose system].

The results obtained by utilizing low-dose plates in xeromammography are reported. Phantom experimental examinations and dosimetric measurements were made before using these plates. The new exposition data were then compared to those obtained with the old plates. Findings prove low-dose plates to be more sensitive--thus granting very good iconographic results with reduction of skin dose (about 30%) at FFD of 110 cm. The skin dose has been compared to that obtained with the most modern mammographs.

Female↗
PubMed1986

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells.

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.

Amino Acids↗
PubMed1986

Hydrophobic adsorbents for the isolation and purification of biosynthetic human growth hormone from crude fermentation mixtures.

Hydrophobic interaction chromatography was used as the initial step for the concentration and partial purification of biosynthetic human growth hormone (hGH). This molecule was extracted from the culture medium of a transformed monkey kidney cell line by batch adsorption either to Octyl-Sepharose or Phenyl-Sepharose. The adsorbate was decanted and packed into a column for elution. This initial work-up procedure rendered a concentrated hGH solution that was further purified by ion-exchange and gel filtration chromatography. The hGH obtained was homogeneous, based on polyacrylamide gel electrophoresis, N-terminal sequence and fingerprint analysis.

Adsorption↗
PubMed1986

Purification to homogeneity and NH2-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line.

A subclone, referred to as 3B6, derived from a DR-negative EBV-transformed B cell line, has been found to spontaneously produce IL 1. 3B6-IL 1 displays a pI of 5 on FPLC chromatofocusing. It has been purified to homogeneity by a sequence of ion-exchange chromatography and affinity chromatography on procion red agarose. The homogeneous material migrated with an apparent m.w. of 13,500 on SDS-PAGE. The overall recovery of IL 1 activity was estimated at 57%. The final material had a specific activity of 7.8 X 10(6) half-maximal units/mg and represented a 50,000-fold purification. A partial NH2-terminal amino acid sequence has been obtained that is different from those reported from monocytic IL 1. However, this molecule can formally be identified as IL 1 on its spectrum of biologic activities. In addition to inducing the proliferation of murine thymocytes in the co-stimulator assay. 3B6-IL 1 is active on both human T and B cells, respectively, in inducing IL 2 synthesis by cells from a subcloned HSB 2 line and promoting the proliferation of anti-IgM-stimulated human peripheral blood B lymphocytes. Furthermore, 3B6-IL 1 acts as a growth factor for normal human fibroblasts and for the 3B6 line itself. However, 3B6-IL 1 is not pyrogenic in rabbits. Thus, the 3B6 cell line was shown to produce a new molecular species of IL 1, with respect to its NH2-terminal sequence, which shared all of the studied biologic activities of monocytic IL 1 except for pyrogenicity.

Amino Acid Sequence↗
PubMed1986

Influence of food intake on bioavailability of oral 6-mercaptopurine in children with acute lymphoblastic leukemia.

Plasma levels of 6-mercaptopurine (6-MP) were measured after oral administration in 17 children with acute lymphoblastic leukemia (ALL). In the fasting state or after a breakfast consisting of 250 ml milk and 50 g biscuits, 6-MP was administered at a dose of 75 mg/m2. In patients studied in a fasting state, the mean time to plasma peak (tmax) level was 1.2 h, whereas in patient studied after breakfast the mean tmax was 2.3 h. This difference is statistically significant (p less than 0.001). Moreover, the 6-MP peak plasma concentration (cmax) and the areas under the plasma concentration time curves (AUC) were significantly reduced when the drug was administered after breakfast. The mean Cmax +/- SD were 0.98 +/- 0.54 microM and 0.63 +/- 0.48 microM, respectively (p less than 0.05). The mean 6-MP AUC +/- SD in patients studied in a fasting state and after breakfast were 143 +/- 69 microM min and 105 +/- 68 microM, respectively (p less than 0.01). These results indicated that 6-MP should be taken in a fasting state to optimize drug absorption in children undergoing chemotherapy for ALL.

Administration, Oral↗
PubMed1985

Beta-endorphin does not protect alkylation of opiate receptor by N-ethylmaleimide.

Rat brain membranes were incubated in N-ethylmaleimide (NEM, 0.5-1.0 mM) in the presence and absence of various concentrations of morphine, Leuenkephalin and human beta-endorphin (beta h-EP). After sufficient washing, the binding of dihydromorphine (DHM), [D-Ala-D-Leu]-enkephalin (DADLE) and tritiated beta h-EP was 10-40% above that of membranes treated with NEM alone. There was no additive effect of morphine and Leu-enkephalin with respect to their effect on recovery of beta h-EP binding. Evaluation of beta h-EP as protecting ligand proved to be difficult since preincubation completely inhibits subsequent DHM and DADLE binding unless a more extensive washing protocol is employed. A protocol for washing beta h-EP preincubated membranes using a Tris-phosphate buffer of pH 6 containing 150 mM NaCl, 20 mM MgCl2 and 10% glycerol was used to recover enough binding potential to evaluate the effects of beta h-EP preincubation towards NEM treatment. Preincubation with beta h-EP itself at 0.1-1.0 microM did not result in any increased recovery of opiate binding, in contrast to the findings with the other two ligands.

Alkylation↗
PubMed1984

Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12.

In Escherichia coli, AK I- HDH I and AK II- HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the common pathway leading to threonine and methionine. An extensive amino acid sequence comparison of both molecules reveals two main features on each of them: (i) two segments, each of about 130 amino acids, covering the first one-third of the polypeptide chain, are similar to each other and (ii) two segments, each of about 250 amino acids and covering the COOH-terminal 500 amino acids also present a significant homology. These findings suggest that these two regions may have evolved independently of each other by a process of gene duplication and fusion previous to the appearance of an ancestral aspartokinase-homoserine dehydrogenase molecule.

Amino Acid Sequence↗
PubMed1984

Beta-endorphin. Binding activity of synthetic analogs with various chain lengths in neuroblastoma x glioma NG108-15 cell membranes.

Inhibition of binding of beta h-endorphin or Leu-enkephalin by beta h-endorphin analogs of various chain lengths in memmbrane preparations of the neuroblastoma x glioma NG108-15 cells has been investigated. The removal of even a single residue from the C-terminus results in the inability of the resulting peptide to completely displace beta h-endorphin. In addition, the proportion of nondisplaceable binding increases with decreasing chain length.

Animals↗
PubMed1983

Structure of the metJBLF cluster in Escherichia coli K12. Sequence of the metB structural gene and of the 5'- and 3'-flanking regions of the metBL operon.

The total nucleotide sequence (1,158 nucleotides) of the metB gene of Escherichia coli coding for cystathionine gamma-synthase (386 amino acid residues, Mr = 41,503/chain) is presented. The nucleotide sequences of the flanking regions of the metB and metL genes are also presented. Analysis of these sequences and identification of a promoter region upstream from the metB gene confirms that metB and metL form an operon. The transcription direction is from metB to metL; the start site of the gene transcription has been determined. There is no structural evidence of a classical attenuation mechanism in the regulation of this operon coding for enzymes implicated in an amino acid biosynthetic pathway. Finally, the overall organization of the metJBLF gene cluster is discussed.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of metF, the E. coli structural gene for 5-10 methylene tetrahydrofolate reductase and of its control region.

The nucleotide sequence of the E.coli metF gene (888 nucleotides), coding for 5-10 methylene tetrahydrofolate reductase, has been determined. The metF gene product was identified in maxicells and found to be a protein of subunit molecular weight 33,000, in agreement with the size of the coding region. The starting point for metF transcription was determined by S1 nuclease mapping. No structural evidence was found for an attenuation mechanism regulating the independent metF transcriptional unit. Comparison of the regulatory region preceding the metF structural gene with the 5' flanking region of the metBL operon shows some homology spanning 24 nucleotides. These homologous sequences could be operator structures belonging to the two transcriptional units, metF and metBL, and recognized by the same regulatory protein.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1983

[Submicroscopic study of brown adipose tissue in young rats by the freeze-fracturing method].

The authors report the results of their ultrastructural study on the rat brown adipose tissue using the freeze-fracturing techniques. It is well known from transmission electron microscopy that lipid vacuoles of the brown adipose tissue cells are bound by a dense line corresponding to a structure as yet totally unknown. The results of the study using freeze-fracturing technics show that the morphology of lipid vacuoles surface is different from that of the typical unit membrane. The possibility is discussed that the particular aspect of the surface is due to the lypolysis induced by tissue fixation.

Adipose Tissue, Brown↗
PubMed1983

Synthesis and properties of beta h-endorphin analogs containing the dynorphin-(1-13) sequence.

Two analogs of beta h-endorphin containing dynorphin-(5-13) or dynorphin-(6-13) sequence have been synthesized by the solid-phase method. Biological activities of the analogs have also been investigated. It was found that the opiate activity is about three times more than beta h-endorphin in the guinea pig ileum assay. Both analgesic potency and opiate receptor-binding activity of the analogs are lower when compared with beta h-endorphin. However, the analogs have interesting behavioral effects in mice.

Analgesics↗
PubMed1982

Construction and physical mapping of plasmids containing the metJBLF gene cluster of E. coli K12.

In vitro recombination techniques were used to clone the E. coli metJBLF gene cluster in a plasmid vector. Several chimeric plasmids were obtained, analyzed by restriction mapping and characterized genetically. The combined results establish that the met gene cluster is contained on an approximately 5.6 kilobase segment of bacterial DNA with metL between metB and metF. The origin of metL was localized precisely by its DNA sequence and its transcription direction was established.

Bacteriophage lambda↗