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Biomedical subjects

S Merli

Publications and source records attributed to S Merli.

At least 19 recordsLinked to original sources
PubMed1996

An expression system for the single-step production of recombinant human amidated calcitonin.

Amidating mouse pituitary cells (AtT-20) have been engineered to secrete human calcitonin (hCT) in the fully active amidated form, without the need of additional enzymatic or chemical modifications. The 141-residue human calcitonin precursor has first been cloned in the eucaryotic expression vector pRc/RSV, and the resulting plasmid pRc/RSV/hCT introduced in AtT-20 cells. After transfection, 122 independent clones resistant to G-418 were selected and screened for calcitonin production using a competitive ELISA specifically designed to detect the amidated form of calcitonin. One of these clones was amplified and showed expression of 17 ng/ml of hCT, with a 70% increase in productivity after cAMP treatment. Calcitonin was partially purified from culture medium by two sequential steps of reverse-phase chromatography and characterized in terms of immunoreactivity and molecular weight by TOF-MALDI mass spectroscopy, which confirmed the intended chemical nature and the presence of the C-terminal amidated residue.

Amino Acid Sequence↗
PubMed1995

Production of soluble tumor necrosis factor receptor type I in Escherichia coli: optimization of the refolding yields by a microtiter dilution assay.

In this study optimization of the soluble tumor necrosis factor receptor type I (sTNF-RI) refolding by the use of a micro-renaturation assay in 96-well microplates is described. Microplate wells were filled with buffers varying in pH and urea and substrate concentration. Denatured and reduced sTNF-RI was then rapidly diluted and allowed to refold for a variable time at different temperatures. The extent of renaturation was measured by a sandwich enzyme-linked immunosorbent assay (ELISA), based on the use of two monoclonal antibodies obtained against urinary sTNF-RI. Among about 100 different combinations tested, a maximum refolding yield of 21.5% has been obtained in 100 mM Tris, pH 8-8.5, 1 mM EDTA, 0.1% bovine serum albumin, 2 M urea, at a denatured protein concentration of 10 micrograms/ml and at 26 degrees C. Folded sTNF-RI was purified by batchwise immunoaffinity chromatography and its activity evaluated by immunological and biological assays. A good correlation was observed between the data obtained with different assays (biological assay, ligand-directed ELISA, and double-determinant sandwich ELISA) indicating that the refolded receptor has gained biological and immunological reactivity comparable to those of the soluble TNF-receptor type I expressed in eukaryotic cells.

Animals↗
PubMed1995

Identification of two forms (31-33 and 48 kD) of the urinary soluble p55 tumor necrosis factor receptor that are differentially N- and O-glycosylated.

The structure and the activity of urinary soluble TNF receptor type 1 (sTNF-R1), isolated from the urine of normal individuals, has been characterized and compared with that of recombinant sTNF-R1 expressed in CHO cells and with that of a nonglycosylated form expressed in Escherichia coli. Urinary sTNF-R1 was resolved in a major band of 31-33 kD and in a 48 kD band (less than 5% of total) by reducing SDS-PAGE; CHO sTNF-R1 was resolved in two bands of 29 and 31 kD. All bands were recognized by various anti-sTNF-R1 antibodies as well as by TNF-alpha in western and ligand blotting assays. No cross-reaction was observed with anti-TNF-R2 antibodies. N- and O-glycosylation studies indicated that (1) the 29-31 kD recombinant form as well as the 31-33 kD urinary form are N-glycosylated; (2) the differences between the 29-31 and 31-33 kD recombinant and natural products are mainly related to differences in the N-linked sugar content; and (3) the 48 kD sTNF-R1 isolated from urine also contains O-linked sugars. The urinary sTNF-R1 antigen mixture was able to inhibit TNF-alpha cytotoxicity with a potency comparable to that of nonglycosylated E. coli sTNF-R1. At variance, urinary sTNF-R1 was able to inhibit TNF-beta sevenfold more efficiently than E. coli sTNF-R1. In conclusion, two subtypes of sTNF-R1 have been isolated from urine: a main N-glycosylated form of 31-33 kD and a N- and O-glycosylated form of 48 kD that appears to be a minor constituent of the urinary sTNF-R1 antigen.

Animals↗
PubMed1995

Quantification of headache disability: a diagnostic-based approach.

The quantification of the social and economic handicaps caused by headache is a complex problem, especially given the great variability of headache patients' clinical pictures. In the present study, 400 patients, consecutively admitted to Headache Centers in Pavia and Milan, were interviewed on the relationship between headache and their work and social activities, in order to evaluate their socioeconomic handicap due to headache. The analysis of the data primarily focused on attack-type headaches (migraine, cluster headache, and episodic tension-type headache) and chronic or daily headaches (chronic tension-type headache and migraine combined with tension-type headache). These latter types were often characterized by the daily use or abuse of analgesics. The overall profile which emerged from the study reveals relatively low levels of handicap or disability in work and social activities. These low levels can be mainly attributed to timely, and at times excessive, use of analgesics.

Absenteeism↗
PubMed1994

Tumor necrosis factor (TNF) alpha quantification by ELISA and bioassay: effects of TNF alpha-soluble TNF receptor (p55) complex dissociation during assay incubations.

It has been previously reported that different quantitative results can be obtained when TNF alpha is measured in biological fluids by bioassay and immunoassay. This is thought to be related to the presence of antigenic forms of TNF alpha that cannot be detected by bioassay, such as complexes with soluble receptors (sTNF-R) or TNF alpha monomers. In this work we have observed discrepancies between antigenic and bioactive TNF alpha even when we used a sandwich-ELISA, unable to detect TNF alpha monomers, based on antibodies that bind epitopes overlapping with the soluble-receptor binding site of TNF alpha. Moreover, we found that antigenic TNF alpha levels in the presence of p55-sTNF-R (sTNF-R1) measured by different immunoassays were variable, depending on the immunoreagents and incubation time. To investigate whether TNF alpha-soluble receptor complex dissociation occurring during assay incubations contributes to the variability of results, we studied the kinetics of TNF alpha-soluble receptor interactions and examined the effect of complex dissociation using different analytical systems. TNF alpha association (k(on)) and dissociation (koff) rate constants with sTNF-R1, measured by real-time biospecific interaction analysis, were 5.01 x 10(5) s-1 M-1 and 2.8 x 10(-4) s-1, corresponding to an equilibrium constant (Kd) of 0.59 nM and to a half life for these complexes of 38 min. Complex dissociation and differential changes in the TNF alpha-sTNF-R1 bound:free ratio, in different analytical systems, markedly affects TNF alpha quantification.

Binding, Competitive↗
PubMed1994

High yield expression and purification of human endothelin-1.

A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%.

ATP-Binding Cassette Transporters↗
PubMed1992

Identification of hydropathically complementary putative contact sequences within epidermal growth factor (EGF) and the EGF receptor.

Hydropathic complementariness (HC) has been proposed as a novel molecular recognition code for how two proteins can recognize one other and thus form a reversible complex. If a protein contains a segment of a few amino acid residues that is surface-exposed, plus in extended conformation, plus composed of residues whose hydropathy pattern is opposite to that of a correspondingly sized segment on the respective other protein, this protein may bind to the other one through such a segment of HC (1). In order to identify in a pair of proteins sequences of HC we have developed the program PUTATIVE SITES SEARCHER (PSS-1) (2), a name that alludes to the possibility that such a segment of HC could represent a putative contact "site". Here we describe the application of PSS-1 to the study of human epidermal growth factor (EGF) and human EGF receptor (EGF-R). Six segments of HC were identified, two of which, designated a and b, fall exactly into experimentally verified contact regions on EGF as well as on EGF-R. Site a consists of residues 25.AEIYMCV.19 of EGF ("half site" aEGF) and of residues 331.NIKHFKN.337 of the EGF-R ("half site" aEGF-R); site b consists of residues 34.VCNCAY.29 of EGF and residues 365.PQELDI.370 of the EGF-R. Most interestingly, both half sites aEGF and bEGF localize in loop B of hEGF which is recognized as being essential for receptor binding. Similar is true for the half sites aEGF-R and bEGF-R that localize in subdomain III (residues 314-445) of the extracellular part of the EGF-R, also identified to be responsible for EGF binding. Thus, each of the two theoretically predicted sites is composed of half sites whose functional importance is experimentally verified. This correspondence supports the principal suitability of PSS-1 and suggests that EGF binds to EGF-R at least in part by means of HC contacts besides using, most probably, also "classical" (i.e. non-HC-type) contacts (e.g. charge interactions or hydrophobic bonds).

Amino Acid Sequence↗
PubMed1990

New biosynthetic anthracyclines related to barminomycins incorporating barbiturates in their moiety.

Three new anthracyclines, FCE 21424 (2), FCE 24366 (3) and FCE 24367 (4), were isolated from culture broths of Streptomyces peucetius and its mutant strains after addition of sodium barbiturates during the fermentation. Structural assignment, achieved through spectroscopic and degradative studies, that the new anthracyclines had a common barminomycin-like structure incorporating different barbiturate moieties. The new anthracyclines were found to display outstanding cytotoxicity and remarkable potency "in vivo" against P388 ascitic leukemia.

Animals↗
PubMed1989

A single amino acid interchange yields reciprocal CTL specificities for HIV-1 gp160.

For the IIIB isolate of human immunodeficiency virus type-1 (HIV-1), the immunodominant determinant of the envelope protein gp160 for cytotoxic T lymphocytes (CTLs) of H-2d mice is in a region of high sequence variability among HIV-1 isolates. The general requirements for CTL recognition of peptide antigens and the relation of recognition requirements to the natural variation in sequence of the HIV were investigated. For this purpose, a CTL line specific for the homologous segment of the envelope from the MN isolate of HIV-1 and restricted by the same class I major histocompatibility (MHC) molecule (Dd) as the IIIB-specific CTLs was raised from mice immunized with MN-env-recombinant vaccinia virus. The IIIB-specific and MN-specific CTLs were completely non-cross-reactive. Reciprocal exchange of a single amino acid between the two peptide sequences, which differed in 6 of 15 residues, led to a complete reversal of the specificity of the peptides in sensitizing targets, such that the IIIB-specific CTLs lysed targets exposed to the singly substituted MN peptide and vice versa. These data indicate the importance of single residues in defining peptide epitopic specificity and have implications for both the effect of immune pressure on selection of viral mutants and the design of effective vaccines.

Amino Acid Sequence↗
PubMed1988

Group-specific, major histocompatibility complex class I-restricted cytotoxic responses to human immunodeficiency virus 1 (HIV-1) envelope proteins by cloned peripheral blood T cells from an HIV-1-infected individual.

Freshly separated unfractionated peripheral blood mononuclear cells (PBMC) and cloned cell lines from a healthy human immunodeficiency virus 1 (HIV-1)-seropositive individual were examined for cytotoxic responses to HIV proteins expressed by recombinant vaccinia viruses. It was found that freshly isolated PBMC recognize variant envelope proteins of HIV-1 but not a more distantly related envelope protein derived from the simian immunodeficiency virus (SIVmac). Although the effector cells were predominantly CD8+, both MHC-matched and -unmatched target cells were lysed. Cytotoxic T lymphocyte (CTL) clones were found to lyse cells expressing HIV-1 envelope or reverse transcriptase. In contrast to the cytotoxic response detected with PBMC, the cloned CTLs were major histocompatibility complex (MHC) class I restricted. Our finding that a cloned CTL line lysed cells expressing highly divergent HIV envelopes strongly suggested that a conserved epitope was recognized. Identification of these shared epitopes may assist in designing a vaccine for HIV-1 that could stimulate MHC-restricted cytotoxic responses.

Acquired Immunodeficiency Syndrome↗
PubMed1984

A molecular study of a family with Greek hereditary persistence of fetal hemoglobin and beta-thalassemia.

A family was studied in which two inherited defects of the non-alpha-globin cluster segregate: Greek hereditary persistence of fetal hemoglobin (HPFH) and beta-thalassemia. Fragments of the non-alpha-globin cluster from two patients were cloned in cosmid and phage lambda vectors, and assigned to either the HPFH or beta-thalassemic chromosome on the basis of the demonstration of a polymorphic BglII site in the HPFH gamma-globin cluster. The thalassemic beta-globin gene carries a mutation at nucleotide 1 of the intervening sequence I, known to cause beta zero-thalassemia; the beta-globin gene from the HPFH chromosome is entirely normal, both in the intron-exon sequence and in 5' flanking regions required for transcription. As the compound HPFH/beta-thalassemia heterozygote synthesizes HbA, these data prove that the HPFH beta-globin gene is functional, although at a decreased rate; its lower activity is likely to be due to a distant mutation. The HPFH A gamma-globin gene shows only two mutations: a T----C substitution in the large intervening sequence (responsible for the BglII polymorphic site) and a C----T substitution 196 nucleotides 5' to the cap site; the 5' flanking sequence is normal up to -1350 nucleotides upstream from the gene. Circumstantial evidence suggests that the mutation at -196 may be responsible for the abnormally high expression of the A gamma-globin gene.

Base Sequence↗
PubMed1982

New anthracycline glycosides: 4-O-demethyl-11-deoxydoxorubicin and analogues from Streptomyces peucetius var. aureus.

The new anthracyclines 4-O-demethyl-11-deoxydoxorubicin, 4-O-demethyl-11-deoxydaunorubicin along with its 13-dihydro and 13-deoxo analogues are the main components of the anthracycline complex produced by cultures of Streptomyces peucetius var. aureus. They were isolated by solvent partition, separated by column chromatography and characterized by chemical and physical methods. Among these new anthracyclines, displaying antibacterial and cytotoxic activity "in vitro", 4-O-demethyl-11-deoxydoxorubicin and the corresponding daunorubicin analogue were also active against experimental tumors.

Animals↗