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Biomedical subjects

S Di Marco

Publications and source records attributed to S Di Marco.

9 recordsLinked to original sources
PubMed1995

Interferon-alpha hybrids.

The alpha-interferons (IFN-alpha) belong to a family of polypeptides comprising several subtypes. Using recombinant DNA technology, it has been possible to create IFN hybrids that provide novel combinations of the amino acid residues from the parental protein sequences. They have been used to study structure-activity relationships of IFN-alpha and interactions with the IFN-alpha receptor, and to create analogs of natural IFNs with novel properties for potential therapeutic application. The biological data obtained with these hybrids are now evaluated in terms of the published structural and homology models of IFN-beta and -alpha.

Animals↗
PubMed1994

Domains of interaction between alpha interferon and its receptor components.

We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.

Animals↗
PubMed1994

Mutational analysis of the structure-function relationship in interferon-alpha.

A number of mutants of the recombinant human hybrid interferon-alpha 8[60]alpha 1[92]alpha 8 have been expressed in Escherichia coli, purified to homogeneity and characterized. The introduction of one or two negative charges results in an anomalously lower electrophoretic mobility than that expected from the molecular mass. The mutations Lys84-->Glu, Cys86-->Tyr, Cys86-->Ser, Thr87-->Ile, Tyr90-->Asp and Lys84-->Glu/Tyr90-->Asp (double mutant) result in a marked decrease of the biological activity in murine cells compared to the unmutated protein. Comparisons with published structural and homology models of interferon-beta and -alpha, imply that these residues are located primarily on the external surface of the carboxylterminus of helix C. We propose a model of interferon-receptor interaction in which these residues define a potential binding site.

Animals↗
PubMed1993

Synovial type (group II) phospholipase A2 in cartilage.

Phospholipase A2 (PLA2) was produced in E. coli by a recombinant technique using a synthetic gene coding for PLA2 found in synovial fluid (SF) of patients suffering from rheumatoid arthritis (RA). Polyclonal antibodies were produced against the recombinant synovial type PLA2 (syn-PLA2) in a rabbit. The IgG fraction of the antiserum was isolated and the specificity was tested by immunoblotting. The antiserum detected a protein with an apparent molecular weight of 15 kDa in extracts of cartilage, but did not react with PLA2 isolated from human pancreas or ascitic fluid. In immunohistochemistry, the anti-syn-PLA2 antibody decorated chondrocytes and matrix of articular, laryngeal and auricular cartilage, but did not decorate synovial tissue or its contained inflammatory cells in RA or other human tissues tested (pancreas, liver, thyroid, tonsil, lung, nasal polyp, skin, placenta, myometrium, bone). The results show that syn-PLA2 is present both in articular and extraarticular cartilage and support the view that PLA2 found in SF might originate from chondrocytes, and not from synovial lining or inflammatory cells.

Aged↗
PubMed1992

Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variants.

Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.

Amino Acid Sequence↗
PubMed1991

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

[Doppler echocardiography in the functional evaluation of patients with pure mitral valve stenosis].

To evaluate the utility of echo-Doppler (ED; PW, CW and color), 67 patients affected by pure mitral stenosis (20 M, 47 F, mean age 52 years) were submitted to ED examination. Right and left cardiac catheterization were performed in 20 patients within 24 hours before ED. Mitral area obtained by Doppler method (Hatle's formula) correlated highly with both echo-2 dimensional and hemodynamic area (r = 0.93, p less than 0.001; r = 0.95, p less than 0.001 respectively). It was possible to calculate systolic pulmonary pressure, in patients with tricuspid incompetence, (43.9 +/- 14.9 mmHg, range 25-80) which correlated significantly (r = 0.95, p less than 0.001) with hemodynamic data (40.2 +/- 12.7 mmHg, range 20-70). The left atrial-left ventricular pressure gradient was 15.6 +/- 6.9 mmHg, range 6-32; the mean pressure gradient was 8.4 +/- 3.7 mmHg, range 3-17; the pressure half time 170.2 +/- 62.3 ms, range 83-330. We observed different types of direction of transmitral jets: centrally directed (n = 34); forward antero-lateral wall (n = 28); toward interventricular septum (n = 5). The transmitral jets presented 4 different appearances: scimitar-shaped (n = 28); candle flame (n = 24); mushroom (n = 9); double-jets (n = 6). No correlation was observed between the different types of transmitral jets (direction and appearance) and the parameters obtained by Doppler (PW and CW): velocities, pressure half-time, gradients. Thus, Doppler echocardiography permits a complete anatomic and functional evaluation of patients with pure mitral stenosis. We have not observed any correlation between the hemodynamic data and the different types of transmitral jets visualized by color Doppler.

Echocardiography, Doppler↗
PubMed1965

Inhibiting effect of the new cytotoxic antibiotic daunomycin on nucleic acids and mitotic activity of HeLa cells.

The effect has been studied of Actinomycin D, Daunomycin (Da.), and Da. N acetyl derivative on mitotic activity and on the nucleic acid synthesis of in vitro HeLa cell cultures. The experiments were carried out by means of the radioautographic technique using stripping films. The relative uptake of thymidine-H(3) and uridine-H(3) was determined by means of the reduced silver grain count present in the nuclei of controls and treated cells. The mitotic activity and thymidine incorporation were noticeably reduced by Daunomycin and Actinomycin, whereas both processes appeared less affected by Da. N acetyl derivative. As regards nuclear RNA synthesis, all three antibiotics at low doses chiefly inhibit nucleolar RNA synthesis. On the other hand, whilst Actinomycin at higher doses causes an almost total inhibition of the synthesis of the whole nuclear RNA, in Daunomycin- and Da. N acetyl derivative-treated cells extranucleolar RNA synthesis is less susceptible to inhibition.

Autoradiography↗