Biomedical subjects

L Calabrese

Publications and source records attributed to L Calabrese.

At least 37 records · Page 2Linked to original sources

PubMed1995

Comparative study of ELISA and indirect immunofluorescence for the detection of anti-neutrophil cytoplasmic antibodies. Evaluation of the SCIMEDX/EURO Diagnostica ELISA assay in a clinical setting.

This study evaluates the performance of a commercial ELISA assay for detection of anti-neutrophil cytoplasmic antibodies (ANCA). Anti-proteinase 3 (anti-PR3) and anti-myeloperoxidase (anti-MPO) ELISA by (SCIMEDX/EURO Diagnostica, Denville, NJ) were compared with the indirect immunofluorescence (IIF) method using ethanol-fixed neutrophils. Three hundred sixty serum samples were examined. There was complete correlation of results in 357 (99.2%) cases between cANCA pattern and anti-PR3 results, and in 356 (98.9%) cases between pANCA pattern and anti-MPO ELISA results. The specificity of ELISA for detection of vasculitis was 95.7% and sensitivity was 60%, whereas for IIF, they were 95.7% and 65%, respectively. The ELISA showed good reproducibility with interassay variation (CV), ranging from 7.1% to 13.2%. Our results show that SCIMEDX/EURO Diagnostica ELISA for ANCA is a reliable and reproducible method that is as good as IIF for detection of ANCA.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed1994

Molecular modeling and electrostatic potential calculations on chemically modified Cu,Zn superoxide dismutases from Bos taurus and shark Prionace glauca: role of Lys134 in electrostatically steering the substrate to the active site.

The three-dimensional structure of Cu,Zn superoxide dismutase (SOD) from the shark Prionace glauca was homology modeled on the structure of the bovine enzyme used as a template. Shark SOD displays the conservative substitution of one of the residues involved in the electrostatic guidance of superoxide toward the active site of the enzyme, i.e., Lys134, which is replaced by arginine. The association rate, calculated by brownian dynamics simulations incorporating electrostatic terms, and the enzyme reaction rate, measured by the pulse radiolysis method, were found to be identical for both bovine and shark SODs. In order to quantify the role of Lys134 in electrostatically steering the superoxide to the active site, chemical modification of the lysine residues by carbamoylation was carried out on both bovine and shark SODs. Experimental determination of the enzymatic reaction rate indicated that the charge located on the residue 134 gives 19% contribution to the guidance of superoxide anion at low ionic strength. This value matches the association rates predicted by brownian dynamics.

Amino Acid Sequence↗

PubMed1993

Age-related changes in human ceruloplasmin. Evidence for oxidative modifications.

Human plasma or serum from donors of age comprised between 15 and 95 years was analyzed for paramagnetic and total copper content, as well as for immunoreactive ceruloplasmin content and oxidase activity. All parameters were essentially unaltered, except the paramagnetic copper content, which increased 2-fold upon aging. A dramatic change of the electron paramagnetic resonance spectrum due to ceruloplasmin occurred in individuals over 65 years old and was associated with both an increase of the type 1 copper signal intensity and the appearance of new resonances of a type 2 copper species. Ceruloplasmin was isolated from either young or old donors. Spectroscopic analyses of the isolated proteins confirmed the tendency of type 1 copper to stay reduced in the "young" and oxidized in the "old" protein. The type 2 copper signal observed in most young ceruloplasmin samples was different from the species invariably present in the old protein. The magnetic parameters of the latter species were more consistent with a partially reduced trinuclear copper site. In vitro limited proteolysis resulted in identical fragmentation patterns and kinetics in both proteins. However, changes of the net electric charge were detected in the fragments of the protein isolated from aged individuals, which exhibited a carbonyl content of 0.6 mol of carbonyl/mol of protein. The same pattern of modifications, including a higher carbonyl content (0.65 versus 0.2 mol of carbonyl/mol of protein), could be reproduced by exposure of the young protein to the metal-catalyzed oxidation system iron/ascorbate. These results suggest that during aging ceruloplasmin is subjected to oxidative modifications which are likely to be the source of conformational changes around the copper sites leading to an intramolecular electron rearrangement among the various copper sites.

Adolescent↗

PubMed1993

The primary structure of turtle Cu,Zn superoxide dismutase. Structural and functional irrelevance of an insert conferring proteolytic susceptibility.

A copper,zinc superoxide dismutase, has been isolated from the marine turtle Caretta caretta and the complete amino acid sequence obtained. The sequence was determined by isolation and analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin or Staphylococcus aureus protease. Turtle superoxide dismutase consists of 166 amino acid residues, which represents the largest number to date for a cytosolic copper,zinc superoxide dismutase. The comparison of this amino acid sequence with that of bovine superoxide dismutase revealed a one-residue C-terminal extension, two single residue insertions and a 12-residue insertion in the N-terminal region, in turtle superoxide dismutase. The new segment consists of a threefold repeating sequence and was found to be the site for selective proteolytic attack by trypsin under native conditions. The biochemical characteristics, the spectroscopic and catalytic properties as well as the thermal stability and the resistance to irreversible denaturation, were carefully examined and were very similar to those of other superoxide dismutases. These results indicate that the presence of the new polypeptide segment does not affect the main folding of the chain and the quaternary structure, nor the functional parameters of turtle superoxide dismutase. The possibility that the new insert constitutes a loop excluded from the protein scaffold providing the framework of the active site is also discussed.

Amino Acid Sequence↗

PubMed1993

The state of the copper sites in human ceruloplasmin.

The state of the various, spectroscopically distinguishable copper sites (type 1, type 2, and type 3 copper) of human ceruloplasmin was investigated by electron spin resonance (ESR) spectroscopy. The ESR measurements were performed at 100 K and at X-band during the reaction of the protein with either ascorbate or with ferricyanide. A method was developed to directly measure the contribution of type 1 and type 2 copper signals to the ESR spectrum of the native protein. A signal arising from an unperturbed type 2 copper site, obtained by aerobically treating the protein with ascorbate, allowed the estimation that the number of type 2 copper centers detectable by ESR was substantially lower than unity. A fraction of type 1 copper sites was found to be in the reduced state and could be reoxidized by treatment with ferricyanide. The data obtained were consistent with the presence of three type 1 copper sites per protein molecule. Based on the experimentally determined stoichiometries, computer simulations of the ESR lineshape were carried out which confirmed the presence of three nonequivalent type 1 copper sites and of a noninteger amount of ESR-detectable type 2 copper in human ceruloplasmin.

Anaerobiosis↗

PubMed1992

Ceruloplasmin in human plasma and its relationships with the copper-albumin complex.

The electron paramagnetic spectrum of human plasma is dominated, in the g = 2 region, by resonances from copper atoms bound to ceruloplasmin, and does not reveal the fraction of copper normally associated with albumin, except in a few cases, where a copper-albumin signal increases with time after blood withdrawal. This copper-albumin complex is responsible for a resonance at a g value below g = 2 in the spectrum of human serum, which has been recently attributed to a modified form of type 2 copper bound to ceruloplasmin [Rylkov, V.V., Tarasiev, M.Y. & Moshkov, K.A. (1991) Eur. J. Biochem. 197, 185-189]. In the plasma, copper associated to albumin comes from ceruloplasmin. Purified ceruloplasmin is unable to exchange copper with albumin, either purified or in plasma. It can not be ruled out that some serum components trigger the metal exchange, in a defence mechanism operating when ceruloplasmin leaks, by unknown processes, its copper content before discharging the metal into the various organs.

Amino Acid Sequence↗

PubMed1992

Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.

Animals↗

PubMed1992

Nitrogen mustard as induction therapy for rheumatoid arthritis: clinical and immunologic effects.

We treated 5 patients diagnosed with rheumatoid arthritis (RA) with nitrogen mustard (HN2) and monitored clinical and immunologic variables. HN2, 0.3 mg/kg ideal body weight was given over 7 days. Disease activity and immune function were monitored during and after treatment. Duration of morning stiffness (p = 0.0044), joint count (p = 0.0140), and assessment of pain (p = 0.0264) and function (p = 0.0057) improved by Day 6. T lymphocytes (p = 0.0060), especially T memory cells (CD4CD29; p = 0.0017) fell dramatically. HN2 is effective for rapidly gaining control of active RA. This effect is T cell specific.

Aged↗

PubMed1992

Chronic fatigue syndrome.

Fatigue is one of the most common complaints among patients seen in the primary care setting. Chronic fatigue syndrome, which has recently been called chronic fatigue immune dysfunction syndrome, is distinctive, with an abrupt onset of symptoms that wax and wane for at least six months. Usually there is low-grade fever, pharyngitis and tender, but not enlarged, lymph nodes. The fatigue can be disabling and is often made worse by physical activity. Some patients with this disorder have also been found to have highly characteristic immunologic abnormalities. Treatment can be rewarding and is based on patient education and support, exercise and symptomatic therapies for abnormal sleep patterns, musculoskeletal pain and other symptoms.

Adolescent↗

PubMed1992

Real risks of HIV transmission to physicians and their staffs. American Osteopathic Association Task Force on AIDS.

This series will provide brief clinical updates and perspectives on the human immunodeficiency virus (HIV). It was developed from the AOA Task Force on AIDS Writers' Workshop, held August 16 to 18, 1991, in New York, and sponsored by an education grant from Burroughs Wellcome. A related editorial appears on page 63. Readers may request tear sheets from the AOA editorial offices.

HIV Infections↗

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↗

PubMed1991

Involvement of the copper in the inhibition of Cu,Zn superoxide dismutase activity at high pH.

The alkaline spectroscopic transition of the copper at the active site of Cu,Zn superoxide dismutase has been reexamined by room temperature EPR, in order to correlate it with the inhibition of the enzyme activity at high pH. The EPR transition is governed by a single prototropic equilibrium, with pK values of 11.3 and 11.1 for ox and shark superoxide dismutase, respectively. This result suggests possible contributions of changes of the copper environment to the higher pK of the activity/pH curve. When Arg141 was chemically modified by phenylglyoxal treatment of the ox protein, a lower pK value (10.8) was obtained, indicating that Arg141 is involved in the observed modifications of the EPR spectra.

Animals↗

PubMed1990

The Cu,Zn superoxide dismutase isoenzymes of Xenopus laevis: purification, identification of a heterodimer and differential heat sensitivity.

The three Cu,Zn superoxide dismutase electromorphs of the amphibian Xenopus laevis were purified by an original procedure. N-terminal sequence analysis demonstrated that they are two different homodimers (AA and BB) and a hybrid heterodimer (AB), arising from the co-expression of duplicated genes. The three forms have the same pI, same enzyme activity and EPR spectra, but different heat-sensitivity, form BB being more resistant than form AA, with form AB showing intermediate sensitivity. Thermostability of BB and the control bovine enzyme was enhanced by a tenfold increase in protein concentration. It is suggested that the higher heat sensitivity of the AA isoenzyme is related to the presence of an extra Cys residue and to an easier dissociation of the protein dimer into monomers.

Amino Acid Sequence↗

PubMed1990

The multidomain structure of ceruloplasmin from calorimetric and limited proteolysis studies.

Differential scanning calorimetry has been used to investigate the thermal stability of three different ceruloplasmins (from sheep, chicken, and turtle) in their native state and after limited proteolysis. The three undegraded proteins showed a similar structural organization in three calorimetric domains, although their temperature of unfolding varied from 57.8 degrees C (turtle) to 71.2 degrees C (sheep) to 82.1 degrees C (chicken). The spectroscopic and the catalytic properties were totally lost at temperatures corresponding to the unfolding of the less thermostable domain in the case of sheep and chicken ceruloplasmins and to the unfolding of the most thermostable domain in the turtle protein. Trypsin, but not plasmin, digestion caused a significant decrease of the thermal stability of sheep and chicken ceruloplasmins. Turtle ceruloplasmin was insensitive to both proteases. Comparing the thermodynamic parameters of the sheep protein in its undegraded and cleaved states revealed a mismatch between the three calorimetric domains and the 3-fold internal replication of the primary structure, which is evident in the highly homologous, fully sequenced human protein. Copper removal caused the rearrangement of the molecule in only two calorimetric domains, suggesting a role of the metal atoms in organizing a new calorimetric domain, which was tentatively assigned to the less thermostable cooperative unit of the native protein.

Animals↗

PubMed1990

Unusual stability properties of a reptilian ceruloplasmin.

Ceruloplasmin from the turtle Caretta caretta was isolated to purity by using the single-step procedure recently developed by us to purify sheep and chicken ceruloplasmin. It has a Mr of ca. 145,000 and a total copper content of 5.1 +/- 0.2 atoms of copper per molecule, 50% of which are detectable by EPR. The spectroscopic features include an absorption maximum at 603 nm in the electronic spectrum and the total absence of any resonance attributable to Type 2 copper in the EPR spectrum. Turtle ceruloplasmin was found to be unusually resistant to aging and proteolysis, when compared to ceruloplasmins isolated from other species. p-Phenyl-endiamine oxidase activity measurements revealed an unusually low catalytic efficiency, while the kinetic parameters of Fe(II) oxidation were consistent with those reported for other species of ceruloplasmin.

Animals↗

PubMed1990

The interaction of ceruloplasmin with Kupffer cells.

The binding and uptake of ceruloplasmin was studied with rat liver cells using gold-labeled probes. Ceruloplasmins from either rat or sheep were used, in which different molecular conformations had been induced according to established biochemical criteria. The native protein from either species could bind not only to the endothelium, but also to Kupffer cells, at variance with previous findings. The proteins which had been converted to the conformation typical of stored molecules--which can be considered aged, but not denatured, according to standard activity and spectroscopic assays--were bound by endothelium irrespective of species, while only rat ceruloplasmin was able to bind to rat Kupffer cells. Internalization of sheep ceruloplasmin occurred with either endothelium or Kupffer cells. This property was lost with isolated suspended Kupffer cells. These findings suggest the presence of receptors for ceruloplasmin on Kupffer cells which are different from those present on endothelial cells.

Animals↗

PubMed1989

Primary structure from amino acid and cDNA sequences of two Cu,Zn superoxide dismutase variants from Xenopus laevis.

A mixture of two different amino acid sequences was discovered in Cu,Zn superoxide dismutase purified from the amphibian Xenopus laevis. No N-terminal post-translational modification was found. The high number of substitutions in the sequence suggested that protein heterogeneity was a product of gene duplication. This was confirmed by isolation of two different cDNA clones. Nucleotide sequence analysis allowed the primary structure of the two peptide chains to be unambiguously assigned. The observed changes (19 in 150 residues) are distributed along the peptide chain to give similar protein net charges although substitutions of the same polarity and/or charge were the exception rather than the rule. The degree of diversity between the two Xenopus variants is comparable to that between mammalian sequences and shows that the putative increase of the rate of mutation for Cu,Zn superoxide dismutase at later evolution stages (Y. M. Lee et al., 1985, Arch. Biochem. Biophys. 241, 577-589; G. J. Steffens et al., 1986, Biol. Chem. Hoppe-Seyler 367, 1017-1024) is observed in amphibians. This is the first time complete sequences for Cu,Zn superoxide dismutase variants from the same organism have been found to be products of divergent genes and not simply allelic mutations.

Amino Acid Sequence↗

PubMed1989

Substitution of arginine for lysine 134 alters electrostatic parameters of the active site in shark Cu,Zn superoxide dismutase.

The complete amino acid sequence was determined for the Cu,Zn superoxide dismutase from the shark Prionace glauca. The active site region shows the substitution of an Arg for Lys at position 134, which is important for electrostatic facilitation of the diffusion of O2- to the catalytically active copper. This change may be related to observed alterations of electrostatic parameters of the enzyme (pK of the pH dependence of the enzyme activity, rate of inactivation by H2O2), although it preserves a high efficiency of dismutation at neutral pH.

Amino Acid Sequence↗