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Biomedical subjects

A Albertini

Publications and source records attributed to A Albertini.

At least 73 records · Page 4Linked to original sources
PubMed1994

The role of the L-chain in ferritin iron incorporation. Studies of homo and heteropolymers.

Mammalian ferritins are 24-meric proteins composed of variable proportions of H and L-subunits. The L-chain, in contrast to the H-chain, lacks detectable ferroxidase activity, and its role in ferritin iron incorporation is unclear. In this study, apoferritins were subjected to iron loading with large iron increments to favour spontaneous iron hydrolysis. The homopolymers of the wild-type H-chain, and of a mutant H-chain with an inactivated ferroxidase centre, formed massive protein aggregates, while the L-chain homopolymers remained mostly soluble. The difference between H and L-ferritins was not related to the rate of iron oxidation or to the presence of preformed iron cores. Heteropolymers were constructed in vitro by co-renaturing different proportions of the H-chain with the L-chain or mutant H-chain with an inactivated ferroxidase centre. After loading with high iron increments, protein aggregation of the heteropolymers was reduced when the L-chain content was above 70 to 80%, either in combination with the wild-type H-chain or with the inactivated mutant H-chain. Under acidic conditions (pH 5.5, 1000 Fe atoms per molecule) the heteropolymers with about 20% H and 80% L-chains incorporated three to fourfold more iron into soluble 24-mers than the homopolymers. The data indicate that ferritins with more than 18 L-chains per molecule have the capacity to lower non-specific iron hydrolysis in bulk solution. This property is possibly due to a specific attraction of the incoming oxidized iron into the cavity and may be related to an effect of the L-chain on the cavity microenvironment. It is concluded that under high iron increments the ferritins with high L:H-chain ratios are the most efficient in incorporating iron, and this goes some way to explain why iron storage tissues contain L-rich isoferritins.

Ceruloplasmin↗
PubMed1994

Biosensor analysis of antigen-antibody interactions as a priority step in the generation of monoclonal bispecific antibodies.

A biosensor system aimed at real-time measuring molecular interactions among label-free reactants has been used for a comparative analysis of the binding features (i.e., association-dissociation rates and affinity constants) as well as epitope mapping between bivalent monoclonal antibodies and the derived monovalent bispecific monoclonal antibody. The results show that observed different affinities between parental and derived bispecific antibodies concern the association rate constant, whereas the dissociation rate constants are unaltered. The apparent affinity-constant values determined by solid-phase radioimmunoassay yielded figures almost overlapping with those obtained with the biosensor instrument. The results of the present work indicate that the biosensor system has gained a key role not only as a tool for the study of antigen-antibody interactions, but also for setting up the reference parameters for the selection of the best candidates in the generation of bispecific monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1994

Usefulness of changes in exercise tolerance induced by nitroglycerin in identifying patients with syndrome X.

Two exercise tests, one under basal conditions and one after sublingual nitroglycerin (NTG), were performed in 39 patients with stable angina pectoris--16 with critical coronary stenoses and 23 with normal coronary arteries (syndrome X). Under basal conditions, times at ischemic threshold, at peak exercise, and at complete ECG recovery were similar in the two groups. Peak ST depression was significantly higher in patients with coronary artery disease (CAD). In a similar proportion of patients, ST-segment depression developed earlier or at a low heart rate. Patterns of heart rate, blood pressure, and rate-pressure product during exercise and recovery were also similar. After NTG an increase in the ischemic threshold was observed in a significantly higher proportion of patients with CAD (93.8% vs 39.1%). Furthermore, a subgroup of patients with syndrome X showed a worsening of exercise performance. This suggests that NTG does not directly affect small coronary vessels. Our results confirm that no relevant differences exist in exercise responses between patients with CAD and those with syndrome X under basal conditions. NTG-induced changes in this response could be useful in identifying patients with normal coronary arteries. Moreover, this test could be used as a guide to therapeutic approaches.

Administration, Sublingual↗
PubMed1994

HCV-RNA detection in ultrasound-guided fine needle biopsies of liver nodules and surrounding tissue.

HCV-RNA was examined in serum and liver tissue obtained from 8 hepatitis B surface antigen (HBsAg) negative patients with liver nodules ranging in size from 2 to 11 cm. Histological examination of ultrasound-guided fine needle biopsies revealed the presence of hepatocellular carcinoma (HCC) in six patients (5 of whom were anti-HCV positive), cholangiocarcinoma in 1 patient (anti-HCV positive) and dysplastic regenerative nodule in 1 patient (anti-HCV negative). The HCCs were surrounded by cirrhosis (3 cases), chronic active hepatitis (CAH) (n = 2) and post hepatitic fibrosis (n = 1), the cholangiocarcinoma by CAH and the regenerative nodule by cirrhotic liver. Total and replicative intermediate HCV-RNA was analyzed by reverse-transcription-nested PCR of the 5'-untranslated region. The five patients with HCC had HCV-RNA in serum, in tumorous and surrounding liver tissues. The viral nucleic acid was also detected in the cirrhotic tissue surrounding the cholangiocarcinoma but not in the tumor. Two out of 5 HCC patients had replicative intermediate RNA (negative strand) in tumorous tissue, 4 in nontumorous tissue and 3 in serum. These results demonstrate that fine needle biopsy can provide sufficient material for both histological examination and HCV-RNA determination and suggest the existence of continuous viral replication during the carcinogenic process.

Aged↗
PubMed1994

Diagnosis of viral hepatitis with a nonisotopic hybridization assay.

The DNA enzyme immunoassay is an efficient method for the screening of PCR products derived from different hepatitis virus genomes, and allows to bypass both agarose gel electrophoresis and Southern blot hybridization with radioactively labeled probes. A wider application of this method will disclose new perspectives for the introduction of PCR in clinical laboratories.

Blotting, Southern↗
PubMed1994

Intracellular localization of full-length and truncated hepatitis C virus core protein expressed in mammalian cells.

The putative hepatitis C virus core protein has a predicted molecular weight of about 22 kD and contains two carboxy (COOH)-terminal hydrophobic domains. The cleavages generating the hepatitis C virus structural proteins (core, E1 and E2) are catalyzed by host signal peptidases. In the present study, we investigated the processing and intracellular localization of the hepatitis C virus core protein expressed in mammalian cells. Expression vectors encoding the entire core protein or COOH-terminal deletion mutants under the control of SV40 regulatory sequences were transfected in COS cells. Immunofluorescent staining with either polyclonal immunoglobulin or monoclonal anti-core antibodies showed that fragments containing the COOH-terminal hydrophobic stretch were retained in the cytoplasm of transfected cells, whereas truncated core proteins deleted of 28 or more residues were located in the nucleus. Our results suggest that a putative nuclear targeting sequence is contained in the first 40 residues of the core protein.

Amino Acid Sequence↗
PubMed1994

Differential pattern of sequence heterogeneity in the hepatitis C virus E1 and E2/NS1 proteins.

The E1 and E2/NS1 genes, encoding the putative hepatitis C virus envelope proteins, show a high rate of sequence variations. We analyzed the degree and distribution of sequence heterogeneity in serum samples from hepatitis C virus-infected subjects. The mutations in the E1 region were mainly type-specific and the rate of variability was apparently not linked to the clinical phase of the infection. The sequence evolution of the E1 region during interferon treatment was low, regardless of the response to therapy. In contrast, an increased degree of variation, apparently related to the stage of viral replication, was present in E2 region derived from patients undergoing interferon treatment. These results are consistent with the hypothesis that the E2 protein represents a major target of the immune response.

Adolescent↗
PubMed1994

Superantigen-reactive human T cells express a biased repertoire of T-cell receptor V beta joining regions.

A major characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T-cell receptor (TCR) V beta chain. Recently, however, reports from several laboratories have also implied a role for non-V beta elements in superantigen binding. The goal of the present study was to determine whether TCR V beta-D beta-J beta joining sequences may influence the interaction of superantigens with their target cells. To ascertain how the actual TCR repertoire of superantigen-triggered cells deviates from the theoretical one, we generated a large panel of joining region sequences from TCR carrying the TCR V beta 12 and TCR V beta 5,1 regions. The 245 sequences analysed represent transcripts of T cells from the same donor triggered either with an anti-CD3 monoclonal antibody or with the Staphylococcus aureus enterotoxins. Comparison of the joining sequences of these different groups demonstrates a skewed J beta usage in the sequences derived from superantigen-triggered cells and also provides evidence that ascribes to the putative CDR3 region of V beta segments a role in superantigen recognition. Finally, the data presented give some hints of the regions of the putative CDR3 loop that may play a major role in this function.

Amino Acid Sequence↗
PubMed1994

High-performance liquid chromatographic determination of oxytetracycline in channel catfish (Ictalurus punctatus) muscle tissue.

A high-performance liquid chromatographic method for the determination of oxytetracycline in channel catfish muscle tissue is presented. Oxytetracycline is extracted three times from muscle tissue with an ethylenediaminetetraacetic acid disodium salt-McIlvaine buffer (pH 4.0) by using an Ultra Turrax. Analysis is carried out by using high-performance liquid chromatography and an acetonitrile-oxalic acid (0.05 mol 1(-1), pH 2.2) mixture (14 + 86, v/v) is used as mobile phase. Oxytetracycline is separated on a Lichrosorb RP-8 125 x 4.0 mm i.d. column and ultraviolet detection at 355 nm is used. The limit of quantification is 10 ng g-1 and the linearity, tested in the spiking range 20-500 ng g-1, is 0.9997. Recovery from muscle spiked at 20, 50, 100, 200 and 500 ng g-1 levels is in the range 70-80%. Precision, expressed as percentage relative standard deviation, is below 7%. The method is applied to muscle tissue from channel catfish fed on a medicated diet.

Animals↗
PubMed1994

Differential priming to programmed cell death of superantigen-reactive lymphocytes of HIV patients.

Programmed cell death or apoptosis has been shown to play a central role in CD4+ T cell depletion following HIV infection. Because most apoptotic signals are delivered through T cell receptor stimulation, we investigated whether T cell depletion in AIDS is a stochastic phenomenon or if it preferentially affects T cell subsets defined by their interaction with superantigens. To address this problem we have taken advantage of the exclusive property of superantigens to trigger T cells expressing selective sets of T cell receptor V beta elements. Here we report that CD4+ T cells from HIV-infected patients can proliferate in vitro to T cell receptor mobilization by some superantigens, but not others. Furthermore, the failure of T cells to respond to some superantigens was shown to be due to an active cell death process that differentially affected T cells capable of interacting with different superantigens. The selective programmed cell death priming of T cells responsive to particular superantigens, observed in this study, suggests that T cell depletion in HIV infection is not simply due to the cytopathic effect of the virus. The possible link between programmed cell death and T cell receptor variable regions suggested by the present experiments may help to better define current models of AIDS pathogenesis.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Characterization of a novel transcription complex required for glucocorticoid regulation of the rat alpha-1-acid glycoprotein gene.

The liver alpha-1-acid glycoprotein (AGP) gene promoter contains several positive cis-acting sequences that are involved in the hormone regulation of its expression. We have characterized a new functionally important sequence located at -155 to -143 upstream from the glucocorticoid regulatory element (GRE, -120 to -105). At least three nuclear proteins bind to this sequence (CTGTGGGAACAG), called the upstream regulatory element (URE). One of these proteins, AGP nuclear factor 4 (ANF-4), is the major component of the DNA-protein complex we detected in footprint and electrophoresis mobility shift assay (EMSA) experiments using rat liver, HTC(JZ-1) rat hepatoma cell extracts and affinity-purified proteins. Another is C/EBP beta, which also binds to three elements downstream from the GRE. The third protein is shown to have a molecular weight of 102 kD. Deletions and site-directed mutagenesis demonstrated that this complex of proteins is involved in the positive hormonal regulation of AGP gene transcription. Binding experiments revealed that ANF-4 and C/EBP beta binding sites are partially overlapping and require the palindromic structure of the URE for high-affinity binding. Southwestern (DNA-protein blot analysis) and cross-linking experiments with nuclear extracts from rat liver and HTC(JZ-1) rat hepatoma cells, revealed two identical constitutive binding activities with molecular masses of 66 and 102 kD. We concluded that this transcription complex is composed of three distinct proteins, ANF-4, C/EBP beta, and a 102-kD protein, and that they play an important role for the hormone regulation of AGP.

Animals↗
PubMed1994

Clinical significance of serum hepatitis C virus (HCV) RNA as marker of HCV infection.

We have evaluated the clinical significance of hepatitis C virus (HCV) RNA determination by analyzing a group of 221 hospitalized patients with abnormal liver function tests. Serum HCV RNA was detected by "nested" PCR amplification followed by nonisotopic hybridization. Of the 200 (90.5%) patients with anti-HCV-positive enzyme-linked immunosorbent assay results, 152 (76%) were RIBA reactive, 47 (23.5%) had indeterminate results, and 1 (0.5%) was nonreactive. Of the 180 (90%) patients positive for anti-HCV and HCV RNA, 138 (76.7%) were RIBA reactive and 42 (23.3%) were RIBA indeterminate. The pattern of RIBA reactivity did not correlate with the presence of HCV RNA. Elevated alanine aminotransferase levels were associated neither with the presence of viremia nor with the RIBA pattern. Histological findings consistent with non-A non-B hepatitis correlated with the presence of HCV RNA but not with the RIBA pattern. HCV RNA was detected in 11 of 21 (52.4%) anti-HCV-negative patients. These 11 patients were either immunosuppressed or in the prodromic phase of acute hepatitis C. Circulating HCV RNA can therefore be described as being predictive of virus-induced liver damage in anti-HCV-positive patients and may be useful in the diagnosis of HCV infection in anti-HCV-negative immunosuppressed patients or in those with early acute infection.

Adult↗
PubMed1994

Distribution of viral genotypes in Italy determined by hepatitis C virus typing by DNA immunoassay.

The distribution of hepatitis C virus (HCV) genotypes in Italy was investigated by PCR amplification of the E1 region and hybridization with type I- and type II-specific nonisotopic probes. Positive PCR results were obtained for 65 of 72 patients (90.3%). Type I was detected in 13 of 72 patients (18%), type II was detected in 39 patients (54.2%), and a mixed type I-type II infection was detected in 7 patients (9.7%). Six amplification products not classified by this method shared a low level of homology with HCV types I and II. HCV type I was significantly associated with human immunodeficiency virus, whereas HCV type II was detected in older subjects who were negative for human immunodeficiency virus markers. These results indicate different epidemiological distributions of HCV types I and II in Italy.

Adult↗
PubMed1993

Human ferritin H-chains can be obtained in non-assembled stable forms which have ferroxidase activity.

We found conditions to obtain the Leu-169-->Arg mutant of human ferritin H chain in a stable and non-assembled state. The protein obtained is an oligomer of subunits with a high degree of structured conformation, and when concentrated it re-assembles into ferritin cages. Functional studies showed that (i) it promotes iron oxidation like the assembled ferritin, but at slower rate, (ii) it is readily precipitated by the oxidised iron unless apotransferrin or L-chain ferritin are added to sequester Fe(III). The results confirm that ferroxidase activity is located within the H-chain, and indicate that the cages of the fully assembled ferritins are important not only in maintaining iron in a soluble form, but also in eliciting the activity of the ferroxidase centres.

Arginine↗
PubMed1993

Production and characterization of recombinant heteropolymers of human ferritin H and L chains.

Vertebrate ferritins are iron storage proteins composed by 24 subunits of one or more types. The recombinant homopolymers of human ferritin H- and L-type chains differ in iron uptake and in physical stability, but the properties of heteropolymers with various proportions of H- and L-type chains cannot be predicted. Present study shows that unfolded human ferritin H- and L- type chains renature under similar conditions to form homopolymers indistinguishable from the native ones and that, when mixed, the unfolded H and L chains renature to form heteropolymers with restricted heterogeneity and with the expected H:L ratios. Seven of these ferritins with different H:L ratios were analyzed; electrophoretic mobility, immunological reactivity, and stability to guanidine denaturation varied as predicted, based on the homopolymers. In contrast, the rate of iron uptake, monitored by the variation of absorbance at 310 nm, increased in the ferritins that ranged in H chain content from 0 to 35%; further increments in H chains had no additional effect. This finding indicates that, under the present conditions, only a limited number of H chains are needed for the maximum rate of ferritin iron uptake. Variations of L- and H-type chains in vivo may thus have biological relevance.

Circular Dichroism↗