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Biomedical subjects

A Albertini

Publications and source records attributed to A Albertini.

At least 19 recordsLinked to original sources
PubMed1998

Monocytes from Wiskott-Aldrich patients display reduced chemotaxis and lack of cell polarization in response to monocyte chemoattractant protein-1 and formyl-methionyl-leucyl-phenylalanine.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by trombocytopenia, eczema, and progressive decline of the immune function. In addition, lymphocytes and platelets from WAS patients have morphologic abnormalities. Since chemokines may induce morphologic changes and migration of leukocytes, we investigated the monocyte response to chemoattractants in cells from WAS patients with an identified mutation in the WAS protein gene. Here, we report that monocytes derived from four patients with molecularly defined typical WAS have a severely impaired migration in response to FMLP and to the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha compared with normal donors. Conversely, neither MCP-1 binding to monocytes nor induction of the respiratory burst by MCP-1 and FMLP is significantly different between WAS patients and normal donors. Within a few minutes of stimulation, monocytes respond to chemokines with increased expression of adhesion molecules and with morphologic changes such as cell polarization. Although up-regulation of CD11b/CD18 expression following stimulation with FMLP or MCP-1 is preserved in WAS patients, cell polarization is dramatically decreased. Staining of F-actin by FITC-phalloidin in monocytes stimulated with chemoattractants shows F-actin to have a rounded shape in WAS patients, as opposed to the polymorphic distribution of F-actin in the polarized monocytes from healthy donors. These results suggest that WAS protein is involved in the monocyte response to the chemokines MCP-1 and macrophage inflammatory protein-1alpha.

Cell Movement↗
PubMed1998

Analysis of ferritins in lymphoblastoid cell lines and in the lens of subjects with hereditary hyperferritinemia-cataract syndrome.

Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal and dominant disease caused by heterogeneous mutations in the iron responsive element (IRE) of the 5' untranslated flanking region of ferritin L-chain mRNA, which reduce the binding to the trans iron regulatory proteins and make L-chain synthesis constitutively upregulated. In the several families identified so far, the serum and tissue L-ferritin levels are fivefold to 20-fold higher than in nonaffected control subjects, iron metabolism is apparently normal, and the only relevant clinical symptom is early onset, bilateral cataract. Some pathogenetic aspects of HHCS remain obscure, with particular reference to the isoferritins produced by HHCS cells, as well as the mechanism of cataract formation. We analyzed lymphoblastoid cell lines obtained from two nonaffected control subjects and from HHCS patients carrying the substitution A40G (Paris-1), G41C (Verona-1), and the deletion of the residues 10-38 (Verona-2) in the IRE structure. Enzyme-linked immunosorbent assays specific for the H- and L-type ferritins showed that L-ferritin levels were up to 20-fold higher in HHCS than in control cells and were not affected by iron supplementation or chelation. Sequential immunoprecipitation experiments of metabolically-labeled cells with specific antibodies indicated that in HHCS cells about half of the L-chain was assembled in L-chain homopolymers, which did not incorporate iron, and the other half was assembled in isoferritins with a high proportion of L-chain. In control cells, all ferritin was assembled in functional heteropolymers with equivalent proportion of H- and L-chains. Cellular and ferritin iron uptake was slightly higher in HHCS than control cells. In addition, we analyzed the lens recovered from cataract surgery of a HHCS patient. We found it to contain about 10-fold more L-ferritin than control lens. The ferritin was fully soluble with a low iron content. It was purified and partially characterized. Our data indicate that: (1) in HHCS cells a large proportion of L-ferritin accumulates as nonfunctional L-chain 24 homopolymers; (2) the concomitant fivefold to 10-fold expansion of ferritin heteropolymers, with a shift to L-chain-rich isoferritins, does not have major effects on cellular iron metabolism; (3) L-chain accumulation occurs also in the lens, where it may induce cataract formation by altering the delicate equilibrium between other water-soluble proteins (ie, crystallins) and/or the antioxidant properties.

Antioxidants↗
PubMed1998

Transient overexpression of human H- and L-ferritin chains in COS cells.

The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.

Animals↗
PubMed1998

Use of the right gastroepiploic artery as a coronary artery bypass graft in 307 patients.

From October 1988 to October 1995 the right gastroepiploic artery was used as a conduit for coronary surgery in 307 patients. Their average age was 56.5 years (range 25-75) and 274 patients (89%) were male. Twenty-six cases (8.5%) were re-operations and 58 patients (19%) were operated upon on an urgent or semi-urgent basis. Target coronary vessels were the right coronary artery and its branches in 280 cases (91.4%), the circumflex artery in 25 cases (8%) and the left anterior descending artery in two cases. The right gastroepiploic artery was used as an in situ graft in 303 cases (98.7%) and as a free graft in 4 (1.3%). A total of 291 patients (94.8%) also received at least one mammary artery graft: both mammary arteries were used in 167 patients (54.4%). An average of 3.6 distal anastomoses were made per patient, three of them with arterial grafts. Eleven (3.2%) right gastroepiploic artery grafts were doubled with saphenous vein intraoperatively because of persistent myocardial ischemia. In-hospital mortality was 1.6% (five patients). Perioperative myocardial infarction occurred in twelve patients (3.9%). Follow-up now averages 26 months (range 6-88). There have been five late deaths (1.6%). A total of 265 (89.2%) patients are angina free. Of the total, 145 patients have been investigated with a maximal-stress test coupled with scintigraphy: residual myocardial ischemia was found in 10 patients, right gastroepiploic artery was related in three. Ninety-six patients have undergone angiographic restudy at a mean of 12 months (range 8-88) postoperatively. Patency of the right gastroepiploic artery grafts was 91.8%. This study confirms that the right gastroepiploic artery can be used as a conduit for coronary artery bypass surgery with minimal mortality or morbidity. Mid-term patency rates and clinical outcome are encouraging.

Adult↗
PubMed1997

High-performance liquid chromatographic determination of polyamines in milk as their 9-fluorenylmethoxycarbonyl derivatives using a column-switching technique.

A high-performance liquid chromatographic method for the determination of polyamines in milk is milk is described. Polyamines were extracted in perchloric acid and derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl). The excess of reagent was reacted with aspartic acid before the analysis on a column-switching system. Linearity of derivatization was calculated for each amine and the coefficient of regression ranged from 0.994 to 0.999. Chromatographic separation of FMOC-polyamines was achieved with a gradient elution programme of water-acetonitrile. The correlation coefficients of the standard curves in the concentration range from 0.5 to 5 nmol ml-1 were higher than 0.991. The repeatability of the method, expressed as R.S.D. for each polyamines ranged from 3.0 to 8.6%. The percent mean recoveries at 1 nmol ml-1 spiking level were 49 +/- 3, 58 +/- 5, 61 +/- 5 and 48 +/- 4 for putrescine, cadaverine, spermidine and spermine, respectively. The limit of detection, calculated on the basis of three times signal-to-noise ratio, was 50 pmol ml-1 for each polyamine.

Animals↗
PubMed1997

Identification of estrogen-responsive genes in neuroblastoma SK-ER3 cells.

To evaluate the role of estrogen receptor in the differentiation of cells of neural origin, we developed a molecular approach aimed at the identification of estrogen target genes by mRNA differential display PCR (ddPCR) in human neuroblastoma SK-ER3 cells. More than 3000 RNAs were examined, a few of which displayed a differential regulation pattern in response to 17beta-estradiol (E2). Sequence analysis of three differentially amplified ddPCR products showed homology with the growth-associated nuclear protein prothymosin-alpha (PTMA), the Bcl2-interacting protein Nip2, and one mRNA previously described by others in fetal human brain. Two ddPCR products, referred to as P4 and P10, corresponded to new DNA sequences. Northern analysis confirmed that estrogen treatment of SK-ER3 cells resulted in the upregulation and downregulation of expression of these messages. In particular, PTMA was found to accumulate at both 1 and 17 hr after E2 treatment, whereas P10 product accumulated only at 1 hr. Conversely, P4, Nip2, and the fetal brain-related mRNAs were significantly decreased by the treatment. Further time course analysis of PTMA and Nip2 mRNAs levels indicated that the hormone exerted a marked biphasic regulatory effect on expression of both messages during the course of cell differentiation. In the present study we report for the first time the identification of a panel of estrogen target genes in neural cells that provide new insights in the molecular mechanism of action of E2 in cells of neural origin.

Base Sequence↗
PubMed1997

Effects of modifications near the 2-, 3- and 4-fold symmetry axes on human ferritin renaturation.

Ferritin is a protein of 24 subunits which assemble into a shell with 432 point symmetry. It can be denatured reversibly in acidic guanidine hydrochloride, with the formation of poorly populated renaturation intermediates. In order to increase the accumulation of intermediates and to study the mechanism of ferritin renaturation, we analysed variants of the human ferritin H-chain altered at the N-terminus (delta(1-13)), near the 4-fold axis (Leu-169 --> Arg), the 3-fold axis (Asp-131 --> Ile + Glu-134 --> Phe) or the 2-fold axis (Ile-85 --> Cys). We also carried out specific chemical modifications of Cys-130 (near the 3-fold axis) and Cys-85 (near the 2-fold axis). Renaturation of the modified ferritins yielded assembly intermediates that differed in size and physical properties. Alterations of residues around the 2-, 4- and 3-fold axes produced subunit monomers, dimers and higher oligomers respectively. All these intermediates could be induced to assemble into ferritin 24-mers by concentrating them or by co-renaturing them with wild-type H-ferritin. The results support the hypothesis that the symmetric subunit dimers are the building blocks of ferritin assembly, and are consistent with a reassembly pathway involving the coalescence of dimers, probably around the 4-fold axis, followed by stepwise addition of dimers until the 24-mer cage is completed. In addition they show that assembly interactions are responsible for the large hysteresis of folding and unfolding plots. The implications of the studies for in vivo heteropolymer formation in vertebrates, which have two types of ferritin chain (H and L), are discussed.

Circular Dichroism↗
PubMed1997

A case-control study on GB virus C/hepatitis G virus infection and hepatocellular carcinoma. Brescia HCC Study.

A new hepatitis-associated RNA virus of the Flaviviridae family has been identified and named GB virus C/ hepatitis G virus (HGV). We carried out a case-control study to evaluate the association of HGV infection with hepatocellular carcinoma (HCC). We recruited 170 patients hospitalized for HCC (143 male and 27 female, mean age 64 years) and 306 patients hospitalized for nonliver diseases (controls) in Brescia, Italy. HGV RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and antibodies against HGV E2 protein (anti-E2) by an immunoassay test. HGV RNA was found in 8 cases (4.7%) and 4 controls (1.3%). The relative risk (RR) for HGV RNA positivity adjusted for demographic variables and hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) RNA, and alcohol was 7.3 (95% confidence interval, 1.7-30.6; P = .009). No HGV RNA-positive subject was also positive for anti-E2. Anti-E2 prevalence did not differ significantly between cases (20%) and controls (15.3%), and no RR increase was found by this marker. Among subjects with HGV exposure (HGV RNA plus anti-E2 positive), a greater proportion of cases (40%) than controls (14%) had transfusion history. The possible role of HGV in HCC etiology seems modest because the population-attributable risk is lower (4%) than those for HBsAg (22%), HCV RNA (36%), and heavy alcohol intake (52%). This study supports the hypothesis of an association between HGV infection and HCC, although at present there are insufficient data on the causality of the association.

Aged↗
PubMed1997

The glucocorticoid receptor regulates the binding of C/EPBbeta on the alpha-1-acid glycoprotein promoter in vivo.

A complex interaction between the Glucocorticoid Receptor (GR), C/EBPbeta, and other transcription factors activate the Alpha-1 Acid Glycoprotein (AGP) promoter in HTC(JZ-1) rat hepatoma culture cells. This effect is mediated by the so-called Steroid Responsive Unit (SRU) of the AGP promoter that contains several binding sites for C/EBP transcription factors, some of which overlap with the Glucocorticoid Responsive Element (GRE). Our in vivo footprinting experiments revealed that the GRE- and the C/EBP-binding sites were already occupied glucocorticoid dependently in HTC(JZ-1) cells 10 min after dexamethasone administration (10(-6) M). Furthermore, local changes in the chromatine structure shown by the appearance of DNAse I hypersensitive sites (HS sites) also took place. These changes were probably dependent on a tissue-specific organization of the chromatine at the SRU because they were not detectable in a different glucocorticoid-responsive cell line (PC12) that did not express AGP. Here, we have also shown that withdrawal of dexamethasone or addition of the anti-glucocorticoid RU486 were able to revert the pattern induced by dexamethasone in vivo. The disappearance of the protected region and the hypersensitive sites, typical of the hormone activated promoter, confirmed the necessity of the GR to be bound by the agonist and the inability of the GR-antagonist complex to bind the DNA. By functional assays, we showed that the occupancy of the SRU by these transcriptional proteins in vivo correlated with the activation of the AGP gene transcription. With these results, we have shown that one of the functions of the GR to activate transcription of the AGP gene is to recruit C/EBPbeta and to maintain it bound at its target DNA sequences (SRU). This process was not accomplished by RU486.

Adrenal Gland Neoplasms↗
PubMed1996

Molecular analysis of mixed infection with hepatitis C virus and human immunodeficiency virus in a patient infected simultaneously.

A case of simultaneous infection with HIV and HCV characterized by a rapidly progressive clinical course was studied retrospectively over 3.5 years. Molecular analysis indicated interference between HIV and HCV and between HCV subtypes 1a and 1b. An ineffective immune response was suggested by the persistence and sequence conservation of the HCV HVR1 variants isolated during the follow-up.

AIDS-Related Opportunistic Infections↗
PubMed1996

Manganese effects on the human neuroblastoma cell line SK-ER3.

SK-ER3 cells were recently demonstrated to represent a valuable model for the study of estrogen-inducible differentiation of neural cells in culture. This system may constitute an important tool also for the analysis of the effects of neurotoxic drugs. The present study demonstrates that short term exposure to Mn causes increased proliferation rate of SK-ER3 cells regardless of their differentiation. Long term treatment causes cell death in undifferentiated cells at concentrations of the metal as low as 100 nM. When the cells are differentiated with estrogens, death is observed only with a Mn concentration two orders of magnitude higher. Measurement of neurite extension and quantitation of tyrosine hydroxylase content after long-term exposure to the metal allow the conclusion that Mn does not alter the state of differentiation of SK-ER3 cells induced by the treatment with the hormone. The study underlines the importance of studying the effect of Mn in proliferating neural cells and demonstrates the toxic role of micromolar concentrations of the metal in fully differentiated neural cells. Since other authors produced evidence of effects of the metal on cell death and proliferation only at millimolar concentrations, and none described its proliferative activity, the model utilized in the present study seems to be of particular interest.

Cell Differentiation↗
PubMed1995

Insertion of a short human immunodeficiency virus (HIV)-2 gp36 sequence into an HIV-1 p24 recombinant protein results in a polypeptide with potent and TCRBV-restricted T cell triggering activity.

In the present work we investigate whether artificial alterations of the structure of an inactive retrovirus-encoded protein could transform it in a superantigen. As a model system we used a recombinant human immunodeficiency virus (HIV)-1 p24 protein and two of its variants in which a short peptide corresponding to sequences of gp41 of HIV-1 (HIV-1 p24*) or gp36 of HIV-2 (HIV-1-2 p24*) has been inserted nearby the carboxy-terminal end of HIV-1 p24. As expected both HIV-1 p24 and HIV-1 p24* were inactive, while HIV-1-2 p24* was a potent inducer of human, but not murine, T cell proliferation. The possibility that the observed activity was due to contaminants was ruled out since the proliferative response could be specifically inhibited by a monoclonal anti-p24 antibody and by a peptide encompassing the area of HIV-1 p24/HIV-2 gp36 junction. Furthermore, the data exclude the possibility that the gp36 insertion is per se responsible for the observed proliferative activity. The analysis of the functional, phenotypic and molecular properties of the responding cells demonstrated that the response was class II dependent and that the activated cells were predominantly CD4+CD8- expressing a strongly biased repertoire of TCRBV segments. Collectively, these data strongly suggest that the HIV-1-2 p24* fusion protein shares common functional properties typical of superantigen molecules. Thus, our demonstration that a viral protein can be transformed into a superantigen simply by the insertion of a short peptide at the carboxy-terminal end has important implications for understanding the mode of action of retrovirus-encoded superantigens.

Amino Acid Sequence↗
PubMed1995

Emergence of hepatitis B virus S gene mutant in a liver transplant recipient.

Immunological and genomic analysis of the "a" determinant was carried out in seven patients with concurrent HBsAg and anti-HBs, four of whom were immunized against hepatitis B virus at liver transplant, two with histologically characterized chronic hepatitis B virus infection, and one HBsAg healthy carrier. The immune reactivity of the HBsAg "a" determinant was evaluated by binding to specific monoclonal antibodies, and the corresponding genomic sequence was studied by differential hybridization in microtiter plates and nucleotide sequence analysis. A double mutation generating an amino acid change (glycine to lysine) at residue 145, able to impair recognition by monoclonal antibodies, was observed in the post-transplant serum from one patient. No significant alteration of the "a" determinant sequence or reactivity was detected in the other patients. Amino acid residue 145 appears therefore to be critical for the recognition by anti-HBs antibodies. A previously undescribed glycine to lysine substitution at this level interferes with the immune reactivity of the "a" determinant.

Amino Acid Sequence↗
PubMed1995

Role of IL-1 beta and corticosteroids in the regulation of the C/EBP-alpha, beta and delta genes in vivo.

We have examined the regulatory effects of interleukin 1 beta (IL-1 beta) on the activation of three different isoforms of the C/EBP family of transcription factors (alpha, beta and delta), in hepatocytes of normal and adrenalectomized (ADX) rats. C/EBP-beta and delta mRNA levels were enhanced by IL-1 beta, whereas that of C/EBP-alpha was not affected by treatment with this interleukin in both normal and adrenalectomized rats. The magnitude of the induction was strikingly higher for C/EBP delta in adrenalectomized animals, indicating a suppressive effect of corticosteroids in the IL-1 beta regulatory pathway. The pattern of C/EBP protein synthesis did not always reflect the mRNA findings. For C/EBP-alpha the protein synthesis was higher than expected in IL-1 beta treated ADX animals compared to normal rats. The pattern of C/EBP synthesis was the one that better reflected the pattern of the mRNA transcription. Differently, the induction of C/EBP-delta was not as pronounced as that of the corresponding mRNA in IL-1 beta treated ADX rats. Hormonal modulation of C/EBP transcription factors was studied in parallel with the hormonal induction of the Alpha-1-Acid Glycoprotein (AGP) gene, which is known to be highly induced in rat liver during the acute phase response. This short report also indicates an important role of corticosteroids in the regulation of transcription factors involved in IL-1 beta signalling during the acute phase response.

Adrenal Cortex Hormones↗
PubMed1995

Different TCRBV genes generate biased patterns of V-D-J diversity in human T cells.

The aim of this work was to assess whether each T-cell receptor (TCR) BV segment generates a random pattern of junctional diversity or if, alternatively, biased patterns of V-D-J rearrangements limit the number of available TCR specificities. Detailed molecular analysis of T-cell receptors expressed by lymphocytes was obtained by generating a large number of junctional regions sequences from TCRBV3, TCRBV4, TCRBV5S1, TCRBV12, TCRBV13S2, TCRBV17, TCRBV20, and TCRBV22 variable genes. The > 800 sequences analyzed have allowed the characterization of the recombination frequencies of each germline-encoded V, D, and J segments, as well as of the magnitude of exonucleolytic nibbling and of the number of N nucleotides inserted for each group of TCRB segments. The data obtained indicate that the extent of junctional diversity varies considerably depending on the TCRBV gene implicated in the recombination event, due to the occurrence of skewed patterns of J and D region usage. Furthermore, our results show that "illegitimate" rearrangements occur with unexpectedly high incidence, specifically at the level of TCRBD to TCRBJ joining. These findings provide additional information for a more accurate estimation of the size of the TCRBV repertoire and for understanding the well-established biased pattern of TCRBV expression in humans.

Base Sequence↗
PubMed1995

Detection of HIV-1 proviral sequences in lymphocytes using a qualitative polymerase chain reaction assay.

The performance and clinical relevance of a qualitative PCR-based assay for the detection of HIV-1 DNA sequences in peripheral blood mononuclear cells (PBMCs) was evaluated by two different laboratories. Four hundred and one samples were obtained from 397 individuals from different risk populations. All blood donors tested had negative results; positive signals were obtained from all infected patients. HIV-1 DNA was detected in 3 of 17 infants born to seropositive mothers; Western blot indeterminate blood donors and exposed health-care workers had negative results. Our results demonstrate that this PCR assay provides both sensitive and specific results and is suitable for testing large numbers of samples and for rapid identification of HIV-1 infection.

Base Sequence↗