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M Giacca

Publications and source records attributed to M Giacca.

At least 55 records · Page 3Linked to original sources
PubMed1998

The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin-mimicking extracellular Tat antagonists.

Heparin binds extracellular HIV-1 Tat protein and modulates its HIV long terminal repeat (LTR)-transactivating activity (M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d'Adda di Fagagna, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313-11320). On this basis, the glutathione S-transferase (GST)-TatR49/52/53/55/56/57A mutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GST-Tat for its capacity to bind immobilized heparin. Dissociation of the GST-TatR49/52/53/55/56/57A.heparin complex occurred at ionic strength significantly lower than that required to dissociate the GST-Tat.heparin complex. Accordingly, heparin binds immobilized GST-Tat and GST-TatR49/52/53/55/56/57A with a dissociation constant equal to 0.3 and 1.0 microM, respectively. Also, the synthetic basic domain Tat-(41-60) competes with GST-Tat for heparin binding. Suramin inhibits [3H]heparin/Tat interaction, 125I-GST-Tat internalization, and the LTR-transactivating activity of extracellular Tat in HL3T1 cells and prevents 125I-GST-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative 14C-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-TatR49/52/53/55/56/57A. Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in selected suramin derivatives originated Tat antagonists whose potency was close to that shown by heparin. In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Tat. Our data demonstrate that tailored polysulfonated compounds represent potent extracellular Tat inhibitors of possible therapeutic value.

Animals↗
PubMed1998

Cell cycle modulation of protein-DNA interactions at a human replication origin.

We followed the variations of protein-DNA interactions occurring in vivo over the early firing replication origin located near the human lamin B2 gene, in IMR-90 cells synchronized in different moments of the cell cycle. In G0 phase cells no protection is present; as the cells progress in G1 phase an extended footprint covering over 100 bp appears, particularly marked at the G1/S border. As the cells enter S phase the protection shrinks to 70 bp and remains unchanged throughout this phase. In mitosis the protection totally disappears, only to reappear in its extended form as the cells move into the next G1. These variations are reminiscent of those corresponding to the formation of the pre- and post-replicative complexes described in yeast and Xenopus cells.

Cell Cycle↗
PubMed1998

In vitro selection of HIV-1 TAR variants by the Tat protein.

Starting from a pool of 10(13) RNA sequences, we isolated a number of TAR RNA variants after nine rounds of selection by binding to recombinant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A and B) were, respectively, represented five and four times. These two groups of sequences constituted approximately 25% of the total number of analyzed clones (9/34). As far as the primary and presumptive secondary structures of the wild-type TAR are concerned, the selected A and B variants showed an almost complete sequence conservation of the Tat-binding domain, but the configuration of this nucleotide region differed within the secondary structure. Despite this difference, as verified by gel retardation and filter binding assays, both the A and B variants bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated trans-activation in vivo when they replaced the wild-type TAR inside the long terminal repeat of HIV_1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) in vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication.

Base Sequence↗
PubMed1998

DNA loop anchorage region colocalizes with the replication origin located downstream to the human gene encoding lamin B2.

The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions.

Binding Sites↗
PubMed1998

Transcriptional interference perturbs the binding of Sp1 to the HIV-1 promoter.

Transcriptional interference between adjacent genes has been demonstrated in a variety of biological systems. To study this process in RNA polymerase II (pol II) transcribed genes we have analysed the effect of transcription on tandem HIV-1 promoters integrated into the genome of HeLa cells. We show that transcriptional activation at the upstream promoter reduces transcription from the downstream promoter, as compared with basal transcription conditions (in the absence of an activator). Furthermore, insertion of a strong transcriptional termination element between the two promoters alleviates this transcriptional interference, resulting in elevated levels of transcription from the downstream promoter. Actual protein interactions with the downstream (occluded) promoter were analysed by in vivo footprinting. Binding of Sp1 transcription factors to the occluded promoter was reduced, when compared with the footprint pattern of the promoter protected by the terminator. This suggests that promoter occlusion is due to disruption of certain transcription factors and that it can be blocked by an intervening transcriptional terminator. Chromatin mapping with DNase I indicates that a factor binds to the termination element under both basal and induced conditions.

Binding Sites↗
PubMed1998

Dynamics of hypervariable region 1 variation in hepatitis C virus infection and correlation with clinical and virological features of liver disease.

Hepatitis C virus (HCV) infection is a dynamic process during which molecular variants are continuously selected as the result of virus adaptation to the host. Understanding the nature of HCV genetic variation is central to current theories of pathogenesis and immune response. We prospectively studied hypervariable region 1 (HVR1) variation in the E2 gene of 36 hepatitis C patients, including 10 asymptomatic carriers, followed up for 1 to 2 years. Sequence changes in single and consecutive serum samples were assessed and correlated with clinical and virological parameters of liver disease. A region of the E1 gene was sequenced for comparison in 3 subjects. HVR1 heterogeneity at single time points widely varied in individual patients, did not increase cumulatively over the follow-up period, and did not correlate with HVR1 evolutionary rates. Conversely, the process of HVR1 sequence diversification, although differed considerably among patients, was stable over time and directly correlated with infections by HCV type 2, lower alanine aminotransferase (ALT) levels, and absence of cirrhosis. HCV carriers showed the highest HVR1 variation rates. Our findings indicate that HVR1 variation has an adaptive significance and is associated with favorable features of liver disease and suggest that prospective, rather than static, observations are required to model the process of HCV variation.

Adult↗
PubMed1998

Advances in molecular genetics of dilated cardiomyopathy. The Heart Muscle Disease Study Group.

In clinical surveys, familial dilated cardiomyopathy (FDC) has been demonstrated in 20% to 30% of patients. In these patients, the cause of the disease lies at the DNA level. Molecular genetic studies represent the tools for the understanding of the etiology of FDC and are currently producing relevant advances: 6 different loci have been mapped so far. The only known disease gene is the dystrophin gene causing X-linked dilated cardiomyopathy, but other cytoskeletal proteins, such as adhalin, could be involved. In familial right ventricular cardiomyopathy (or arrhythmogenic right ventricular dysplasia) characterized by isolated or prevalent right ventricular involvement, three further disease loci have been identified.

Autoimmune Diseases↗
PubMed1998

Human chorionic gonadotropin in the treatment of HIV-related Kaposi's sarcoma.

To evaluate the antineoplastic activity of human chorionic gonadotropin (hCG) in the treatment of HIV-related Kaposi's sarcoma (KS), two clinical trials focusing on two different schedules of administration and types of hCG preparation were conducted. In the low-dose group, hCG (Profasi-HP) was administered three times a week intramuscularly at a dose ranging from 4000 to 32,000 IU for 4 months and no objective response was observed among 5 evaluable patients. In the high-dose group, hCG (Gonadotrafon) was given intramuscularly three times a week at a dose ranging from 100,000 to 300,000 IU for 3 months with 1 partial response among 8 evaluable patients. In 6 patients evaluated for HIV viral load, no significant reduction in HIV viraemia was observed. In conclusion, hCG showed very limited activity against KS and no influence on HIV viral load, along with emerging dose-limiting toxicity and high cost of the therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Gene therapy of chronic granulomatous disease (CGD) by gene transfer into hematopoietic stem cells.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency characterized by severe recurrent bacterial and fungal infections of several organs. The disease is due to the inability of phagocytic leukocytes to generate reactive oxygen species upon phagocytosis. The defect arises as a consequence of mutations of the genes encoding for the subunits of a membrane NADPH oxidase, which catalyzes the production of superoxide anion (O2-). CGD represents an ideal candidate disorder for gene therapy, since the disease has a recessive inheritance, its phenotype is exclusively expressed in phagocytic cells, and a partial correction is likely to be effective. Given the short half-life of mature phagocytes, the optimal target cell population for gene transfer is the pluripotent hematopoietic stem cell. Transduction of CD34+ hematopoietic progenitors with retroviral vectors carrying the cDNA of the defective gene results in the correction of the enzymatic defect in myeloid cells differentiated in vitro. Still, the effective development of a clinical gene therapy protocol for this disease will await a substantial improvement in our current technology for the identification and manipulation of hematopoietic stem cells, and in our understanding of their biological and molecular properties.

Adenoviridae↗
PubMed1997

Effects of subcutaneous interleukin-2 therapy on CD4 subsets and in vitro cytokine production in HIV+ subjects.

HIV infection is characterized by the reduction of the CD4+, CD45RA+, CD26+, and CD28+ lymphocyte subsets and of the in vitro production of IL-2, IL-4, and interferon-gamma; on the contrary, chemokine production is usually increased. These abnormalities are only partially restored by antiretroviral chemotherapy. Therapy with interleukin-2 has been proposed to restore the functions of the immune system, but the mechanisms by which IL-2 exerts its activities are unknown. The aim of this study was to define the effects of rIL-2 administration on CD4+, CD45RA+, CD45R0+, and CD26+ lymphocytes and on the in vitro production of IL-2, IL-4, IL-10, IFN-gamma, RANTES, and sCD30 in HIV+ patients. 10 HIV+ patients with CD4 cell counts between 200 and 500 cells/mm3 were treated with six cycles of subcutaneous recombinant IL-2 administration, in combination with zidovudine and didanosine. This therapeutic regimen resulted in a remarkable increase in the number of CD4+ cells and in the prolonged reduction of the levels of viremia. CD45R01 cells were expanded during the first cycle of therapy, while CD45RA+/CD26+ cells predominated after the third cycle. At this time, the in vitro production of IL-2, IL-4, IFN-gamma, and sCD30 were significantly upregulated. These results demonstrate that rIL-2 in HIV+ patients induces the reconstitution of the CD4/CD45RA lymphocytes subtype. This expanded cell population recovered the ability to produce in vitro IL-2, IL-4, and IFN-gamma. These effects may be beneficial to HIV+ patients by improving their immune response to microorganisms or vaccines.

CD4 Lymphocyte Count↗
PubMed1997

Functional properties of the separate subunits of human DNA helicase II/Ku autoantigen.

The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.

Adenosine Triphosphatases↗
PubMed1997

Dynamics of HIV-1 mRNA expression in patients with long-term nonprogressive HIV-1 infection.

A large number of evidences indicate that progression of HIV disease is driven by an increase in viral burden. It is still unclear, however, to what extent this is contributed by the dysregulation of the molecular mechanisms governing virus gene expression at the transcriptional or posttranscriptional levels. To address this issue, several quantitative virologic parameters (including provirus transcriptional activity and splicing pattern) were analyzed in individuals with nonprogressive HIV infection and compared with those of a matched group of progressor patients. Exact quantification was achieved by a competitive PCR procedure using a multicompetitor template. Nonprogressors were characterized by striking differences in the levels of viremia, provirus copy number, and overall levels of all viral mRNA classes in peripheral blood mononuclear cells. Additionally, the transcriptional activity of the proviral DNA in these patients was mainly engaged in the production of multiprocessed transcripts, with a pattern resembling the early phases of the experimental infection. Taken together, these results show that both viral load and provirus transcription pattern are remarkably different in infected individuals nonprogressing toward overt disease, and further support the notion that disease progression is accompanied by a change in the kinetics of HIV gene expression.

Adult↗
PubMed1997

Transcription and polyadenylation in a short human intergenic region.

The poly(A) signal of the human Lamin B2 gene was previously shown to lie 600 bp upstream of the cap site of a gene of unknown function (ppv 1). However, using RNase protection analysis, we show that ppv 1 has two clusters of multiple initiation sites, so that the 5"cap site lies only approximately 280 nt downstream of the Lamin B2 poly(A) signal. We analysed nascent transcription across this unusually short intergenic region using nuclear run-on analysis of both the endogenous locus and of transiently transfected hybrid constructs. Surprisingly, transcription of the Lamin B2 gene does not appear to terminate prior to any of the mapped ppv 1 start sites, although pausing of the elongating polymerase complexes is observed downstream of the Lamin B2 poly(A) signal. We suggest that this pausing may be sufficient to protect the downstream gene from transcriptional interference. Finally, we have also investigated the sequences required for efficient recognition of the Lamin B2 poly(A) signal. We show that sequences upstream of the AAUAAA element are required for full activity, which is an unusual feature of mammalian poly(A) signals.

Base Sequence↗
PubMed1997

Interaction of HIV-1 Tat protein with heparin. Role of the backbone structure, sulfation, and size.

Human immunodeficiency virus type 1 (HIV-1) Tat protein is released from infected cells. Extracellular Tat enters the cell where it stimulates the transcriptional activity of HIV-long terminal repeat (LTR) and of endogenous genes. Heparin modulates the angiogenic (Albini, A., Benelli, R., Presta, M., Rusnati, M., Ziche, M., Rubartelli, A., Paglialunga, G., Bussolino, F., and Noonan, D. (1996) Oncogene 12, 289-297) and transcriptional (Mann, D. A., and Frankel, A. D. (1991) EMBO J. 10, 1733-1739) activity of extracellular Tat. Here we demonstrate that heparin binds specifically to recombinant HIV-1 Tat produced as glutathione S-transferase (GST) fusion protein and immobilized on glutathione-agarose beads. Heparin and heparan sulfate (HS), but not dermatan sulfate, chondroitin sulfates A and C, hyaluronic acid, and K5 polysaccharide, competed with 3H-labeled heparin for binding to immobilized GST-Tat and inhibited HIV-LTR transactivation induced by extracellular GST-Tat. Selective 2-O-, 6-O-, total-O-desulfation, or N-desulfation/N-acetylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-O-desulfated heparins also showed a reduced capacity to inhibit the transactivating activity of GST-Tat. Very low molecular weight heparins showed a significant decrease in their capacity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kDa heparin was affinity chromatographed on immobilized GST-Tat to isolate binding and non-binding subfractions, the Tat-bound fraction was >/=1,000 times more potent than the unbound fraction in inhibiting the transactivating activity of GST-Tat. The results demonstrate that Tat interacts in a size-dependent manner with heparin/HS and that high affinity Tat-heparin interaction requires at least some 2-O-, 6-O-, and N-positions to be sulfated. The Tat binding activity of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating the possibility to design specific heparin/HS-like structures with Tat-antagonist activity.

Binding Sites↗
PubMed1997

C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

Cells, Cultured↗
PubMed1997

Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction.

A procedure was developed for mapping origins of DNA replication in mammalian cell chromosomes based on determining the relative abundance of nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from recently activated origins and the quantification, within this sample, of the relative abundances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin mapping procedure (i) allows analysis of single-copy genomic regions, (ii) can be performed on cultured and primary cells in the absence of any chemical treatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of the cis- and trans-acting elements required for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an approximately 500-bp segment in the human lamin B2 gene domain and within an approximately 800-bp segment in the hamster dihydrofolate reductase gene locus.

Animals↗
PubMed1997

Molecular genetics of dilated cardiomyopathy.

A major advance in the study of the pathogenesis of dilated cardiomyopathy (DC) has been the identification of a familial trait in a relevant proportion of cases (more than 25%), which indicates that, at least in these cases, a mutated gene is the cause of the disease. Familial dilated cardiomyopathy is a genetically heterogeneous disorder, most frequently with autosomal-dominant inheritance. Five different loci that cosegregate with the disease have been mapped so far; the identification of the disease genes is still in progress. The only disease gene known so far is the dystrophin gene, which causes X-linked DC. By analogy with dystrophin, it is believed that other cytoskeletal proteins could be involved in the pathogenesis of DC. Finally, in right ventricular cardiomyopathy, a peculiar form of cardiomyopathy that is frequently familial as well, several disease loci have been described. Also in this case, no disease gene has been yet identified. The advances in clinical and molecular genetics of DC have relevant clinical and therapeutic implications.

Animals↗