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Biomedical subjects

A Caputo

Publications and source records attributed to A Caputo.

At least 19 recordsLinked to original sources
PubMed1995

The human immunodeficiency virus type-1 Tat protein upregulates Bcl-2 gene expression in Jurkat T-cell lines and primary peripheral blood mononuclear cells.

The regulatory Tat protein of human immunodeficiency virus type-1 (HIV-1) exerts a pleyotropic activity on the survival and proliferation of different cell types in culture. In this report, we investigated the effect of either endogenous or exogenous Tat on Bcl-2 proto-oncogene expression and cell survival in Jurkat T-cell lines and primary peripheral blood mononuclear cells. Stable and transient transfections of Jurkat cells with the cDNA of tat and a plasmid containing Bcl-2 promoter in front of CAT (Bcl-2 Pr/CAT) stimulated CAT activity and showed an increase of Bcl-2 mRNA and protein expression. This effect was specifically related to tat, because Jurkat cells transfected with the cDNA of tat in antisense orientation, tat carrying a mutation in the amino acid cys22-gly22, or the control vector alone (pRPneo-SL3) did not show any significant difference in Bcl-2 promoter activity with respect to parental Jurkat cells. We also observed a specific correlation between tat-induced Bcl-2 gene expression and inhibition of apoptosis induced by serum withdrawal. Our results suggest that the structural integrity of the activation domain of Tat was required for the promotion of the Bcl-2 promoter and Jurkat cell survival, because a single mutation in the aminoacid cys22 was sufficient to completely block the upregulation of Bcl-2 and inhibition of apoptosis. Moreover, picomolar concentrations of native or recombinant Tat were able to upregulate Bcl-2 expression both in Jurkat and primary peripheral blood mononuclear cells, suggesting that extracellular Tat, actively released by infected cells, may also play a significant role in suppressing apoptosis. An aberrant cell survival of lymphoid cells consequent to the upregulation of Bcl-2 may represent an additional pathogenetic mechanism that could help explain both the dysregulated immune response and the frequent occurrence of hyperplastic/neoplastic disorders in HIV-1-seropositive individuals.

Apoptosis↗
PubMed1995

Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A.

The ability of cytolytic cells to cause apoptosis in target cells is in part due to the action of the serine proteinase granzyme B. We demonstrate that granzyme B is inhibited, with an association rate constant of 2.9 x 10(5) M-1 s-1, by the cowpox viral serpin cytokine response modifier A (CrmA). Previously we have shown CrmA to be an inhibitor of the cysteine proteinase interleukin-1 beta-converting enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability to efficiently inhibit proteinases from two distinct catalytic classes, in this case serine and cysteine proteinases. Granzyme B and ICE are both used to combat viral infection, and we propose that cowpox virus uses CrmA to evade the contribution of these two proteinases. Thus, through CrmA, the virus may influence two of the pathways normally used to kill virus-infected cells: acting on endogenous proteinases such as ICE and on exogenous proteinases delivered by cytotoxic lymphocytes to infected cells.

Binding, Competitive↗
PubMed1995

Site and mechanics of failure in normal and dystrophin-deficient skeletal muscle.

Partial or complete tearing of skeletal muscle occurs both in acute muscle injury and in some pathological muscle conditions. Despite the impact of such tearing on normal muscle function, few studies have examined the site or mechanism of muscle injury at cellular or subcellular levels of organization. The present study determines the ultrastructural location and mechanical conditions of tensile failure in normal mouse extensor digitorum longus muscles. All of these muscles failed near a myotendinous junction (MTJ), but within the muscle fibers, in a transverse plane coincident with the edge of an A-band. The breaking stress averaged 5.71 x 10(5) N/m2 for muscles stimulated tetanically during lengthening, and 5.01 x 10(5) N/m2 for unstimulated muscles. Breaking strain averaged 135%, and showed no dependence on the state of activation of the muscle. The energy absorbed by each muscle averaged 362.5 mJ/g in unstimulated muscle, and 613.2 mJ/g in the stimulated samples. In addition to the failure properties of normal muscle, the effect of dystrophin deficiency on the site and conditions of failure was determined using muscle from mdx mice, which lack dystrophin. The absence of dystrophin had no detectable effect on the stress, strain, or energy absorbed, regardless of the state of muscle activation. Unstimulated mdx muscle failed in the plane of an A-band, but tetanic stimulation produced failure in the reticular lamina of the tendon, just external to the MTJ, in 75% of the fibers in the mdx muscles. Although dystrophin's cytoplasmic location makes it unlikely that this unique mode of failure is due directly to the absence of the protein, failure in the reticular lamina may result from a difference in load distribution that accompanies the response of mdx muscle to dystrophin deficiency.

Animals↗
PubMed1995

Language and thought disorder in schizophrenia: brain morphological correlates.

In this magnetic resonance imaging study, the authors analyzed the relationships between frontal and temporal lobe volumes, volumes of ventricular system subdivisions and clinical and neuropsychological aspects of language and thought disorder in a group of 19 young schizophrenic patients. Schizophrenics showed enlargement of lateral ventricles, especially of the central and occipital segments compared with 15 age and sex matched healthy controls but no differences were present in prefrontal, temporal lobe and superior temporal gyrus volumes. Prefrontal volume was inversely correlated with Thought, Language and Communication (TLC) scale total scores; left superior temporal gyral (STG) volume was positively correlated with verbal fluency test performance; higher total ventricular volume was significantly correlated with poor performance to a sentence generation test; STG laterality index was correlated with global TLC scores, the more severe the thought and language disorders, the relatively smaller the left and larger the right STG. These results suggest a complex neuroanatomical substrate for thought and language disorders in schizophrenia.

Adult↗
PubMed1995

Tat-expressing Jurkat cells show an increased resistance to different apoptotic stimuli, including acute human immunodeficiency virus-type 1 (HIV-1) infection.

Human CD4+ T lymphoblastoid Jurkat cells were stably transfected with two different plasmid vectors containing the cDNA of human immunodeficiency virus-type 1 (HIV-1) tat gene under the control of either the promoter of simian virus 40 (pRPneo/tat) or the long terminal repeat region of SL3 murine leukaemia virus (pRPneo/SL3/tat). Both pRPneo/tat and pRPneo/SL3/tat Jurkat cell lines showed a constant and high production of bioactive Tat in transient co-transfection assays with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) reporter plasmid. Tat-positive and mock-transfected Jurkat cells were cultured with various cytotoxic agents, which have been associated to the progressive loss of CD4 T-lymphocytes characteristic of HIV-1 disease. In the presence of recombinant tumour necrosis factor-alpha (TNF-alpha), anti-fas antibody, Leu3a anti-CD4 antibody, the percentage of apoptosis, evaluated in a 24-72 h short-term assay, was lower (P < 0.05) in tat-positive Jurkat cells than in mock-transfected controls. The low susceptibility to the cytotoxic activity of TNF-alpha and anti-fas antibody of tat-transfected cells was confirmed by counting viable cells up to 15 d of culture. Also, recombinant Tat protein was able to prevent the increase of apoptosis induced in mock-transfected Jurkat by TNF-alpha. Of note, tat-expressing cells showed a better survival with respect to mock-transfected control cells even when acutely infected with high doses (500,000 cpm of reverse transcriptase) of HIV-1 (strain IIIB) or treated with heat-inactivated HIV-1. These data demonstrate that the expression of the regulatory HIV-1 Tat protein is able to rescue Jurkat lymphoblastoid cells from apoptosis induced by a variety of cytotoxic agents. Since Tat protein expression is restricted to the initial phases of an active HIV-1 replication, the anti-apoptotic effect of Tat could have the physiological significance of selectively protecting HIV-1 producing cells from death, at least for the time necessary to allow virus production and spreading.

Antibodies, Monoclonal↗
PubMed1995

Transcriptional silencing of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by the Krüppel-associated box repressor domain targeted to the transactivating response element.

The evolutionarily conserved protein domain, called the Krüppel-associated box (KRAB), present in the amino termini of a large number of Krüppel-type zinc finger proteins is a strong repressor domain. In order to develop novel strategies to control human immunodeficiency virus type 1 (HIV-1) gene expression, we constructed a series of expression vectors expressing the wild-type Tat or Tat transdominant negative mutants fused to the KRAB repressor domain. We found that the KRAB domain tethered to the transactivating response element is able to suppress both basal and Tat-mediated activity of HIV-1 long terminal repeat-driven gene expression. These results suggest that the KRAB repressor domain fused to the Tat transdominant negative mutants can be successfully employed to control HIV-1 gene expression.

Amino Acid Sequence↗
PubMed1994

The cytotoxic T cell proteinase granzyme B does not activate interleukin-1 beta-converting enzyme.

Murine granzyme B (cytotoxic cell proteinase-1 (CCP1)) is a member of a family of seven serine proteases found in cytoplasmic granules of cytotoxic T lymphocytes (CTLs). Evidence has suggested that it is involved in target cell DNA fragmentation during CTL-mediated cytotoxicity, although intracellular substrates for granzyme B have not yet been identified. The substrate specificity of granzyme B, requiring an aspartic acid residue at site P1, is unique among eukaryotic serine proteases and is shared with only one other known eukaryotic protease, interleukin-1 beta-converting enzyme (ICE). ICE is responsible for processing pro-interleukin-1 beta to produce biologically active interleukin-1 beta and is itself synthesized as an inactive precursor. Recent evidence has suggested a role for ICE in programmed cell death, which led to a model for CTL-mediated cytotoxicity. In this proposal granzyme B activates ICE in the target cell by proteolytically processing the ICE precursor, resulting in active ICE heterodimer that induces apoptosis in the target cell. We have isolated the cDNA encoding murine ICE and generated in vitro translated ICE precursor. Using lysates from COS cells expressing granzyme B we show that ICE precursor is not a substrate for granzyme B and propose an alternate mechanism for CTL-mediated cytotoxicity.

Animals↗
PubMed1994

Conversion of the substrate specificity of mouse proteinase granzyme B.

Mouse granzyme B is the prototypic member of a subfamily of serine proteinases expressed in cytolytic lymphocytes. Molecular modelling of granzyme B indicated that the side chain of Arg 208 partially fills the specificity pocket, thus predicting the preference of this enzyme for substrates containing acidic side chains, a feature unique among eukaryotic serine proteinases. Replacement of Arg 208 with glycine results in an enzyme lacking this activity, but which is able to hydrolyze hydrophobic substrates. These results demonstrate unequivocally that the substrate preference of granzyme B is determined by a positive charge in the specificity pocket and also represent one of the few examples of rational and efficient alteration of serine proteinase substrate-specificity following a single amino acid substitution.

Amino Acid Sequence↗
PubMed1994

[The importance of esthetics in orthognathic surgery].

The authors deal with an analytic method utilized in the diagnosis and treatment of dysmorphosis of the oral-maxillo-facial area. The aesthetic improvement that patients obtain as a result of surgical operation is also pointed out. An aesthetic improvement that offers the patient the conditions of a more comfortable social life. The psychological troubles which are a consequence of their outward appearance are also eliminated in this way.

Adult↗
PubMed1993

Activation of recombinant murine cytotoxic cell proteinase-1 requires deletion of an amino-terminal dipeptide.

Murine cytotoxic cell proteinase-1 (granzyme B) is a member of a family of novel serine proteinases that have been implicated to participate in destruction of target cells by cytotoxic T lymphocytes. Comparison of the sequence of the cDNA with the sequence of the protein isolated from cytoplasmic granules of cytotoxic T lymphocytes suggested that this protein may be synthesized as a preproenzyme containing an amino-terminal activation dipeptide. Here we show that this activation dipeptide regulates the activity of the enzyme in hydrolysis of its preferred substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester. Lysates of COS cells transfected with a vector expressing the unmodified cytotoxic cell proteinase-1 cDNA were unable to hydrolyze this substrate, whereas lysates of cells transfected with a construct in which the activation dipeptide codons has been deleted were able to hydrolyze the substrate. In each case Western blotting of the lysates revealed a form of the proteinase with an apparent molecular weight of 27,000. We conclude that the activation dipeptide regulates activity of the enzyme. This is the first report of production of an enzymatically active recombinant cytotoxic T cell serine proteinase. The strategy for successful expression of an activated form of cytotoxic cell proteinase-1 may be applicable to other members of this proteinase family.

Amino Acid Sequence↗
PubMed1993

Inhibition of human immunodeficiency virus reactivation from latency by a tat transdominant negative mutant.

A BK virus (BKV) expression vector, specific for human cells, was engineered to express antisense human immunodeficiency virus type 1 (HIV-1) tat cDNA (tat-AS) or a tat mutant in cysteine 22 (tat22). Cysteine residues in the cysteine-rich domain of tat are necessary for tat transactivation of the HIV-1 long terminal repeat (LTR). Both the AS tat and the tat mutant significantly inhibited transactivation by tat when assayed in cells cotransfected with an expression vector where the reporter gene for chloramphenicol acetyl transferase was driven by the HIV-1 LTR. Infection of Jurkat cell clones stably expressing tat22 (Jurkat/tat22) or tat-AS (Jurkat/tat-AS) with HIV-1 did not show differences in virus titer in comparison to HIV-1-infected control cells. However, in two Jurkat/tat22 cell clones, entrance of HIV-1 into latency was accelerated significantly and reactivation of HIV-1 from latency induced by tumor necrosis factor-alpha (TNF-alpha) or tat was blocked. These results suggest that, in a combined and integrated approach to the treatment of acquired immunodeficiency syndrome (AIDS), anti-tat genetic therapy could be successfully applied to maintain virus in latency, thereby extending the duration of the asymptomatic phase preceding full-blown AIDS.

BK Virus↗
PubMed1992

Reexamination of the coding potential of the HTLV-1 pX region.

The human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia (ATL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). In addition to tax and rex genes eight additional open reading frames are present in the pX region of HTLV-1. The possibility that these open reading frames can encode a protein in human cell lines persistently infected by HTLV-1 or in COS-1 cells transfected with a pX expressor plasmid was investigated. The results presented here indicate that tax and rex are the only proteins detected. Moreover, antibodies against proteins in vitro translated by five of the additional open reading frames of the pX region were not found in sera of ATL and TSP/HAM patients, further indicating that those proteins are not usually made in infected cells.

Base Sequence↗
PubMed1992

Biomechanical validation of the solitary lag screw technique for reducing mandibular angle fractures.

The purpose of this study was to determine whether the solitary lag screw technique meets the biomechanical demands of angle fractures. Simulated angle fractures in four cadaveric mandibles were reduced using solitary lag screws and the load-displacement relationships determined under functional loading of the individual mandibles. Subsequently, an angle fracture in a photoelastic mandible analog was fixed with a lag screw and subjected to similar loading. The mechanisms by which the solitary lag screw transferred functional loading were observed and recorded in a circular polariscope. Concomitant distraction tendencies were monitored and measured using displacement transducers. Correlation of the force-displacement measurements to photoelastic observation substantiated that the solitary lag screw functions as a tension band to provide a sufficient degree of interfragmentary compression and stability to withstand functional loading of the mandible.

Biomechanical Phenomena↗
PubMed1992

Pyridoxol L,2-pyrrolidon-5 carboxylate prevents active fibroplasia in CCl4-treated rats.

In the present study we evaluated the protective activity of pyridoxol L,2-pyrrolidon-5 carboxylate (metadoxine) against CCl4 intoxication in rats, especially in relation to liver fibrosis. After 6 consecutive weeks of CCl4 treatment, the animals developed liver fibrosis and inflammation as revealed by histological analysis which also included semiquantitative scoring of these features. In addition the serum levels of the immunoreactive prolyl hydroxylase (SIRPH), an enzyme involved in the hydroxylation of the procollagen molecule, were significantly higher (44.2 +/- 16.3 micrograms/ml; P less than 0.005) in this group of animals than in controls (26.1 +/- 8.06). On the contrary, animals treated with CCl4 + metadoxine (200 mg/kg i.p.) had less severe liver fibrosis and normal SIRPH levels (21.5 +/- 14.6). These data suggest that metadoxine may be an effective pharmacological tool for preventing the progression of liver disease in rats exposed to CCl4 to cirrhosis.

Animals↗
PubMed1991

Reciprocal in vitro interactions between human herpesvirus-6 and HIV-1 Tat.

The transactivation induced by human herpesvirus 6 (HHV-6) on the HIV-1 promoter was studied both in the presence and in the absence of the retroviral transactivator protein (Tat) constitutively expressed in Jurkat cells. Jurkat-tat cells were infected with HHV-6, transfected with a plasmid containing HIV-1 long terminal repeat (LTR)/chloramphenicol acetyltransferase (CAT), and CAT assays were performed. HHV-6 infection in the presence of Tat resulted in significantly higher LTR activation than that observed by Tat or HHV-6 alone, indicating that HHV-6 and Tat interact synergically. Furthermore, it was demonstrated that expression of HIV-1 tat inhibits HHV-6 replication, as shown by a 3.6-15.4-fold reduction in infectious yield. We suggest that HHV-6 could trigger HIV reactivation in HIV-seropositive patients which, in turn, could inhibit HHV-6 production.

Cell Line, Transformed↗