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Biomedical subjects

A Cochrane

Publications and source records attributed to A Cochrane.

At least 19 recordsLinked to original sources
PubMed2006

Use of modified U1 snRNAs to inhibit HIV-1 replication.

Control of RNA processing plays a central role in regulating the replication of HIV-1, in particular the 3' polyadenylation of viral RNA. Based on the demonstration that polyadenylation of mRNAs can be disrupted by the targeted binding of modified U1 snRNA, we examined whether binding of U1 snRNAs to conserved 10 nt regions within the terminal exon of HIV-1 was able to inhibit viral structural protein expression. In this report, we demonstrate that U1 snRNAs complementary to 5 of the 15 regions targeted result in significant suppression of HIV-1 protein expression and viral replication coincident with loss of viral RNA. Suppression of viral gene expression is dependent upon appropriate assembly of a U1 snRNP particle as mutations of U1 snRNA that affect binding of U1 70K or Sm proteins significantly reduced efficacy. However, constructs lacking U1A binding sites retained significant anti-viral activity. This finding suggests a role for these mutants in situations where the wild-type constructs cause toxic effects. The conserved nature of the sequences targeted and the high efficacy of the constructs suggests that this strategy has significant potential as an HIV therapeutic.

Anti-HIV Agents↗
PubMed2003

Professional discourse and service cultures: an organisational typology developed from health and welfare services for people with learning disabilities.

This study focuses upon the effect of social policy upon a particular area of service provision. It is influenced by the Foucauldian concept of governmentality and the proposition by Lewis et al. that social policy needs to be understood in local contexts. Only through understanding the partial and fragmented impact of policy can we gain a clear insight into the outcomes for users. The study is undertaken through an exploration of the micro politics of organisations providing health and welfare services for people with learning disabilities. It involves an approach to discourse analysis that focuses upon text developed from interviews with service providers, which is brought into contact with published literature in an iterative process. The interpretation of the text produces four themes: power, trust, citizenship and managerialism. The development of these themes and a further holistic interpretation of the text suggest an emerging organisational typology. A typology based upon different articulations of the themes noted that work to produce particular outcomes for service users.

Community Health Services↗
PubMed2001

Alterations in HIV-1 Rev transport in response to cell stress.

Movement of HIV-1 Rev between the nucleus and cytoplasm is essential to its function. While normally nuclear, the protein can be induced to accumulate in the cytoplasm upon inhibition of RNA polymerase I/II. Nuclear accumulation of Rev in the presence of these inhibitors was found to be rescued upon addition of leptomycin B, an inhibitor of Rev nuclear export. This finding, in conjunction with kinetic data on nuclear import, indicates that the effect of the RNA polymerase inhibitors is due to an inversion of the rates of nuclear import versus export possibly achieved by increasing the rate of Rev nuclear export. We also examined whether changes in Rev localization could be due to a stress response. While neither ultraviolet radiation nor heat shock affected Rev subcellular localization, both oxidative and osmotic shocks induce changes in Rev localization comparable to that observed with the RNA polymerase inhibitors. The ability of certain serine/threonine kinase inhibitors, including CKI/II inhibitors, to cause cytoplasmic accumulation of Rev suggested that the alteration in Rev distribution could be due to changes in Rev or CRM1 phosphorylation. However, no change in extent of phosphorylation of either protein is observed upon treatment of cells with any of the agents tested, indicating involvement of another cellular factor.

Active Transport, Cell Nucleus↗
PubMed2000

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of Fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein.

BACKGROUND: In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. METHODS AND RESULTS: Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 21/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC(50) [mol/L] NE 5.5+/-0.1, n=12; EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC(50) [mol/L] NE 5.8+/-0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC(50) [mol/L] NE 5.7+/-0.2; EPI 5.8+/-0.1). Ventricular membranes from Fallot infants, labeled with (-)-[(125)I]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. CONCLUSIONS: Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

Calcium-Binding Proteins↗
PubMed2000

The amino acid sequence of the PKR-eIF2alpha phosphorylation homology domain of hepatitis C virus envelope 2 protein and response to interferon-alpha.

A region of the hepatitis C virus (HCV) envelope 2 protein, the protein kinase, PKR and early initiation factor 2alpha phosphorylation homology domain (PePHD), may be important in interferon (IFN)-alpha resistance. The PePHD was amplified by polymerase chain reaction and sequenced, and the amino acid sequence derived from pretreatment serum of 14 genotype 3-infected patients with a range of responses to IFN-alpha therapy. Only 1 patient had a PePHD variant. IFN-resistant PePHD variants present at low titers in pretreatment serum should be selected by therapy; therefore, the PePHD amino acid sequence was also obtained from serum collected during or after treatment in 5 patients with breakthrough or relapse of HCV RNA positivity. No difference was found between the pre- and posttreatment PePHD sequences. Thus, it appears that pretreatment sequencing of the PePHD would not enable clinicians to predict the treatment response. There was no evidence that IFN therapy exerts selection pressure in this region.

Amino Acid Sequence↗
PubMed1999

Regulated expression of HIV-1 Rev function in mammalian cell lines.

In order to facilitate further investigation of Rev function, we have generated two systems for the inducible expression of Rev in mammalian cell lines (HeLa and U937) using either a tetracycline-regulated promoter or fusion of Rev to a modified form of the hormone binding domain of the estrogen receptor. In the case of the fusion of Rev to the modified hormone binding domain of the estrogen receptor, we demonstrated induction of Rev function in response to tamoxifen administration to levels comparable to that of the unmodified Rev protein. Subsequently, U937 lines were generated that retained the observed pattern of hormone-dependent function of the Rev fusion protein. In the case of the tetracycline-regulated system, cell lines (both HeLa and U937) were generated that displayed tight regulation of Rev. In the case of the HeLa cell lines, they were used for the subsequent generation of stable cell lines expressing either HIV-1 env or chloramphenicol acetyl transferase (CAT) in a Rev-dependent fashion. Using the latter cell lines, we demonstrate the ability to control Rev expression over a broad concentration range and find that, as soon as Rev expression is detectable, induction of Rev-dependent gene expression is also observed.

Gene Expression Regulation, Viral↗
PubMed1998

Control of human immunodeficiency virus type 1 RNA metabolism: role of splice sites and intron sequences in unspliced viral RNA subcellular distribution.

In the course of examining the various factors which affect the metabolism of human immunodeficiency virus type 1 (HIV-1) RNA, we examined the role of intron sequences and splice sites in determining the subcellular distribution of the RNA. Using in situ hybridization, we demonstrated that in the absence of Rev, unspliced RNA generated with an HIV-1 env expression construct displayed discrete localization in the nucleus, coincident with the location of the gene and not associated with SC35-containing nuclear speckles. Expression of Rev resulted in a disperse signal for the unspliced RNA throughout both the nucleus and the cytoplasm. Subsequent fractionation of the nucleus revealed that the majority of unspliced viral RNA within the nucleus is associated with the nuclear matrix and that upon expression of Rev, a small proportion of the unspliced RNA is found within the nucleoplasm. Mutations which altered splice site utilization did not alter the sequestration of unspliced RNA into discrete nuclear regions. In contrast, a 2.2-kb deletion of intron sequence resulted in a shift from discrete regions within the nucleus to a disperse signal throughout the cell, indicating that intron sequences, and not just splice sites, are required for the observed nuclear sequestration of unspliced viral RNA.

Animals↗
PubMed1997

Novel exonic elements that modulate splicing of the human fibronectin EDA exon.

Three exons in the fibronectin primary transcript are alternatively spliced in a tissue- and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3'-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric beta-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context.

Alternative Splicing↗
PubMed1997

Cyclic-AMP modulates downstream events in CD40-mediated signal transduction, but inhibition of protein kinase A has no direct effect on CD40 signaling.

The role of cAMP/protein kinase A (PKA) in CD40 signal transduction is controversial, with evidence both for and against its importance. In this study we have used a tetracycline-repressible expression system to reversibly express a dominant-negative form of the PKA regulatory subunit type I (PKA-R(G324D)) in a B lymphoma line, M12. Expression of PKA-R(G324D) in M12 lymphomas inhibits both cAMP-mediated growth inhibition and cAMP-mediated induction of B7-2. This inhibition is reversed by tetracycline treatment of the cells to turn off inhibitor expression. In contrast, the expression of the PKA-R(G324D) subunit has no effect on CD40-mediated growth inhibition in M12 cells, nor on CD40-mediated induction of B7-1, CD23, Fas, ICAM-1, or LFA-1. Thus, our data do not support a direct role for cAMP/PKA in CD40-mediated signal transduction. However, we do observe that cAMP can regulate CD40 signaling both positively and negatively. Cyclic-AMP synergizes with CD40-mediated B7-1 induction in M12 lymphomas, while inhibiting CD40-mediated CD23, Fas, and ICAM-1 induction.

Adjuvants, Immunologic↗
PubMed1997

Management strategy and long-term outcome for truncus arteriosus.

OBJECTIVE: Evaluation of a consistent policy of elective repair of truncus arteriosus at 2-3 months of age for the patients who are independent of hospital ward care, and long-term outcome. METHODS: Retrospective study of 82 patients with truncus arteriosus who underwent total repair at the Victorian Paediatric Cardiac Surgical Unit between 1979 and December 1995. The timing was based on a consistent policy of elective repair at 2-3 months of age for patients who were independent of hospital ward care. Earlier repair was performed when the patients were in uncontrolled congestive heart failure. RESULTS: Follow-up was complete for all patients with a mean of 76 months (1-183). There were 11 hospital deaths (13.4% CL 9-18.5), and five late deaths, actuarial survival at 80 months was 81% (CL 70-88%) with 39 patients uncensored at that point. For the purpose of this presentation, patients have been grouped according to their age at repair; 1, neonates n = 17 (hospital mortality = 5); 2, infants 1-6 months of age n = 48 (hospital mortality = 4); 3, patients beyond 6 months n = 17 (hospital mortality = 2). This series includes 10 patients with interrupted aortic arch with no mortality, and 10 patients with discontinuous pulmonary artery (hospital mortality = 2). Thirty-seven patients have had 54 conduits replaced to date. Using multiple regression, body weight < 3 kg was the only significant independent risk factor for hospital mortality. CONCLUSIONS: Our management policy tended to gather patients with risk factors described elsewhere into presentation group 1 and low risk truncus patients into presentation group 2. Deferral of surgery to 2-3 months of age is possible and lowers the surgical risk.

Actuarial Analysis↗
PubMed1997

Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis.

The nef gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression in vivo. However, the precise biological function of Nef remains poorly characterized in vitro, with previous reports suggesting that Nef might be either cytotoxic or cytostatic. As a result of difficulties encountered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in stable Nef expression in various mammalian cell lines. Tetracycline-regulated Nef expression was achieved in HeLa cells but could not be established in human T cell lines. Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated nef gene were generated using a system in which Nef expression was controlled by a mutated version of the heavy metal-inducible human metallothionein IIA promoter. Induction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef cells resulted in a moderate (2-fold) and a dramatic (10-fold) retardation of cell growth respectively, supporting the contention that Nef may be a cytotoxic or cytostatic factor. This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clones (5-fold reduction in growth) and was associated with an altered morphological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expression systems provide tools for investigating the molecular basis of Nef function, including Nef-mediated cytopathogenicity, CD4 down-regulation and enhancement of virus infectivity.

Animals↗
PubMed1997

Arterial switch operation. Surgical solutions to complex problems.

The arterial switch operation is appropriate treatment for most forms of transposition of the great arteries. In this review we analyze indications, techniques, and outcome for various subsets of patients with transposition of the great arteries, including those with an intact septum beyond 21 days of age, intramural coronary arteries, aortic arch obstruction, the Taussig-Bing anomaly, discordant (corrected) transposition, transposition of the great arteries with left ventricular outflow tract obstruction, and univentricular hearts with transposition of the great arteries and subaortic stenosis.

Cardiac Surgical Procedures↗
PubMed1996

Human immunodeficiency virus type 1 Rev function requires continued synthesis of its target mRNA.

Synthesis of human immunodeficiency virus structural proteins is dependent on expression of the virus-encoded Rev protein due to the constitutive nuclear sequestration of mRNAs coding for the structural proteins. The pathway by which Rev, through interaction with the Rev-responsive element (RRE) within the mRNA, achieves export of the mRNA remains unclear. To probe the mechanism by which Rev induces nuclear export of its target mRNAs, the effect of inhibiting mRNA synthesis on the function of Rev was examined. Two approaches to address this issue were pursued: (i) the use of general transcription inhibitors such as 5,6-dichlorobenzimidazole riboside (DRB) and actinomycin D, and (ii) the more selective modulation of target gene transcription permitted by the use of a tetracycline-regulated promoter. Addition of either DRB or actinomycin D inhibited Rev action despite the presence of significant quantities of the target mRNA throughout the course of drug treatment. Furthermore, prolonged DRB treatment was found to improve rather than diminish the induction observed. Subsequent analysis using the tetracycline-modulated promoter demonstrated that Rev function was dependent on the transcription rate of the target mRNA and independent of target mRNA concentration. These data strongly indicate that Rev functions through interaction with newly synthesized target mRNA, facilitating its export by preventing its interaction with the host factors that effect nuclear sequestration.

Cell Line↗
PubMed1995

Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1.

The requirement of human immunodeficiency virus type 1 to generate numerous proteins from a single primary transcript is met largely by the use of suboptimal splicing to generate over 30 mRNAs. To ensure that appropriate quantities of each protein are produced, there must be a signal(s) that controls the efficiency with which any particular splice site in the RNA is used. To identify this control element(s) and to understand how it operates to generate the splicing pattern observed, we have initially focused on the control of splicing of the tat-rev intron, which spans the majority of the env open reading frame. Previous analysis indicated that a suboptimal branchpoint and polypyridimine tract in this intron contribute to its suboptimal splicing (A. Staffa and A. Cochrane, J. Virol. 68:3071-3079, 1994). In this report, we identify two additional elements within the 3'-terminal exon, an exon-splicing enhancer (ESE) and an exon splicing silencer (ESS), that modulate the overall efficiency with which the 3' tat-rev splice site is utilized. Both elements are capable of functioning independently of one another. Furthermore, while both the ESE and ESS can function in a heterologous context, the function of the ESS is extremely sensitive to the sequence context into which it is placed. In conclusion, it would appear that the presence of a suboptimal branchpoint and a polypyrimidine tract as well as the ESE and ESS operate together to yield the balanced splicing of the tat-rev intron observed in vivo.

Animals↗
PubMed1994

HIV-1 Rev is capable of shuttling between the nucleus and cytoplasm.

The mechanism by which Rev facilitates the export, and consequently, the translation of the structural protein mRNAs of the human immunodeficiency virus type 1 remains undefined. Previous immunolocalization has determined that Rev is predominantly in the nucleus with significant accumulation in the nucleolus, a localization consistent with the assumed site of Rev action. To determine whether the subcellular distribution is more dynamic than what was indicated by the original studies, the capacity of Rev to shuttle between the nucleus and cytoplasm was examined. It was observed that treatment of cells with DRB or actinomycin D resulted in a dramatic alteration in Rev distribution, the majority of the protein being found in the cytoplasm. Removal of the drug resulted in a rapid accumulation of Rev in the nucleus indicating that the block to nuclear import was reversible. Subsequent studies indicated that the movement of Rev into the cytoplasm was a passive process while its accumulation in the nucleus was an active one, given that only the latter displayed sensitivity to temperature. Finally, it was demonstrated that, while extensive redistribution of Rev could be attained by inhibition of RNA polymerase I alone, Rev was still capable of inducing expression of HIV structural gene expression under these conditions. Consequently, Rev activity does not appear to be dependent on either an intact nucleolus or the accumulation of the protein in the nucleus.

Biological Transport↗
PubMed1994

The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site.

Proportional expression of retroviral genes requires that splicing of the viral primary transcript be an inefficient process. Much of our current knowledge about retroviral suboptimal splicing comes from studies with Rous sarcoma virus. In this report, we describe the use of chimeric introns composed of human beta-globin and human immunodeficiency virus type 1 (HIV-1) splice sites to establish the basis for inefficient splicing of the intron which comprises most of the HIV-1 env coding sequences (referred to as the tat/rev intron). S1 RNA analysis of transfected COS-7 cells revealed that the 3' splice site (3' ss) of this region was significantly less efficient than the 3' ss of the first intron of beta-globin. Deletion of sequences flanking the tat/rev intron 3' ss demonstrated that the requirements for its inefficiency reside within the region that is expected to comprise the essential signals for splicing (i.e., the branchpoint region, the polypyrimidine tract, and the AG dinucleotide). Introduction of an exact copy of the efficient beta-globin branchpoint sequence within a highly conserved region rendered the tat/rev intron 3' ss highly efficient. Improvement of the polypyrimidine tract also increased the splicing efficiency, but to a degree slightly less than that obtained with the branchpoint mutation. Subsequent examination of the tat/rev intron 5' splice site in a heterologous context revealed that it is efficiently utilized. These results indicate that both a poor branchpoint region and a poor polypyrimidine tract are responsible for the low splicing efficiency of the HIV-1 tat/rev intron. It is of fundamental interest to establish the basis for inefficient splicing of the HIV-1 tat/rev intron since it may provide the key to understanding why nuclear export of mRNAs encoding HIV-1 structural proteins is Rev dependent.

Base Sequence↗