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Biomedical subjects

A Cochrane

Publications and source records attributed to A Cochrane.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Plasmodium cynomolgi: the hsp 70 gene.

The hsp 70 gene of Plasmodium cynomolgi was isolated and characterized. As expected the gene is highly similar to that of the hsp 70 gene of Plasmodium falciparum (98% at the protein level, 82% at the nucleotide level). Surprisingly, the hsp 70 gene appears to be present in a single copy in all the P. cynomolgi strains tested, a finding that has implications for the parasite's ability to undergo a heat shock response.

Amino Acid Sequence↗
PubMed1992

A study on the pathogenesis of human cerebral malaria and cerebral babesiosis.

Cerebral complications are important, but poorly understood pathological features of infections caused by some species of Plasmodium and Babesia. Patients dying from P. falciparum were classified as cerebral or non-cerebral cases according to the cerebral malaria coma scale. Light microscopy revealed that cerebral microvessels of cerebral malaria patients were filled with a mixture of parasitized and unparasitized erythrocytes, with 94% of the vessels showing parasitized red blood cell (PRBC) sequestration. Some degree of PRBC sequestration was also found in non-cerebral malaria patients, but the percentage of microvessels with sequestered PRBC was only 13%. Electron microscopy demonstrated knobs on the membrane of PRBC that formed focal junctions with the capillary endothelium. A number of host cell molecules such as CD36, thrombospondin (TSP) and intercellular adhesion molecule I (ICAM-1) may function as endothelial cell surface receptors for P. falciparum-infected erythrocytes. Affinity labeling of CD36 and TSP to the PRBC surface showed these molecules specifically bind to the knobs. Babesia bovis infected erythrocytes produce projections of the erythrocyte membrane that are similar to knobs. When brain tissue from B. bovis-infected cattle was examined, cerebral capillaries were packed with PRBC. Infected erythrocytes formed focal attachments with cerebral endothelial cells at the site of these knob-like projections. These findings indicate that cerebral pathology caused by B. bovis is similar to human cerebral malaria. A search for cytoadherence proteins in the endothelial cells of cattle may lead to a better understanding of the pathogenesis of cerebral babesiosis.

Animals↗
PubMed1992

Cardiopulmonary transplantation service establishment in the 1990s: Australia--a developing country?

1. A National Transplantation Service has been established at the Alfred Hospital performing more than 50 transplants per year. 2. Increased access to transplantation technology has facilitated an improvement in national population servicing from 2.7 to 6.2 transplants per million population per year. National funding of A$4.062 million per year has been secured. 3. Basic research into organ preservation has facilitated transcontinental organ procurement. 4. An active lung transplantation program has been established synchronous with the cardiac transplant service activities. 5. MCS program establishment has paralleled clinical transplantation activities. 6. Budget management and cost containment has been achieved through rationalisation of management protocols.

Australia↗