Molecular biology & medicine最新文献

Understanding the molecular basis by which cells of the heart and blood vessels adapt to physiological stress conditions is an important goal for cardiovascular investigators. The ubiquitous heat shock response provides a model for cellular adaptations to metabolic stresses that are encountered in cardiac disease. Stress-induced synthesis of a family of highly conserved proteins serves to protect cells from injury. In addition, members of this family have essential roles in protein processing and assembly of macromolecular complexes, and in regulation of gene expression, even in unstressed cells. Research concerning the regulation and function of stress proteins potentially is pertinent to the pathophysiology of myocardial hypertrophy, remodeling, and failure, to age-related changes in the cardiovascular system, as well as to ischemic heart disease.

Since the first patient was treated with recombinant tissue plasminogen activator (t-PA) in 1984, there has been remarkable progress in our understanding of optimal methods for administration of this thrombolytic agent. As a background and foundation to clinical trials, the experimental data for bolus t-PA, adjunctive treatments and new plasminogen activators for more optimal thrombolysis are reviewed. The major findings in clinical evaluation for acute myocardial infarction to date include (1) substantial mortality reduction and improvement in cardiac function; (2) an excess of serious bleeding complications at high doses (150 mg) of t-PA; (3) rapid infarct vessel recanalization with an accelerated "front-loaded" regimen; (4) the importance of conjunctive intravenous heparin; and (5) the potential for new, combined plasminogen activator therapies. The recent data, collectively, have set the stage for a new greater than 30,000 patient mortality reduction trial entitled Global Utilization of Streptokinase and t-PA for Occluded Coronary Arteries (GUSTO).

Over the last few years several groups have used retroviral vectors to achieve stable gene transfer into endothelial cells. In vitro experiments include transduction of cultured cells with genes of potential therapeutic interest, such as growth hormone and tissue plasminogen activator (t-PA). Animal studies have demonstrated the feasibility of in vivo recombinant gene expression from transduced endothelial cells, but have thus far been accomplished only with the lacZ marker gene. All studies to date have been oriented primarily toward the use of transduced endothelial cells to provide gene therapy. Numerous issues remain to be addressed with experimental data prior to the initiation of a clinical protocol using transduced endothelial cells. These issues include the introduction of larger numbers of transduced cells into the vasculature and the achievement of appropriate regulation of transgene expression. The use of retroviral vectors to study basic endothelial cell biology has been relatively ignored. The tool of retroviral vector-mediated gene transfer is available for use in answering both therapeutic and pathophysiological questions in endothelial cell biology.

Coronary thrombolysis is the treatment of choice for patients with acute Q wave myocardial infarcts who have no contraindication to such therapy. However, the time required for thrombolysis to occur and the possibility of reocclusion of the infarct-related artery following thrombolytic therapy are problems. The time required for thrombolysis to occur with currently available agents ranges from 40 to 60 minutes and the frequency of reocclusion of the infarct-related artery after tissue-type plasminogen activator is 10 to 20%. We review experimental studies and clinical evaluations in which attempts have been made to develop adjunctive therapies that when coupled with available thrombolytic interventions might shorten the time to thrombolysis and delay or prevent reocclusion. From the studies done to date, it appears that a combination of thromboxane synthesis inhibitor and receptor antagonist with a serotonin receptor antagonist and heparin shortens the time to thrombolysis and delays or prevents coronary artery reocclusion in experimental canine models with copper coil-induced coronary artery thrombi. A monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor given with tissue plasminogen activator and heparin also shortens the time to thrombolysis and delays or prevents reocclusion in experimental canine models. A mutant tissue plasminogen activator with a glycosylation defect and prolonged systemic clearance delays coronary artery reocclusion following lysis of three-hours coronary thrombi, induced by a copper coil. Thrombin inhibitors, including heparin, and synthetic inhibitors, given with tissue plasminogen activator and aspirin, appear to shorten the time to thrombolysis and delay or prevent coronary artery reocclusion in experimental canine models.(ABSTRACT TRUNCATED AT 250 WORDS)

The data reviewed above show that the ideal thrombolytic or thrombolytic plus anticoagulant regimen does not exist. Nor is it clear to me that one regimen is unequivocally better than another in regards to clinical outcome. Publication of the full results of the ISIS-3 study and completion of the TAPS study, the GUSTO study, the TIMI-4 study plus others only now in the planning phases, should help. This review will not stay current very long. These data do, however, give some guides to certain circumstances in which one regimen might be preferred over others. If economics is a compelling issue, as it may be in public hospitals on a fixed budget or in the developing world, streptokinase may be the best choice. For early application of thrombolytic therapy, such as at the site of infarct occurrence and in automotive and aerial ambulances, anistreplase may be preferred because of its ease of administration. Previous administration of streptokinase or anistreplase (within the period of 48 h to 6 months after prior use) militate against their use as does a recent streptococcal infection. Heightened concerns about bleeding risk, except intracranially, in the absence of absolute contraindication of fibrinolytic therapy, e.g. remote gastrointestinal hemorrhage or the expected imminent need for an invasive procedure, may lead to preference for alteplase over streptokinase or anistreplase. On the other hand, heightened concerns about intracranial hemorrhage may lead to preference for streptokinase over alteplase or anistreplase. Alteplase may be preferred over non-fibrin-selective agents in the treatment of patients when administration is begun more than three hours after the presumed onset of infarction. These considerations notwithstanding, it is crucial that debates over the best choice of a regimen must not be allowed to prolong the time before administration of an effective thrombolytic agent to a patient with evolving Q-wave infarction who is a good candidate for this therapy. This review may also become dated in the not-too-distant future because of expected further advances in thrombolytic regimen. Application of new antithrombotic regimens was noted above. Future thrombolytic and antithrombotic regimens may be "cocktails" of one or more thrombolytic agents plus more powerful antithrombotic and antiplatelet agents. New generations of thrombolytic agents may replace the current first and second generation agents now used. Combination thrombolytic and anti-fibrin antibody agents and mutant tissue-type plasminogen activators with lower affinity for plasminogen activator inhibitor and longer half-lives are being developed.(ABSTRACT TRUNCATED AT 400 WORDS)

Hybrid molecules containing the catalytic domain of either tissue plasminogen activator (tPA) or single chain urokinase-type plasminogen activator (scuPA), and the fibrin binding domain of a murine antifibrin monoclonal antibody were constructed using either cDNA or genomic DNA encoding the plasminogen activator and genomic DNA encoding antifibrin monoclonal antibody 59D8. In order to optimize expression of these fusion proteins in hybridoma cells, we compared plasminogen activator 3' UT domains (which decrease mRNA stability) with immunoglobulin and beta globin 3' UT domains (which increase mRNA stability). The presence of the plasminogen activator 3' UT domain resulted in approximately tenfold lower steady-state mRNA levels, and 300 to 500-fold lower levels of expressed functional protein. The initial goal of these studies was to increase the fibrinolytic potency and selectivity of tPA or scuPA. Fusion proteins comprising an antifibrin antibody domain and the catalytic domain of either tPA or scuPA were expressed and shown to have very different properties. The fusion protein that comprised the Fab portion of an antifibrin antibody and the catalytic domain of tPA, while displaying antigen binding properties indistinguishable from those of the parent antibody and amidolytic activity similar to that of tPA, was not more efficient than tPA in an in vitro clot lysis assay. In contrast, it had been shown that tPA chemically coupled to the same antibody was four- to sixfold more efficient in fibrinolysis both in vitro and in vivo. A recombinant scuPA-antifibrin antibody hybrid, however, was sixfold more potent than scuPA in vitro and 20-fold more potent in a rabbit thrombolysis model. An explanation for this apparent discrepancy may relate to the requirement for stimulation by fibrin in order for tPA to achieve its maximal catalytic activity, a property that was demonstrated to have been lost in the antifibrin-tPA fusion protein. In contrast, the activity of urokinase is independent of the presence of fibrin. This may explain the greater success achieved in enhancing catalytic activity in the urokinase-antifibrin fusion protein. It is of additional interest that fibrin or soluble fibrin fragments stimulate the catalytic activity of both tPA and the isolated tPA B chain, demonstrating that at least part of the enhanced catalytic activity of tPA observed in the presence of fibrin is independent of fibrin binding either by the tPA kringles or finger domain (or any heavy chain domain). These data indicate that it is possible to construct recombinant hybrid molecules in which both plasminogen activator catalytic function and antibody binding are preserved.(ABSTRACT TRUNCATED AT 400 WORDS)

Familial Hypertrophic Cardiomyopathy (FHC) is a genetically inherited disorder of heart muscle. Over the past 40 years many studies have been done to describe in detail the clinical presentation of this disease and its associated pathophysiological consequences. The primary focus of this review is to discuss more recent studies involving the genetic mapping of one locus on chromosome 14, which causes FHC, and then to summarize studies demonstrating that this locus contains mutations in the cardiac myosin heavy chain genes. The chromosomal location of other putative FHC loci will also be considered. Finally, the implications of results that demonstrate that cardiac myosin heavy chain defects produce the pathophysiology of FHC will be considered from both clinical and basic research perspectives.

In cardiac muscle, the selectivity and specificity of gene regulation by heparin-binding and transforming growth factors resembles the characteristic program of fetal gene induction during myocardial hypertrophy produced by load. Shared by isolated cardiac myocytes and intact hearts, these complex and heterogeneous responses provide intriguing systems, which are distinct from other lineages and models of cell growth, for the study of trophic signal transduction by cellular oncogenes. A functional role for peptide growth factors and other oncogene-encoded proteins in myocardial hypertrophy suggests biological pathways which might usefully be exploited to promote compensatory growth following infarction or to interfere with maladaptive changes during a hemodynamic load.

Transcription of the alpha 2-macroglobulin gene (alpha 2M) in rat hepatocytes is strongly induced during acute inflammations by interleukin 6 (IL6). An IL6-response region has previously been mapped in the promoter upstream sequence of this gene. The region consists of two adjacent elements (IL6-REs), the IL6-RE core (CTGGGAA, -164 to -158 bp) and the core homology (CTGGAAA, -184 to -178 bp), elements, that are located 20 bp apart. Both elements bind nuclear factors with very similar protein-DNA contact patterns when they are contained in their original sequence context. A protein-DNA complex III was obtained in gel mobility shift experiments using a probe individually representing the core site. With probes containing both the core and core homology sites, a hormone inducible complex II of slower mobility was obtained. Complex II consisted of multiple copies of the same protein or proteins with very similar molecular masses bound at both sites. The core homology site was the weaker binding site. With a probe containing two tandem copies of the core site, binding at the second site occurred with 81 times greater affinity when the first site was occupied, than when it was free. Thus, the factor binding at the IL6-REs, the IL6-RE binding protein (IL6 RE-BP), was capable of co-operatively interacting with itself. Another factor, IL6-DBP/LAP, has recently been shown to be involved in the regulation of a major subgroup of acute phase genes by IL6. Using recombinant IL6-DBP/LAP and corresponding antisera, we demonstrated here that the IL6 RE-BP of the alpha 2M gene was distinct from IL6-DBP/LAP and from the related factor DBP. Thus, two major IL6-response elements can be distinguished: type 1 elements occurring in the human C-reactive protein, hemopexin and haptoglobin genes and utilizing IL6-DBP/LAP; and type 2 elements occurring in the rat alpha 2M, and alpha 1-acid glycoprotein genes, and utilizing a different IL6 RE-BP. The IL6 RE-BP of the alpha 2M gene was also shown to be distinct from the transcription factor NF kappa B. The IL6RE-BP had relative molecular mass of Mr = 46,000, distinct from IL6-DBP/LAP (Mr = 32,000) and NF kappa B (Mr = 50,000) and its overall DNA binding capacity was induced under acute phase conditions.

Human prolidase (PEPD, iminodipeptidase, EC 3.4.13.9) and related deficiencies were analyzed in terms of the nature and molecular biology of the enzyme and the molecular events seen in patients with this deficiency. The analyses were based on findings concerning isolation of the enzyme, development of specific antibodies and molecular cloning of cDNA and genome DNA of human prolidase. The studies revealed that human prolidase is a homo-dimer of an identical subunit 492 amino acid residues. The gene for prolidase (PEPD gene) was localized on chromosome 19, spanned more than 130 x 10(3) base-pairs and split into 15 exons. Molecular defects in prolidase deficiency were then analyzed. Two patients with the polypeptide-positive phenotype of the disease carried a mis-sense mutation of exon 12. Two siblings with a polypeptide-negative phenotype carried a gene deletion that encompassed exon 14. These mutations were not found in ten other patients with the disease, hence the molecular defects in prolidase deficiency are apparently highly heterogeneous.