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Biomedical subjects

L Alexander

Publications and source records attributed to L Alexander.

At least 55 records · Page 3Linked to original sources
PubMed1995

Interaction between the 5'-terminal cloverleaf and 3AB/3CDpro of poliovirus is essential for RNA replication.

On the basis of sequence alignments and secondary structure comparisons of the first 100 nucleotides of enterovirus and rhinovirus RNAs, chimeric constructs in which this region of poliovirus type 1 Mahoney [PV1(M)] is replaced with that of human rhinovirus type 2 (HRV2) or HRV14 have been engineered. These chimeric constructs contain the internal ribosomal entry site of either poliovirus or encephalomyocarditis virus. Independent of the internal ribosomal entry site elements, only the constructs containing either the PV1(M) or HRV2 cloverleaf sequences yielded viable viruses. The secondary structures of all three cloverleaves are quite similar. However, highly purified polioviral proteins 3CDpro and 3AB together bound to the PV1(M) and HRV2 cloverleaves, albeit with different affinities, whereas the HRV14 homolog did not interact with these proteins to any appreciable extent. These results support a mechanism of poliovirus genomic replication in which the formation of a complex between the cloverleaf structure and the 3CDpro/3AB proteins of poliovirus plays an essential role.

3C Viral Proteases↗
PubMed1995

Construction and genetic analysis of dicistronic polioviruses containing open reading frames for epitopes of human immunodeficiency virus type 1 gp120.

On the basis of previous studies of dicistronic (dc) polioviruses that carried two internal ribosomal entry sites (L. Alexander, H.-H. Lu, and E. Wimmer, Proc. Natl. Acad. Sci. USA 91:1406-1410, 1994; A. Molla, S. K. Jang, A. V. Paul, Q. Reuer, and E. Wimmer, Nature [London] 356:255-257, 1992), we have constructed a variety of dc polioviruses which express foreign genetic elements that were inserted either between two internal ribosomal entry site elements upstream of the poliovirus open reading frame (pPNENPO derivatives) or upstream of the open reading frame for the poliovirus proteinase 2Apro (pDI-E2A derivatives). Surprisingly, the addition of an N-terminal secretory pathway signal sequence to the open reading frame of the inserted foreign sequences (specifying either truncated versions of human immunodeficiency virus type 1 [HIV-1] gp120 or chloramphenicol acetyltransferase) resulted in a null phenotype, whereas removal of the signal sequence led to the production of viable viruses. Constructs that carried a foreign gene with a signal sequence were negative in RNA synthesis, an observation that suggested a very early block in viral replication. The insertion of transmembrane sequences downstream of the leader sequence did not reverse the replication block. Studies of dc polioviruses that encoded the truncated versions of HIV-1 gp120 showed an increase in genetic stability that correlated with a decrease in the size of the insert. A dc construct that contained a minigene encoding the principal neutralization determinant of HIV-1 produced a stable virus that retained the foreign sequence through multiple passages in cultured cells. These data indicate that dc polioviruses have potential as vaccines for the expression of small foreign epitopes.

Amino Acid Sequence↗
PubMed1994

Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding.

Poliovirus proteinase 3CDpro by itself is not an RNA-binding protein. Two cellular proteins have been purified from HeLa cells (p50 and p36) which interact with purified 3CDpro but only p36-3CDpro bind to the 5'-terminal 110 nucleotides of polioviral RNA genome, an RNA segment whose secondary structure resembles a cloverleaf. The identity of these factors was determined by microsequencing tryptic digests of the purified proteins. Host protein p50 is the eukaryotic elongation factor EF-1 alpha, and p36 an N-terminal fragment thereof. p36, referred to as host factor, did not appear to interact with purified 3Cpro or 3Dpol. Significantly, the formation of a 3CDpro-cloverleaf complex was also observed in the presence of purified poliovirus polypeptide 3AB, the precursor of VPg. 3AB by itself does not stably bind to the cloverleaf. Competition experiments have demonstrated that the RNA-protein interactions are specific for the full-length cloverleaf. UV cross-linking studies were employed to examine the protein components of the cloverleaf ribonucleoproteins. RNA footprinting was used to determine the site on the cloverleaf where the viral and cellular factors bind. Finally, we have discovered that 3AB-3CDpro also interacts with the 3'-terminal sequence of poliovirus RNA. In contrast to the 5'-terminal cloverleaf, the 3'-terminal RNA can bind 3AB in the absence of other proteins. A model for initiation of poliovirus RNA synthesis is presented.

3C Viral Proteases↗
PubMed1994

Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene.

A picornavirus hybrid genome was constructed in which the internal ribosomal entry site (IRES) of encephalomyocarditis virus was inserted between the 5' non-translated region and the open reading frame of poliovirus (PV), type 1 (Mahoney). Upon transfection into HeLa cells, the hybrid RNA replicated and yielded a derivative of PV (W1-PNENPO). The PV IRES could be removed from pPNENPO, which resulted in a hybrid picornavirus (W1-P108ENPO) in which the translation of the PV open reading frame normally promoted by the type 1 IRES of PV was promoted by the type 2 IRES of encephalomyocarditis virus. This result indicates that these elements are not likely to contain cis-acting elements necessary for PV replication or encapsidation. A foreign gene (bacterial chloramphenicol acetyltransferase, CAT) was inserted into pPNENPO cDNA between the PV and encephalomyocarditis virus IRES elements. The dicistronic RNA replicated in HeLa cells and yielded a derivative of PV (W1-DICAT) with a genome 17% longer than that of wild-type PV. CAT assays and immunoblot analyses showed that the viral RNA efficiently expressed the foreign gene in cell culture. The CAT activity diminished somewhat with each passage of the dicistronic virus, an observation which suggested that the inserted gene had a deleterious effect on viral replication. However, even after five virus passages, a significant quantity of the foreign gene was still expressed. Insertion of the open reading frame of luciferase (67 kDa) resulted in an RNA species that replicated and expressed luciferase for up to 20 hr after transfection. However, this elongated RNA was not encapsidated.

Base Sequence↗
PubMed1994

Dicistronic polioviruses as expression vectors for foreign genes.

We have made use of certain novel genetic elements of picornaviruses termed internal ribosomal entry sites (IRES) to construct a viral RNA with the following genetic order: PV 5' NTR-EMCV IRES-PV ORF-3' NTR (PV, poliovirus; NTR, nontranslated region; EMCV, encephalomyocarditis virus; ORF, open reading frame). Transfection of this RNA into HeLa cells yielded a poliovirus (W1-PNENPO) that contained two heterologous IRES elements (type 1 IRES of PV; type 2 IRES of EMCV) in tandem. The insertion of foreign coding sequences into the genome of W1-PNENPO between the IRES elements yielded viable polioviruses with the gene order PV 5' NTR-foreign ORF-EMCV IRES-PV ORF-3' NTR. The foreign ORFs we have employed in this study included the coding region for chloramphenicol acetyltransferase (CAT), or segments of either luciferase or the HIV-1 envelope glycoprotein gp120. W1-PV/V3-3, a dicistronic poliovirus that contained HIV-1-specific sequences that included the V3 domain of gp120, was used to infect transgenic mice (PVR+) that were engineered to express the poliovirus receptor. The genetic stability of the dicistronic viruses and the HIV-1-specific immune response in PVR+ mice after infection with these novel agents are discussed.

AIDS Vaccines↗
PubMed1993

Colour flow mapping in the diagnosis of the calf deep vein thrombosis.

This prospective trial was organized to evaluate further the role of colour flow Doppler ultrasound techniques in the diagnosis of deep vein thrombosis (DVT) with particular reference to the isolated calf lesion. In 100 patients ultrasound was compared against the recognized gold standard of ascending venography. Fifty venograms were positive for DVT compared with 49 on ultrasound, a sensitivity of 98%. The specificity of ultrasound for DVT in the 50 patients without DVT on venography was 100%. Forty-five venograms demonstrated calf thrombus with varying proximal extent. Sixteen proved to be isolated calf lesions and these were all identified by ultrasound (100% sensitivity and specificity respectively for isolated calf DVT). In five of these isolated calf thrombus cases, results suggested that ultrasound better diagnosed the presence of thrombus. Ultrasound diagnosed significant pathology in 13 of the normal venogram patients giving an overall diagnostic yield of 62% as compared to 50% at venography.

Adolescent↗
PubMed1993

Psychological health-sickness (PHS) as a predictor of outcomes in dynamic and other psychotherapies.

This is the first dedicated review of quantitative studies on Sigmund Freud's proposition that the poorer the psychological health, the more limited are the benefits from treatment. Since observer-rated scales for psychological health-sickness were developed in 1949, many studies have applied them, and the majority show significant prediction of outcomes of psychotherapy, with correlations between .2 and .35. This article reviews (a) the main methods of measurement, (b) the record of predictive success, (c) validity studies, (d) the relation to psychiatric diagnosis, (e) prediction in forms of treatment other than psychotherapy, and (e) theories of why psychological health predicts outcomes of psychotherapy.

Humans↗
PubMed1992

Analysis of deaths in patients with an implantable cardioverter defibrillator.

The cause of death and clinical characteristics of 26 patients that died after implantable cardioverter defibrillator placement were reviewed and compared to the 145 patients still living after a mean follow-up of 17 months. Operative mortality was 4% (7/171) and resulted from postoperative ventricular arrhythmias (four patients), heart failure (two patients), and respiratory failure (one patient). Operative mortality was significantly higher (1.7% vs 9.6%, P less than 0.05) following concomitant surgical procedures. Total late mortality was 11% (18/171). Thirteen deaths (75%) occurred in-hospital from progressive deterioration of left ventricular function (nine patients), arrhythmia (two patients), and noncardiac causes (two patients). Outpatient mortality was 3.5% (6/171) and resulted from presumed sudden cardiac death in five of six patients; two of the five had devices that were inactive, one had high defibrillation thresholds, and two had suspected bradyarrhythmic deaths. One postoperative death and one late in-hospital death were also considered sudden cardiac deaths for a total of seven patients with defibrillation system failures. By multivariant analysis, preoperative clinical characteristics associated with a worse prognosis following defibrillator implantation were identified: presentation as ventricular tachycardia (P less than 0.02), induction of sustained monomorphic ventricular tachycardia (P less than 0.05), poor left ventricular performance (P less than 0.01), poor functional status (P less than 0.001), and the use of diuretics (P less than 0.01). Frequent device discharges (P less than 0.001) and concomitant antitachycardia pacing systems (P less than 0.001) were markers for greater arrhythmia recurrence and were potent predictors of a worse prognosis and particularly sudden death.

Arrhythmias, Cardiac↗
PubMed1992

Oesophageal propulsive force and its relation to manometric pressure.

A fixed volume capsule incorporating a force transducer and a side hole for manometric measurements was constructed and calibrated. Simultaneous measurements of the propulsive (aboral) force and the manometric pressure (intraluminal pressure) were made at 5, 10, and 15 cm above the lower oesophageal sphincter and in response to dry and wet (5, 10, and 15 ml) swallows. The propulsive force and manometric pressure waves had a simultaneous onset and were of similar duration. Peak values of propulsive force for wet swallows increased significantly as measurements were made progressively more distally within the oesophagus and were greatest in the distal oesophagus. The association between manometric pressure and propulsive force is not strong (r = 0.61) suggesting that intraluminal pressure is a poor predictor of propulsive force and hence an unreliable measure of oesophageal 'function'.

Adult↗
PubMed1991

The power of the therapeutic relationship.

A review of the psychotherapy research literature of the last decade shows that considerable advances of clinical significance have been made toward defining and measuring components of the treatment relationship. The relevance of the therapeutic alliance for predicting outcome in diverse models of treatment is emphasized, and the implications of the findings for clinical training, practice, and research are discussed.

Adaptation, Psychological↗
PubMed1991

Production of the mouse whey acidic protein in transgenic pigs during lactation.

The mouse whey acidic protein (WAP) gene was introduced into the genome of pigs and its expression was analyzed in the mammary gland. Mouse WAP was detected in milk of lactating females from five lines at levels between .5 and 1.5 g/liter, thereby representing as much as 2% of the total milk proteins. The corresponding mRNA was expressed in mammary tissue at levels similar to those of pig beta-lactoglobulin and beta-casein. The pattern of WAP secretion in three pigs over a period of 6 wk was quantitatively similar to that of pig beta-lactoglobulin. From the eight transgenic pigs analyzed, three successfully completed one lactational period, but five pigs stopped lactating a few days after parturition. Our results show that it is possible to produce large quantities of a foreign protein in milk of pigs over a full lactational period. However, expression of WAP can compromise the mammary gland and render it nonfunctional.

Animals↗
PubMed1990

Transcriptional regulation of the murine TSH subunit genes.

These studies have demonstrated that the murine TSH subunit genes are sensitively regulated by thyroid hormone. The T3 receptor complex interacts with both the TSH beta and alpha-subunit gene either in or near the 5' flanking region in close proximity to the transcriptional start sites. This interaction interrupts transcription from the start sites, thus decreasing transcription of the two genes. As a result steady state mRNA levels of both TSH beta and alpha-subunit genes are decreased in the cytoplasm of thyrotropic cells. This series of interactions explains most of the effects of T3 on TSH biosynthesis.

Animals↗
PubMed1989

Chromatographic separations of serum proteins on immobilized metal ion stationary phases.

The separation of proteins on stationary phases consisting of a bound organic chelator and a chelated divalent transition metal has been studied as a function of (A) metal ion species; (B) mobile phase composition and pH; and (C) anion and cation concentration. Optimum separation was observed at alkaline pH on chelated nickel stationary phases. Ammonium and Tris salts reduced the affinity of the metal chelate packing for serum proteins. Halide ions caused the proteins to be more strongly bound to the stationary phase. High salt concentrations had only a small effect on the binding of serum proteins in the absence of amine containing buffers or salts. It was also observed that the ease of elution and the recovery of protein were dependent on pH and upon the presence of halides. The general order of elution of serum proteins, based on isoelectric focusing, was independent of metal ion species and elution conditions, suggesting that a single mechanism or a unique sequence of mechanisms was operative. The results suggest that ligand exchange is the major mechanism of separation under basic conditions and that hydrophobic effects are the result of the competition of nonnitrogen ions with ammonium ions or amines for ligand binding sites modifying or participating in protein binding. Protein binding studies under weak acidic conditions are also presented although the mechanism responsible for protein binding is unclear.

Blood Protein Electrophoresis↗
PubMed1989

Ibuprofen, acetaminophen, and placebo treatment of febrile children.

A double-blind, parallel-group, triple-dummy-designed, single-oral-dose study compared the efficacy, tolerability, safety, and dose-response of 5 mg/kg (n = 32) and 10 mg/kg (n = 28) ibuprofen suspension, 10 mg/kg acetaminophen elixir (n = 33), and placebo liquids (n = 34) in 127 children (2 to 11 years of age) with fever (101 degrees to 104 degrees F). Blood samples, oral temperatures, pulse, blood pressure, and respiration were obtained before and 1/2, 1, 2, 3, 4, 5, 6, and 8 hours after the dose was administered. The study was terminated early if oral temperature was greater than 104 degrees F or if it increased 1 degree F above baseline. All agents were well tolerated and more effective than placebo (p less than 0.05) for fever control. Ibuprofen, 10 mg/kg, was favored over 10 mg/kg acetaminophen (p less than 0.05). For temperatures greater than 102.5 degrees F, a dose-response relationship for 5 and 10 mg/kg ibuprofen was demonstrated in terms of percentage of fever reduction and in terms of the initial 2-hour rate of decrease in temperature. Antipyretic efficacy for temperatures greater than 102.5 degrees F was 10 mg/kg ibuprofen greater than 5 mg/kg greater than 10 mg/kg acetaminophen greater than placebo. All treatments were well tolerated. No significant clinical or laboratory abnormalities were noted. Ibuprofen suspension may be a safe and effective antipyretic in children.

Acetaminophen↗