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Biomedical subjects

B J Levine

Publications and source records attributed to B J Levine.

15 recordsLinked to original sources
PubMed2000

C-peptide inhibits leukocyte-endothelium interaction in the microcirculation during acute endothelial dysfunction.

C-peptide is a cleavage product that comes from processing proinsulin to insulin that induces nitric oxide (NO) -mediated vasodilation. NO modulates leukocyte-endothelium interaction. We hypothesized that C-peptide might inhibit leukocyte-endothelium interaction via increased release of endothelial NO. Using intravital microscopy of the rat mesentery, we measured leukocyte-endothelium interactions after administration of C-peptide to the rat. Superfusion of the rat mesentery with either thrombin or L-NAME consistently and significantly increased the number of rolling, adhering, and transmigrated leukocytes. C-peptide significantly attenuated either thrombin- or L-NAME-induced leukocyte-endothelium interactions in rat mesenteric venules. A control scrambled sequence of C-peptide characterized by the same amino acid composition in a randomized sequence failed to inhibit leukocyte-endothelium interactions. These effects of C-peptide were associated with decreased surface expression of the cell adhesion molecules P-selectin and ICAM-1 on the microvascular endothelium. Endothelial nitric oxide synthase (eNOS) mRNA levels were increased in rats injected with C-peptide. This enhanced eNOS expression was associated with a marked increase in basal NO release from the aorta of C-peptide-treated rats. We conclude that C-peptide is a potent inhibitor of leukocyte-endothelium interaction and that this effect is specifically related to inhibition of endothelial cell adhesion molecules via maintenance of NO release from the vascular endothelium.

Animals↗
PubMed1999

Challenges faced in recruiting patients from primary care practices into a physical activity intervention trial. Activity Counseling Trial Research Group.

BACKGROUND: Special challenges are encountered when clinical trial recruitment targets a physician practice-based population, as opposed to recruiting from the community. Since most published information about recruitment has focused on the latter group, summation of successful primary-care-based recruitment strategies could prove useful for future trials recruiting from this population. METHODS: The Activity Counseling Trial (ACT) is a multicenter, randomized clinical trial that evaluated approaches to primary care-based interventions to increase physical activity in sedentary adults 35-75 years of age. Fifty-four clinicians from eight practices recruited 874 participants from three U.S. sites. Recruitment challenges that related, in great part, to the primary care setting included: (1) focusing on patients from ACT physician practices who had regularly scheduled or intend-to-schedule appointments within the next year; (2) placing trial staff in the clinical offices for recruitment purposes; and (3) placing trial interventionists in the physicians' offices. Other challenges were related to recruitment of minorities and men. RESULTS: Patient mailing yielded 43.4% of all randomized participants, followed by office-based questionnaires (32.5%) and direct telephone contact (21.6%). Based on a retrospective cost-effective analysis (indirect costs excluded), the self-administered office-based questionnaire was the least costly strategy for one site ($14/randomized participant), followed by patient mailing at another site ($58). The direct telephone contact method utilized at one site serving primarily a minority population yielded a per randomized participant cost of $80. CONCLUSIONS: Recruitment of clinical trial participants from practice-based populations requires modification of the strategies used to recruit from the community. Multiple strategies should be employed, followed closely for their respective yields, and adapted as needed.

Adult↗
PubMed1996

Precise limitation of concerted evolution to ORFs in mosquito Hsp82 genes.

Two Hsp82 genes were isolated from the malaria vector Anopheles albimanus in a single lambda phage clone. The two genes are in a head-to-head arrangement separated by approx. 0.9 kbp. Northern hybridizations and 5' RACE demonstrate that both genes are transcribed, have moderate levels of constitutive transcription, and are also heat-inducible with maximum transcript accumulation occurring after 40 degrees C heat shocks. Both genes have typical heat-shock promoters and conserved intron boundaries in the untranslated leaders. The open reading frames are 99.6% identical differing in only nine silent nucleotide positions in the 2166 bp ORFs. However, precisely outside the ORFs, the flanking DNA of the two genes shows no evidence of common derivation. The high degree of identity between the two ORFs appears to be a result of gene conversion occurring by a process similar to that previously suspected in the A. albimanus Hsp70 genes and several D. melanogaster genes arranged as palindromes. This process probably involves a stem-loop intermediate and is restricted in extent by flanking sequence divergence. These Hsp82 genes clearly demonstrate the extreme precision with which gene conversion can lead to protein-coding-region homogeneity yet allow flanking DNA divergence.

Amino Acid Sequence↗
PubMed1995

An RNA tertiary structure of the hepatitis delta agent contains UV-sensitive bases U-712 and U-865 and can form in a bimolecular complex.

Genomic RNA of the hepatitis delta agent has a highly conserved element of local tertiary structure. This element contains two nucleotides which become covalently crosslinked to each other upon irradiation with UV light. Using direct RNA analysis, we now identify the two nucleotides as U-712 and U-865 and show that the UV-induced crosslink can be broken by re-exposure to a 254 nm peak UV light source. In the rod-like secondary structural model of delta RNA, nucleotides U-712 and U-865 are off-set from each other by 5-6 bases, a distance too great to permit crosslinking. This model needs to be modified. Our data indicate that bases U-712 and U-865 closely approximate each other and suggest that the smooth helical contour proposed for delta RNA is interrupted by the UV-sensitive element. The nucleotide sequence shows that the UV-sensitive site does not have a particularly high density of conventional Watson-Crick base pairs compared to the rest of the genome. However, this element may have a number of non-Watson-Crick bonds which confer stability. Following UV-crosslinking and digestion with 1 mg/ml of RNase T1 at 37 degrees C for 45 min in 10 mM Tris-HCl, 1 mM EDTA (conditions expected to give complete digestion), this element can be isolated as part of a 54 nucleotide long partial digestion product containing at least 16 internal G residues. UV-crosslinking analysis shows that this unusual tertiary structural element can form in a bimolecular complex.

Base Sequence↗
PubMed1992

Transcripts of the viroid central conserved region contain the local tertiary structural element found in full-length viroid.

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural element, a small RNA oligonucleotide containing the CCR of the viroid genome was synthesized. The tertiary structure of these CCR transcripts was examined by UV-crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and classical RNA secondary analysis. The CCR transcript was found to undergo UV-crosslinking between the same two bases as in full-length viroid, indicating that the tertiary structure is the same and that the CCR transcript will be useful for the affinity purification of host components.

Base Sequence↗
PubMed1991

The novel tertiary structure in delta RNA may function as a ribozyme control element.

The viroid-like domain making up the lefthand end of the delta hepatitis genome (Branch et al., 1989) has structural elements whose interactions may be essential for replication. This portion of the genome is highly conserved in primary sequence and contains two well-defined types of structural features: sites capable of self-cleavage, and those forming a UV-sensitive element of local tertiary structure which is very stable and contains non-Watson:Crick bonds that may form a distinctive surface for binding specific proteins. The proximity of the tertiary structure to the genomic self-cleavage site suggests that the photoreactive element may regulate the ribozyme. This element's stability would limit "breathing" in this region of the circular genome, maintaining the ribozyme in the "off" conformation; its tight structure could be relieved by protein binding at the time of replication. We previously mapped a novel local tertiary structure to a highly conserved portion of the viroid genome (Branch et al., 1985). The many additional properties shared by viroids, related infectious circular RNAs of plants, and the delta agent are discussed in a recent article (Branch et al., in press-a).

Base Sequence↗
PubMed1989

The efficiency of 3'-end formation contributes to the relative levels of different histone mRNAs.

Sequences at both the 5' and 3' ends of mouse histone genes contribute to the expression of individual genes. The 3' sequences required for high expression of the mouse H2a-614 gene are the same as the sequences required for 3'-end formation. When these sequences were substituted for the 3' end of the poorly expressed H2a-291 gene, expression of the H2a-291 gene was increased fivefold. A 65-nucleotide fragment containing the H2a-614 3' processing signal increased expression of the H2a-291 gene when it was placed in the proper orientation downstream of the H2a-291 3' end. The only mRNAs that accumulated from this gene ended at the H2a-291 3' end, which suggests that the transcript is sequentially processed. In an in vitro processing system, the different histone 3' ends showed different processing efficiencies, which correlated with their expression in cells. These results suggest that the efficiency of processing is important in determining the steady-state levels of individual mouse histone mRNAs.

Animals↗
PubMed1988

Differential expression of individual members of the histone multigene family due to sequences in the 5' and 3' regions of the genes.

Histone proteins are encoded by a multigene family. The H3.2(614) and H2a(614) genes are present as single copies which are expressed at high levels, accounting for 30 to 40% of the H3 and H2a mRNAs, respectively, in different types of mouse cells. The other genes which have been isolated each contribute only a very small amount to the total type-specific mRNA pool. We demonstrate here that the differences in the level of expression of these genes are partly due to differences in their transcription rates. To investigate the sequences responsible for these differences in expression among the members of each family, we carried out DNA-mediated gene transfer experiments with both intact and chimeric histone genes. The 5' region of a highly expressed gene [H3.2(614) or H2a(614)] was attached to the 3' region of a histone gene which was expressed at low levels (H3-221 or H2a-291) and vice versa. The results show that sequences in both the 5' and 3' regions of the H3.2(614) and H2a(614) genes contribute to their high level of mRNA production by two independent mechanisms. The effect of the 3' sequences on mRNA accumulation has been narrowed to a 65-base-pair region including the 3'-terminal palindrome and downstream signal implicated in mRNA processing.

Animals↗
PubMed1987

Coupling of replication type histone mRNA levels to DNA synthesis requires the stem-loop sequence at the 3' end of the mRNA.

The role of the 3' end of mRNA in coupling between the level of histone mRNAs and DNA synthesis was examined. We introduced modified mouse histone H3 genes into mouse fibroblasts and studied the regulation of several different H3 mRNAs that are not terminated with a normal histone 3' end. In two cases, the stem-loop sequences were deleted from the mRNAs and replaced either by 3' sequences flanking the H3 gene or by globin 3' untranslated region sequences including the polyadenylylation signal. In the former case, approximately equal to 50% of the modified mRNA was polyadenylylated, whereas in the latter case all of the mRNA had a polyadenylylated terminus. In contrast to the normal histone mRNAs, these mRNAs, including the nonadenylylated form, were stable when DNA synthesis was inhibited with several drugs. The levels of two other histone mRNAs, each containing the stem-loop sequences as an internal part of the mRNA, also were stable when DNA synthesis was inhibited. These results indicate that the posttranscriptional coupling of histone mRNA levels to DNA synthesis requires the presence of the stem-loop sequences at the 3' end of the mRNA.

Animals↗
PubMed1987

Expression of mouse histone genes: transcription into 3' intergenic DNA and cryptic processing sites downstream from the 3' end of the H3 gene.

Introduction of the mouse histone H3.1 gene into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene resulted in the production of two mRNAs from the transfected gene, one with a normal 3' end and the other one with a longer 3'-untranslated region, ending at site X, which was poly(A)+. In contrast, the endogenous histone H3.1 gene only produced a single mRNA. The cryptic poly(A)+ site was only used when the histone H3.1 gene was transfected. To localize possible downstream cryptic processing sites, the hairpin loop at the end of the histone gene was deleted and the resulting deletions were introduced into L cells. Two major mRNAs were produced from this gene, one ending at site X and the major one ending at site Y, which was located 150 nucleotides before site X. Transcription extended downstream of site X efficiently in the endogenous gene, as judged by the extent of transcription of downstream sequences in isolated nuclei. Transcription extended downstream of site X in the transfected gene because the placement of a normal histone 3' end downstream of site X resulted in transcripts that ended at site X and longer transcripts that ended with the new histone 3' end. These results indicate that transcription may normally proceed a substantial distance past the hairpin loop (greater than 500 bases). The formation of the different 3' ends in these transfected genes was due to competition between different processing mechanisms.

Animals↗
PubMed1986

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.

Acetyltransferases↗
PubMed1980

Physicochemical properties and interactions of Escherichia coli ribonucleic acid polymerase holoenzyme, core enzyme, subunits, and subassembly alpha 2 beta.

We have investigated several physicochemical properties of Escherichia coli DNA-dependent RNa polymerase, its constituent subunits alpha, beta, beta', and sigma, and the subassembly alpha 2 beta. These included ultraviolet (UV) absorption, isoelectric points, sulfhydryl content, extinction coefficients, and circular dichroism (CD). Among the most notable results is the observation, based on CD measurements, that the sigma subunit, free and combined in holoenzyme, is a highly structured protein with approximately 75% of its residues folded in alpha-helical conformation and little or no detectable beta sheet. No secondary structure changes in either sigma or core accompany formation of holoenzyme. In contrast to the conformational independence of the subunits in assembly of holoenzyme, the protein and its components exhibit conformational flexibility as glycerol concentration is varied and in their interaction with DNA. The effect of glycerol on the conformation of sigma, core, and holoenzyme was monitored by circular dichroism measurements. In the far-ultraviolet, the residue ellipticity at 220 nm ([theta]220) increased approximately 15% from 0 to 10% glycerol for both core and holoenzyme. In the near-ultraviolet, the residue ellipticity at a peak near 280 nm also varied with glycerol concentration, decreasing in intensity by about 50% with holoenzyme, when glycerol was raised from 5 to 10%, then increasing at still higher glycerol contents. Electrophoretic and molecular sieve anaysis showed core and sigma to have greater affinity for each other in 50% glycerol than in 10% glycerol. The presence of 10% glycerol in the assay buffer increased the activity of the enzyme. The effect of various DNA templates on the conformations of core, holoenzyme, alpha 2 beta subassembly, and beta' subunit was also monitored by far-ultraviolet circular dichroism. All the protein samples showed between 10 and 20% decrease in secondary structure upon the addition of the DNA.

Bacterial Proteins↗
PubMed1980

Cardiovascular complaints. Correlation with cardiac arrhythmias on 24-hour electrocardiographic monitoring.

Long-term ambulatory electrocardiographic (Holter) monitoring is frequently used to evaluate patients with various cardiovascular complaints, including palpitations, dyspnea, discomfort in the chest, dizziness, and syncope. In the present study, 518 consecutive 24-hour electrocardiographic recordings were reviewed to determine correlations between cardiac diagnoses, presenting complaints, and specific electrocardiographic abnormalities. Two hundred seventy-four patients (53 percent) had significant arrhythmias; 212 (41 percent) had significant ventricular arrhythmias, and 106 (20 percent) significant atrial arrhythmias, including 44 patients (8 percent) with both. No presenting complaint or cardiovascular diagnosis correlated closely with any specific cardiac arrhythmia. Major arrhythmias, including supraventricular and ventricular tachycardias, often occurred asymptomatically (in 44/54 and 37/40 patients, respectively); however, among 371 patients with accurate historic logs, only 176 (47 percent) had long-term electrocardiographic studies in which their typical symptoms occurred during the monitoring period. Fifty (13 percent) of the 371 patients had concurrence of their presenting complaints with an arrhythmia, and 126 patients (34 percent) had their typical symptoms associated with a normal electrocardiogram, which was helpful in excluding an abnormality of rhythm or conduction as the primary cause for their complaints.

Arrhythmias, Cardiac↗
PubMedDate not supplied

Another false alarm? apnea monitor activation in a Neonatal Intensive Care Unit graduate.

Neonatal emergencies have become more common as increasingly sophisticated Neonatal Intensive Care Units graduate lower birth-weight babies born at younger gestational ages. These patients present a number of challenges to emergency physicians. They are often discharged with apnea monitors, which generate a high number of false alarms. Neonatal Intensive Care Unit graduates, however, are predisposed to a number of conditions that can result in true episodes of apnea. We present such a case and will discuss the unusual underlying cause of apnea, the utility of apnea monitors, and the need for emergency physicians to be prepared to evaluate and treat these potentially complicated patients.

Apnea↗