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Biomedical subjects

M Belfort

Publications and source records attributed to M Belfort.

At least 37 records · Page 2Linked to original sources
PubMed1998

Normative oxygen saturation values for pregnant women at sea level.

Objective: To develop normative ranges of maternal arterial oxygen saturation (SpO(2)) for women residing at sea level throughout gestation and postpartum.Materials and Methods: Healthy, nonsmoking, non-laboring pregnant women, with no obstetrical risk factors, were recruited into this longitudinal study performed at The Stamford Hospital (elevation sea level). Maternal SpO(2) was measured in the dominant index finger while breathing room air with a Nelcor N-200 pulse oximeter and DS-100A sensor (Nellcor Puritan Bennett, Pleasanton, CA) at set antepartum (AP) and postpartum (PP) intervals. Oxygen saturation was measured in the following positions: sitting, lying on the left then right side, knee-chest, and supine. Five minutes were allowed between measurements. Measurements were taken at 12, 24, and 36 weeks of gestation and then at 12 weeks postpartum for each patient.Results: Thirty-six patients were initially enrolled. Three were excluded from this study, secondary to loss of follow-up (1), preterm delivery at 35 weeks (1), and complicated placenta previa (1). Of the 33 patients who remained in the study 5 have delivered and undergone postpartum evaluation. SpO(2) values (%) are summarized as mean +/- 2 SD in the Table.Conclusion: Preliminary data in this ongoing descriptive longitudinal study suggest that maternal SpO(2) is at least 97% throughout pregnancy for healthy, non-smoking women residing at sea level. These normative data may be useful in managing pregnant women at risk for hypoxia.

Journal Article↗
PubMed1998

Effect of glycosylated hemoglobin on endothelium-dependent relaxation of omental arteries from pregnant women.

OBJECTIVE: This study was intended to test the hypothesis that glycosylated hemoglobin adversely affects endothelium-dependent vascular relaxation during pregnancy. STUDY DESIGN: Omental artery rings (3 mm) obtained from patients undergoing cesarean delivery at term were suspended in organ chambers for isometric tension recording in Krebs bicarbonate solution. They were bubbled with 5% carbon dioxide in air (37 degrees C, pH 7.4). After equilibration at 1-g passive tension and contraction with endothelin 1, concentration-relaxation curves in response to bradykinin were determined in the presence or absence of oxyhemoglobin, glycosylated hemoglobin, or nitric oxide synthase inhibitors (N(omega)-nitro-L-arginine methyl ester or N(omega)-nitro-L-arginine). RESULTS: Oxyhemoglobin and glycosylated hemoglobin attenuated the endothelium-dependent relaxation induced by bradykinin. The inhibition did not differ significantly between the 2 nitric oxide scavengers and was of the same magnitude as that noted with the nitric oxide synthase inhibitors. CONCLUSION: Glycosylation of hemoglobin does not change its effect on endothelium-dependent relaxation in human omental vessels during pregnancy. These data do not support the hypothesis that glycosylated hemoglobin may be the reason for abnormal endothelial function in diabetes.

Adult↗
PubMed1998

Domain structure and RNA annealing activity of the Escherichia coli regulatory protein StpA.

The Escherichia coli regulatory protein StpA bears striking similarity to the chromatin-associated protein H-NS. These two proteins have many structural, functional and mechanistic parallels. Although H-NS is more abundant in the cell, both proteins act as transcriptional regulators, both bind to curved DNA and both restrain DNA supercoils. However, StpA is better able to promote RNA annealing and trans-splicing in vitro. In this study, phylogenetic analyses and experiments to examine the protease sensitivity of StpA and H-NS suggest a similar structure for the two proteins. Both proteins consist of two structured domains separated by an exposed protease-sensitive linker. The N-terminal (StpA-NterL) and C-terminal (StpA-CterL) domains of StpA, as well as the full-length StpA and H-NS proteins, were cloned, overproduced in E. coli and purified to homogeneity. StpA-CterL, but not StpA-NterL, promotes strand annealing of complementary RNA oligonucleotides and in vitro trans-splicing of a model group I intron. Both StpA and StpA-CterL exhibited stronger RNA-modulating activity than H-NS. Phylogenetic analyses showed that the N-terminal and C-terminal domains can exist autonomously. The phylogenetic and experimental data are compatible with a two-domain model for StpA and H-NS, with independently functioning modules joined by a non-conserved linker, and with the observed RNA-related activities residing entirely within the C-terminal domain.

Amino Acid Sequence↗
PubMed1997

A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron.

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.

Bacterial Proteins↗
PubMed1997

Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution.

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Amino Acid Sequence↗
PubMed1997

Homing endonucleases: keeping the house in order.

Homing endonucleases are rare-cutting enzymes encoded by introns and inteins. They have striking structural and functional properties that distinguish them from restriction enzymes. Nomenclature conventions analogous to those for restriction enzymes have been developed for the homing endonucleases. Recent progress in understanding the structure and function of the four families of homing enzymes is reviewed. Of particular interest are the first reported structures of homing endonucleases of the LAGLIDADG family. The exploitation of the homing enzymes in genome analysis and recombination research is also summarized. Finally, the evolution of homing endonucleases is considered, both at the structure-function level and in terms of their persistence in widely divergent biological systems.

Base Sequence↗
PubMed1997

Two-domain structure of the td intron-encoded endonuclease I-TevI correlates with the two-domain configuration of the homing site.

I-TevI, the T4 td intron-encoded endonuclease, catalyzes the first step in intron homing by making a double-strand break in the intronless allele within a sequence designated the homing site. The 28 kDa enzyme, which interacts with the homing site over a span of 37 bp, binds as a monomer, contacting two domains of the substrate. In this study, limited proteolysis experiments indicate that I-TevI consists of two domains that behave as discrete physical entities as judged by a number of functional and structural criteria. Overexpression clones for each domain were constructed and the proteins were purified. The carboxy-terminal domain has DNA-binding activity coincident with the primary binding region of the homing site and binds with the same affinity as the full-length enzyme. The isolated amino-terminal domain, contains the conserved GIY-YIG motif, consistent with its being the catalytic domain. Furthermore, site-directed mutagenesis of a conserved arginine residue within the extended motif rendered the full-length protein catalytically inactive, although DNA-binding was maintained. This is the first evidence that the GIY-YIG motif is important for catalytic activity. An enzyme with an N-terminal catalytic domain and a C-terminal DNA-binding domain connected by a flexible linker is in accord with the bipartite structure of the homing site.

Amino Acid Sequence↗
PubMed1996

Exon coconversion biases accompanying intron homing: battle of the nucleases.

Intron homing in phage T4 occurs in the context of recombination-dependent replication, by virtue of intron-encoded endonucleolytic activity. After the td intron endonuclease I-TevI cleaves the intronless recipient 23 and 25 nucleotides upstream of the intron insertion site, exonucleolytic degradation is required for recombination to proceed. This resection process results in coconversion of exon sequences flanking the intron. In a genetic system designed to study coconversion of flanking markers, we demonstrate that although there is a bidirectional polarity gradient, coconversion can be highly asymmetric. Furthermore, we show that the coconversion of flanking markers favors exon I sequences, upstream of the I-TevI cleavage site. These data are consistent with the asymmetric features of the homing pathways that have been invoked for intron mobility in phage T4. Moreover, these results are in accord with the finding that once the td homing-site substrate is cleaved, I-TevI remains bound to the downstream cleavage product, protecting against exonucleolytic degradation, and thereby limiting the extent of coconversion into exon II. The results suggest that recombination events are influenced by a competition between the homing endonuclease and exonucleases for sequences downstream of the I-TevI cleavage site, thereby implying a role for the homing endonuclease in the repair process.

Bacteriophage T4↗
PubMed1996

Escherichia coli protein analogs StpA and H-NS: regulatory loops, similar and disparate effects on nucleic acid dynamics.

Expression of the Escherichia coli StpA protein was investigated and a functional comparison undertaken with the structurally analogous nucleoid protein H-NS. Analysis of stpA and hns expression indicated that although stpA transcript levels are much lower than those of hns, the two gene products are capable of both negative autogenous control and cross-regulation. Examination of cellular proteins in stpA, hns, or stpA-hns backgrounds revealed that StpA can repress and activate a subset of H-NS-regulated genes. Mechanistic parallels in regulation of gene expression are indicated by the ability of both proteins to inhibit transcription from promoters containing curved DNA sequences, and to form nucleoprotein structures that constrain DNA supercoils. Despite their functional similarities, each molecule is capable of independent activities. Thus, H-NS regulates a class of genes that are unaffected by StpA in vivo, whereas StpA has much stronger RNA chaperone activity in vitro. We therefore propose that in addition to its role as a molecular back-up of H-NS, StpA's superior effect on RNA may be exploited under some specific cellular conditions to promote differential gene expression.

Bacterial Outer Membrane Proteins↗
PubMed1996

Intron mobility in phage T4 occurs in the context of recombination-dependent DNA replication by way of multiple pathways.

Numerous group I introns in both prokaryotes and eukaryotes behave as mobile genetic elements. The functional requirements for intron mobility were determined in the T4 phage system using an in vivo assay to measure intron homing with wild-type and mutant derivatives. Thus, it was demonstrated that intron mobility occurs in the context of phage recombination-dependent replication, a pathway that uses overlapping subsets of replication and recombination functions. The functional requirements for intron homing and the nature of recombinant products are only partially consistent with the accepted double-strand-break repair (DSBR) model for intron inheritance, and implicate additional homing pathways. Whereas ambiguities in resolvase requirements and underrepresentation of crossover recombination products are difficult to rationalize strictly by DSBR, these properties are most readily consistent with a synthesis-dependent strand annealing (SDSA) pathway. These pathways share common features in the strand invasion steps, but differ in subsequent repair synthesis and resolution steps, influencing the genetic consequences of the intron transfer event.

Bacteriophage T4↗
PubMed1996

Intron-encoded endonuclease I-TevII binds across the minor groove and induces two distinct conformational changes in its DNA substrate.

I-TevII is the homing endonuclease encoded by the sunY intron of bacteriophage T4. The enzyme cleaves an intronless sunY gene near the exon I-exon II junction, thereby initiating intron homing into its cognate intronless allele. Specifically, I-TevII cleaves its DNA target 13 to 15 nucleotides (nt) downstream of the sunY intron insertion site, generating 2-nl 3'-OH extensions. Here, we present evidence that I-TevII makes predominantly minor groove contacts in two regions of its recognition sequence, as does I-TevI, the other homing endonuclease encoded by phage T4. Following cleavage, I-TevII was shown to remain bound to one of its DNA products, suggesting possible additional roles for the endonuclease in the mobility process. Interestingly, two distinct conformational changes were detected by gel analysis in the DNA substrate following binding by I-TevII, one occurring in the absence of Mg2+, the second being dependent on the presence of Mg2+. The Mg(2+)-induced distortion accompanies a nick in one strand, and may serve to bring the cleavage site on the other strand into proximity with the catalytic domain of the protein.

Bacteriophage T4↗
PubMed1996

Homology requirements for double-strand break-mediated recombination in a phage lambda-td intron model system.

Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage lambda model system was developed to analyze exon homology effects on intron homing and determine the role of the lambda 5'-3' exonuclease complex (Red alpha beta) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type lambda, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.

Bacteriophage lambda↗
PubMed1995

Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.

I-TevI, the intron-encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence-tolerant fashion. We demonstrate here that the 28 kDa I-TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate. In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I-TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site. Formation of the bent I-TevI-DNA complex is proposed to promote top-strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base-substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I-TevI-induced conformational change. These results, combined with genetic, physical and computer-modeling studies, form the basis of a model, wherein I-TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top-strand cleavage site. The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine-containing DNA, as exists in the T-even phages.

Base Sequence↗
PubMed1995

Selection of a remote cleavage site by I-tevI, the td intron-encoded endonuclease.

I-TevI, a double-strand DNA endonuclease involved in the mobility of the td intron of phage T4, is highly unusual in that it binds and cleaves intronless td alleles (td homing sites) in a site-specific but sequence-tolerant manner. The endonuclease binds to sequences flanking the intron insertion site and near the remote cleavage site, located 23 and 25 nucleotides away on the top and bottom strands, respectively. Mapping studies indicate that I-TevI has both sequence and distance sensors that function during cut-site selection. Although I-TevI cleavage of many insertion and deletion variants of the homing site is impaired, double-strand breaks are generated at positions that collectively span two turns of the helix, indicating that the interaction is extraordinarily flexible. However, the endonuclease does exhibit spacing preferences between its binding domains, and sequence preferences near the cleavage site, with the G:C pair at -23 implicated as a cleavage determinant. Furthermore, I-TevI appears to function through interactions across the minor groove at the cleavage site, as it does at the intron insertion site, and to be capable of cleaving sequentially, first on the bottom and then on the top strand. These properties of I-TevI are incorporated in a model wherein the endonuclease effects distant cleavage via a flexible hinge.

Bacteriophage T4↗