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Biomedical subjects

M Belfort

Publications and source records attributed to M Belfort.

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

Successful urokinase treatment of massive pulmonary embolism in pregnancy.

BACKGROUND: Thrombolytic agents have been used successfully to treat patients with massive pulmonary embolism and cardiorespiratory insufficiency, but experience with these drugs in pregnancy is limited. CASE: A 20-year-old woman at 21 weeks' gestation was admitted with a massive pulmonary embolism. She was initially given intravenous heparin therapy but because of worsening clinical condition, urokinase was used. After two 12-hour periods of therapy, the urokinase was discontinued and the heparin restarted. She remained on subcutaneous heparin therapy for the remainder of her pregnancy, which was otherwise uncomplicated. She delivered a healthy male infant at term without complications and was discharged on warfarin therapy. CONCLUSION: Thrombolytic therapy can be life-saving and should be considered in the treatment of hemodynamically significant pulmonary embolism in pregnancy.

Adult↗
PubMed1995

Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro.

An Escherichia coli gene, stpA, has been identified and cloned based on its ability to suppress the Td- phenotype of a resident, splicing-defective phage T4 td (thymidylate synthase) gene. The stpA gene, which was localized to 60.24 min on the E. coli chromosome, encodes a 15.3-kDa protein. Overproduction of StpA in vivo led to an increase in td pre-mRNA levels and modest enhancement of td mRNA:pre-mRNA ratios. Consistent with its in vivo effect, purified StpA promoted RNA splicing in vitro, and facilitated RNA annealing and strand exchange with model substrates. These results suggest that StpA promotes splicing of the intron by binding RNA nonspecifically, resolving misfolded precursor molecules and facilitating association of critical base pair elements. Furthermore, proteinase K treatment of StpA-assembled precursors prior to the initiation of the splicing reaction still resulted in splicing enhancement, indicating that StpA is not required for the catalytic step, unlike the Neurospora splicing effector CYT-18, whose presence was necessary for catalysis to proceed. Together these results suggest that StpA has chaperone activity in vitro, with the property of promoting assembly of the precursors into an active conformation, in contrast to splicing effectors that stabilize the catalytically active intron structure.

Amino Acid Sequence↗
PubMed1994

Evolution of mobile group I introns: recognition of intron sequences by an intron-encoded endonuclease.

Mobile group I introns are hypothesized to have arisen after invasion by endonuclease-encoding open reading frames (ORFs), which mediate their mobility. Consistent with an endonuclease-ORF invasion event, we report similarity between exon junction sequences (the recognition site for the mobility endonuclease) and intron sequences flanking the endonuclease ORF in the sunY gene of phage T4. Furthermore, we have demonstrated the ability of the intron-encoded endonuclease to recognize and cleave these intron sequences when present in fused form in synthetic constructs. These observations and accompanying splicing data are consistent with models in which the invading endonuclease ORF is provided safe haven within a splicing element. In turn the intron is afforded immunity to the endonuclease product, which imparts mobility to the intron.

Bacteriophage T4↗
PubMed1994

Purification and characterization of two forms of I-DmoI, a thermophilic site-specific endonuclease encoded by an archaeal intron.

The archaeal intron in the 23 S rRNA gene of the hyperthermophile Desulfurococcus mobilis has previously been shown to encode a site-specific DNA endonuclease that contains the LAGLIDADG motif. The enzyme, I-DmoI, has been shown to be active in two forms when expressed in vitro, from RNAs representing either the linear (I-DmoIl) or circular (I-DmoIc) intron. In this study we have overexpressed I-DmoIl and I-DmoIc and purified the enzymes from Escherichia coli. The optimal conditions for the enzymatic activity in vitro were determined, and the enzyme was used to delimit the recognition boundary on its DNA substrate (14-20 nucleotides), an intronless 23 S rRNA gene. Despite belonging to the archaeal kingdom, and being the product of a hyperthermophile, I-DmoI shares many properties with LAGLIDADG intron and intein endonucleases in other kingdoms. These results support the view that these phylogenetically diverse enzymes, which function to mobilize the DNA sequences that encode them, share a common ancestry.

Amino Acid Sequence↗
PubMed1994

Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.

To address the effect of host proteins on the self-splicing properties of the group I introns of bacteriophage T4, we have purified an activity from Escherichia coli extracts that facilitates both trans- and cis-splicing of the T4 introns in vitro. The activity is attributable to a number of proteins, several of which are ribosomal proteins. Although these proteins have variable abilities to stimulate splicing, ribosomal protein S12 is the most effective. The activity mitigates the negative effects on splicing of the large internal open reading frames (ORFs) common to the T4 introns. In contrast to proteins shown previously to facilitate group I splicing, S12 does not bind strongly or specifically to the intron. Rather, S12 binds RNA with broad specificity and can also facilitate the action of a hammerhead ribozyme. Addition of S12 to unreactive trans-splicing precursors promoted splicing, suggesting that S12 can resolve misfolded RNAs. Furthermore, incubation with S12 followed by its proteolytic removal prior to the initiation of the splicing reaction still resulted in splicing enhancement. These results suggest that this protein facilitates splicing by acting as an RNA chaperone, promoting the assembly of the catalytically active tertiary structure of ribozymes.

Amino Acid Sequence↗
PubMed1994

The effect of acute volume expansion and vasodilatation with verapamil on uterine and umbilical artery Doppler indices in severe preeclampsia.

UNLABELLED: Preeclampsia is associated with increased peripheral, uterine, and umbilical artery resistance. Acute blood pressure reduction may result in shunting of blood and sudden fetal distress. We therefore investigated the effects of volume expansion and verapamil therapy on uteroplacental and umbilical resistance during treatment of preeclampsia. MATERIALS AND METHODS: Five severe preeclamptics underwent volume expansion and subsequent vasodilatation with an infusion of verapamil. Invasive hemodynamic monitoring and Doppler ultrasonography were used to study changes in maternal, uterine, and umbilical hemodynamics. RESULTS: Volume expansion and subsequent verapamil therapy was associated with significant changes in maternal hemodynamics without significant change in uteroplacental or umbilical resistance. Uterine artery waveform changes were noted, with disappearance of notching in some cases. CONCLUSIONS: Volume expansion and verapamil therapy effectively reduces maternal blood pressure in preeclampsia, without adversely affecting uteroplacental or umbilical artery resistance. Uterine artery waveform changes may be associated with improved fetal outcome.

Adult↗
PubMed1993

A site-specific endonuclease encoded by a typical archaeal intron.

The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA. However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions. Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron endonucleases of eukaryotes. These observations support the independent evolutionary origin of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes.

Amino Acid Sequence↗
PubMed1993

The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.

I-TevI, a double-strand DNA endonuclease encoded by the mobile td intron of phage T4, has specificity for the intronless td allele. Genetic and physical studies indicate that the enzyme makes extensive contacts with its DNA substrate over at least three helical turns and around the circumference of the helix. Remarkably, no single nucleotide within a 48 bp region encompassing this interaction domain is essential for cleavage. Although two subdomains (DI and DII) contain preferred sequences, a third domain (DIII), a primary region of contact with the enzyme, displays much lower sequence preference. While DII and DIII suffice for recognition and binding of I-TevI, all three domains are important for formation of a cleavage-competent complex. Mutational, footprinting and interference studies indicate predominant interactions of I-TevI across the minor groove and phosphate backbone of the DNA. Contacts appear not to be at the single nucleotide level; rather, redundant interactions and/or structural recognition are implied. These unusual properties provide a basis for understanding how I-TevI recognizes T-even phage DNA, which is heavily modified in the major groove. These recognition characteristics may increase the range of natural substrates available to the endonuclease, thereby extending the invasive potential of the mobile intron.

Base Sequence↗
PubMed1993

Group I and group II introns.

Group I and group II introns are two types of RNA enzymes, ribozymes, that catalyze their own splicing by different mechanisms. In this review, we summarize current information about the structures of group I and group II introns, their RNA-catalyzed reactions, the facilitation of RNA-catalyzed splicing by protein factors, and the ability of the introns to function as mobile elements. The RNA-based enzymatic reactions and intron mobility provide a framework for considering the role of primordial catalytic RNAs in evolution and the origin of introns in higher organisms.

Amino Acid Sequence↗
PubMed1993

The cardiovascular and intracranial effects of laryngoscopy and endotracheal intubation in hypercarbic neonatal piglets.

Laryngoscopy and endotracheal intubation is a potent sympathetic stimulus in adults. Neonates are frequently intubated, but few data exist on the cerebral effects of this intervention. The cardiovascular and intracranial effects of laryngoscopy and endotracheal intubation were studied in 17 hypercarbic neonatal piglets. The mean arterial pressure in the study group (11 piglets) increased significantly within 2 minutes of the stimulus, and remained elevated for almost 14 minutes. The intubated animals showed significantly more haemorrhage in the basal area of the brain than the 6 control animals. The distribution suggests bleeding in the choroid plexus of the 4th ventricle. The significance of such bleeds is not immediately apparent, since none of the animals was grossly neurologically affected by the intervention. However, subtle long-term neurological deficits cannot be excluded and this aspect requires further study. Laryngoscopy and endotracheal intubation may cause non-lethal haemorrhage in the choroid plexus and central canal of the hindbrain in hypercarbic, neonatal piglets.

Animals↗
PubMed1992

The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core.

The Neurospora CYT-18 protein, a tyrosyl-tRNA synthetase, which functions in splicing group I introns in mitochondria, promotes splicing of mutants of the distantly related bacteriophage T4 td intron. In an in vivo assay, wild-type CYT-18 protein expressed in E. coli suppressed mutations in the td intron's catalytic core. CYT-18-suppressible mutations were also suppressed by high Mg2+ or spermidine in vitro, suggesting they affect intron structure. Both the N- and C-terminal domains of CYT-18 are required for efficient splicing, but CYT-18 with a large C-terminal truncation retains some activity. Our results indicate that CYT-18 interacts with conserved structural features of group I introns, and they provide direct evidence that a protein promotes splicing by stabilizing the catalytically active structure of the intron RNA.

Base Sequence↗