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A Rothman

Publications and source records attributed to A Rothman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Probing the conformation of NhaA, a Na+/H+ antiporter from Escherichia coli, with trypsin.

One of the most striking features of NhaA, an Escherichia coli Na+/H+ antiporter, is its extreme sensitivity to pH. The activity of NhaA increases 2000-fold between pH 6.5 and 8.5. In this work, we investigated whether the activation of NhaA by pH is accompanied by conformational changes which can be detected using trypsin as a probe. We have found that NhaA is susceptible to proteolytic digestion at the pH range where it is activated, suggesting that these two events may be related; at alkaline pH, the protein becomes active and adopts an "open" conformational state in which more domains are exposed to the enzyme. This idea was further supported by results from two mutants of NhaA in which His-225, a residue involved in pH sensing, has been replaced by either Arg or Asp. The mutant H225R is activated at more acidic pH values, while H225D at more alkaline pH. In accordance with the results described for the wild-type protein, H225R was susceptible to digestion by trypsin at the pH at which it undergoes main activation. NhaA has many potential tryptic cleavage sites. However, analysis of the tryptic digestion fragments suggests that at alkaline pH, the protein is exposed to cleavage mainly at hydrophilic loops 6, 7, and 8. Thus, upon activation, NhaA appears to undergo a change in conformation that is reflected in specific regions of the protein.

Amino Acid Sequence↗
PubMed1997

Familial occurrence of pulmonary atresia with intact ventricular septum.

Pulmonary atresia with intact ventricular septum (PA/IVS) is a rare disease, accounting for less than 3% of all congenital heart lesions. The cause of PA/IVS is unknown. We report the occurrence of two first cousins with PA/IVS, suggestive of autosomal dominant inheritance with incomplete penetrance. The study of such families should ultimately lead to the identification of the gene(s) that cause congenital heart disease.

Adult↗
PubMed1997

Histidine 225, a residue of the NhaA-Na+/H+ antiporter of Escherichia coli is exposed and faces the cell exterior.

Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli. The functional C-less NhaA has provided the groundwork to study further histidine 225 of NhaA which has previously been suggested to play an important role in the activation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schuldiner, S. and Padan, E. (1995) J. Biol. Chem. 270, 26813-26817). C-less H225C was constructed and shown to possess an antiporter activity 60% of that of C-less antiporter and a pH profile similar to that of both the C-less or wild-type antiporters. Remarkably, whereas neither the wild-type nor the C-less antiporters were affected by N-ethylmaleimide, C-less H225C was inhibited by this reagent. To determine the degree of alkylation of the antiporter protein by N-ethylmaleimide, antiporter derivatives tagged at their C termini with six histidines residues were constructed. Alkylation of C-less H225C was measured by labeling of everted membrane vesicles with [14C]N-ethylmaleimide, affinity purification of the His-tagged antiporter, and determination of the radioactivity of the purified protein. This assay showed that H225C is alkylated to a much higher level than any of the native cysteinyl residues of NhaA reaching saturation at alkyl/NhaA stoichiometry of 1. The wild-type derivative showed at least 10-fold less alkylation even at higher concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residues of NhaA. Since H225C residues both in right-side out and inside-out membrane vesicles were quantitatively alkylated by N-ethylmaleimide, this assay was used to determine the accessibility of H225C to other SH reagents by titrating the H225C left free to react with N-ethylmaleimide, following exposure of the membranes to the reagents. Furthermore, since membrane-impermeant probes can react with residues in membrane-embedded protein only if accessible to the medium containing the reagent, the assay was used to determine the membrane topology of H225C. As expected for a membrane-permeant probe, p-chloromercuribenzoate reacted with H225C as efficiently as N-ethylmaleimide in both membrane orientations. Similar results were obtained with methanethiosulfonate ethylammonium supporting the recent observations that this probe is membrane-permeant. On the other hand, both membrane-impermeant reagents p-chloromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl ammonium bromide reacted with H225C 10-fold more in right-side out than in inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells. These results strongly suggest that H225C is exposed at the periplasmic face of the membrane.

Alkylation↗
PubMed1997

Balloon angioplasty of native aortic coarctation in infants 3 months of age and younger.

The use of balloon dilation to treat native aortic coarctation is controversial, particularly in infants. Between January 1991 and September 1996, 12 patients < or = 3 months of age with native coarctation of the aorta (CoA) underwent balloon angioplasty (BA). All 12 lesions were dilated successfully with a mean reduction in peak systolic gradient from 49.3 +/- 16.5 mm Hg to 6.8 +/- 4.0 mm Hg (p < 0.001) and a mean increase in minimum CoA diameter from 2.4 +/- 0.6 mm to 5.5 +/- 1.3 mm (p < 0.001). Intimal flaps or tears were detected immediately after BA in 4 (33%) of 12 patients by angiography and in 8 (89%) of 9 patients by intravascular ultrasonography. No deaths or major complications related to the BA occurred. One patient had documented asymptomatic femoral artery obstruction, and one patient with hydrops fetalis and congenital pleural effusions died with gram-negative sepsis 1 week after the procedure. Follow-up was available for 10 patients (1 was lost to follow-up) between 2 months and 4.1 years (mean 2.4 +/- 1.3 years) after BA. No patient had an aortic aneurysm. Restenosis occurred in 5 (50%) of 10 patients, requiring reintervention a mean of 2.6 +/- 2.1 months after BA. One patient underwent surgical repair. Repeat BAs were performed in the other four patients; three were successful, and one with partial gradient relief required surgical repair. Five patients have not required reintervention a mean of 2.9 +/- 1.0 years after the initial BA. Among these five patients, follow-up intravascular ultrasound performed in three patients a mean of 2.0 +/- 1.9 years after BA showed favorable endovascular remodeling. There was a tendency for early reintervention in patients < 1 month of age and coexistence of a patent ductus arteriosus at the time of BA. In conclusion, selected infants < or = 3 months of age with discrete native CoA may be treated initially with balloon dilation. Most patients who have restenosis respond successfully to repeat BA.

Age Factors↗
PubMed1997

Percutaneous coil occlusion of patent ductus arteriosus.

OBJECTIVE: To determine the success rate and safety of percutaneous patient ductus arteriosus (PDA) coll occlusion. DESIGN: Thirty consecutive pediatric patients with small to moderate-size PDAs (minimum diameter < or = 4 mm) underwent percutaneous coll occlusion. The results were assessed by angiography and echocardiography. The mean age was 5.1 +/- 4.2 years (range, 0.8 to 18.8 years); mean weight was 19.2 +/- 10.3 kg (range, 8.1 to 40.0 kg). The mean minimum diameter of the PDA was 1.8 +/- 0.8 mm (range, 1.0 to 4.0 mm). RESULTS: PDA occlusion was achieved with one coil in 24 patients, 2 coils in 3 patients and 3 coils in 3 patients. The mean coil/PDA diameter ratio was 2.5 +/- 0.5. Immediately after coil occlusion, 29 PDAs had no flow by anglography; one had a small residual shunt. There were no significant complications. In the first 24 hours after coil implantation, echocardiography showed complete occlusion in 28 patients, a small left-to-right shunt in the same patient that had a residual shunt by anglography, and a trace shunt in one additional patient. In the two patients with residual flow by echocardiography, follow-up ultrasonography revealed no residual shunt 1 and 3 months later. At a mean follow-up of 11.8 +/- 9.3 months (range, 0 to 36.0 months), there was no PDA flow by color Doppler echocardiography in any of the 30 patients. CONCLUSION: Coil occlusion is a safe and effective method of percutaneous closure of small to moderate-size PDAs. The largest PDA that can be closed with this technique remains to be determined.

Child, Preschool↗
PubMed1997

Physical mapping of the 5 Mb D9S196-D9S180 interval harboring the basal cell nevus syndrome gene and localization of six genes in this region.

The basal cell nevus syndrome (Gorlin syndrome) is characterized by multiple basal cell carcinomas and diverse developmental defects. The gene responsible for this syndrome has been mapped previously to a 2 cM interval between D9S196 and D9S 180 at 9q22.3, and very recently mutations of a candidate gene in this region--the human homolog of the Drosophila patched gene have been identified. We report here on physical mapping studies integrating a contig of yeast artificial chromosomes and bacterial artificial chromosomes with a long-range map spanning approximately 5 Mb between the recombination-determined flanking markers. Six genes have been mapped to this interval.

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

Topological analysis of NhaA, a Na+/H+ antiporter from Escherichia coli.

Analysis of the hydropathic profile of the amino acid sequence of NhaA, a Na+/H+ antiporter from Escherichia coli has previously suggested the existence of 11 putative transmembrane segments (Taglicht, D., Padan, E., and Schuldiner, S. (1991) J. Biol. Chem. 266, 11289-11294). In the present work to test the location of the C terminus, right-side-out and inside-out membrane vesicles were digested with carboxypeptidase B and probed with an antibody raised against a synthetic peptide whose sequence was based on the C terminus sequence. The results demonstrate that the C terminus is facing the cell interior because it is available for digestion only from the inside. Previous evidence from an NhaA-beta-galactosidase fusion to loop 5 of NhaA indicated that this loop is also facing the cytoplasm (Karpel, R., Alon, T., Glaser, G., Schuldiner, S., and Padan, E. (1991) J. Biol. Chem. 266, 21753-21759) and therefore was not consistent with the position of the C terminus in an 11-transmembrane segment model. Therefore, the model was re-evaluated. For this purpose, 10 nhaA'-'phoA gene fusions were constructed and assayed for alkaline phosphatase activity. The results support a 12-transmembrane segment model with the N and C termini located in the cytoplasm. The evidence indicates that two very short segments, 14 and 16 amino acids long, must cross the membrane in an unknown conformation.

Alkaline Phosphatase↗
PubMed1996

Comparison of results of stent implantation in small (<20 kg) children versus larger children with pulmonary artery stenoses.

We compared the results of stent implantation for pulmonary artery stenoses in patients weighing <20 kg (group 1, 17 patients, 21 stents) versus those weighing > or = 20 kg (group 2, 11 patients, 13 stents). There was no significant difference in the mean percent increase in diameter or mean percent gradient reduction acutely and at short-term follow-up between group 1 and 2 patients.

Adolescent↗
PubMed1996

Successful balloon dilation of an abdominal coarctation of the aorta in patient with presumed Takayasu's aortitis.

An 11-year-old male with a severe abdominal aortic coarctation, presumably secondary to aortitis, underwent successful percutaneous balloon dilation that resulted in near-complete relief of the obstruction. Intravascular ultrasound imaging showed a major endovascular tear immediately following dilation and satisfactorily excluded significant branch (superior mesenteric) artery compromise. Arterial remodeling was demonstrated with persistence of the gradient relief over a 12-month follow-up period.

Aorta, Abdominal↗
PubMed1996

Adenovirus-mediated increase of exogenous and inhibition of endogenous fosB gene expression in cultured pulmonary arterial smooth muscle cells.

Modification of gene expression in pulmonary arterial smooth muscle cells (PASMCs) could be a valuable tool for investigating the role of specific gene products in normal and pathological PASMC growth, and a novel potential therapy for pulmonary vascular diseases. To examine the direct role of fosB protein in PASMC growth, adenovirus (Ad) vectors were used to transfer sense or antisense full-length fosB cDNAs to cultured PASMCs to modify fosB expression, and investigate the effects of this modification on PASMC growth. The full-length fosB cDNA under the control of the cytomegalovirus (CMV) early gene promoter was constructed into an E1 region-deleted, replication-deficient human type 5 Ad vector in either sense or antisense orientation. Forty-eight hours after infection with the sense construct (Ad.S. fosB) at 3 plaque-forming units per cell (pfu/cell). PASMCs expressed abundant fosB mRNA and fosB protein. FosB protein was detected by immunohistochemistry in approximately 95% of the infected cells. PASMCs infected with Ad.S.fosB at ratios of 0.1, 0.2, 0.5, 1.3, and 10 pfu/cell showed a dose-dependent increase in fosB mRNA expression, with half-maximal and maximal expression at 1 and 10 pfu/cell, respectively. The increase in fosB mRNA expression was detected as early as 8 h and persisted for 25 days after infection. Forty-eight hours after infection with the antisense construct (Ad.A.fosB) at 3 pfu/cell, very low levels of fosB mRNA were detected by Northern blotting, in which the double-stranded fosB cDNA was labeled and used as a hybridization probe. FosB protein was undetectable by Western blotting or immunocytochemical analyses in the Ad.A.fosB infected cells. Cytopathical effects were observed when PASMCs were infected with either Ad.S.fosB or Ad.A.fosB at ratios of 10 pfu/cell or higher. Infection of serum-deprived PASMCs with Ad.S.fosB or Ad.A.fosB alone at 3 pfu/cell did not affect cellular growth. These results show that adenoviral vectors containing sense or antisense fosB cDNA expression units can be used to effectively modify fosB gene expression. Although changes in fos-B gene expression did not affect cellular growth, this model system offers a very effective method for elucidating the biological roles of other gene products and studying the pathways of PASMC gene regulation and signal transduction.

Actins↗
PubMed1996

Transvenous procurement of pulmonary artery smooth muscle and endothelial cells using a novel endoarterial biopsy catheter in a canine model.

OBJECTIVES: The aim of this study was to evaluate the performance of a new arterial biopsy catheter in obtaining pulmonary endovascular samples in a canine model. BACKGROUND: Percutaneous endomyocardial biopsy is a widely used and valuable procedure in the management of posttransplant rejection and selected cardiomyopathies. A similar method of obtaining endoarterial biopsy samples would aid in the study, diagnosis and management of arterial diseases. METHODS: Catheterization was performed in 19 dogs, each weighing 20 to 30 kg, through an 8F sheath in the external jugular vein to obtain pulmonary endoarterial samples. The catheter consists of two sliding tubes: an inner one with a beveled opening that accommodates endoarterial tissue by means of a vacuum and an outer tube with a sharp distal edge that cuts the tissue when activated. RESULTS: Overall, a total of 266 separate biopsy attempts were performed, and 161 tissue samples were obtained (success rate 61%). With modifications in technique in the last nine dogs, 54 (93%) of 58 attempts were successful. There were no deaths, extravasation of contrast material on angiography or thrombi. Of 20 vessels with prebiopsy and postbiopsy angiograms, 1 developed transient spasm (5%). On microscopic examination of cross sections of 50 separate pulmonary endoarterial biopsy samples, all had smooth muscle cells and 30 contained endothelial cells (60%). The arteries of origin showed small intimal and medial tears and mild perivascular hemorrhage. Angiographic and pathologic examination of previously biopsied arterial segments 2 weeks (two dogs) and 8 weeks (two dogs) after the procedure showed patent vessels and no thrombi. Histologically, the biopsy sites revealed mild neointimal and medial proliferation. CONCLUSIONS: This new endoarterial biopsy catheter is safe and effective in obtaining pulmonary artery samples in normotensive dogs.

Angiography↗
PubMed1996

High incidence of cranial ultrasound abnormalities in full-term infants with congenital heart disease.

The objective of this retrospective study was to determine the incidence and types of cranial ultrasound abnormalities in full-term infants with congenital heart disease (CHD). We reviewed the cranial ultrasound scans of 49 full-term infants with CHD and compared them to 42 healthy full-term control infants. The relationship of each abnormality with the type of CHD, the presence of cyanosis, and cardiac catheterization and cardiac surgery were examined. We found that infants with CHD had a higher incidence of cranial ultrasound abnormalities than control infants (59% versus 14%; p < 0.001). Cerebral atrophy and linear echodensities in the basal ganglia and thalamus were the most common sonographic findings in infants with CHD, particularly in those with coarctation of the aorta or ventricular septal defect. Intraventricular hemorrhage occurred more often in infants with acryanotic CHD than in those with cyanotic CHD. Cardiac catheterization and cardiac surgery had no significant effects on cranial ultrasound findings. We conclude that cranial ultrasound abnormalities are very frequent in full-term infants with CHD. These findings emphasize the importance of cranial ultrasonography and long-term neurodevelopmental follow-up of infants with CHD.

Aortic Coarctation↗
PubMed1996

Who should rate candidates in an objective structured clinical examination?

PURPOSE: To determine who is the better rater of history taking in an objective structured clinical examination (OSCE): a physician or a standardized patient (SP). METHOD: During the 1991 pilot administration of an OSCE for the Medical Council of Canada's qualifying examination, five history-taking stations were videotaped. Candidates at these stations were scored by three raters: a physician (MD), an SP observer (SPO), and an SP rating from recall (SPR). To determine the validity of each rater's scores, these scores were compared with a "gold standard", which was the average of videotape ratings by three physicians, each scoring independently. Analysis included both correlations with the standard and a repeated-measures analysis of variance (ANOVA) comparing raters' mean scores on each station with mean scores of the gold standard. RESULTS: Ninety-one videotapes were scored by the "gold-standard" physicians. Correlations with the standard showed no clear preference for MD, SPO, or SPR raters. ANOVAs revealed significant differences from the standard on three stations for the SPR, two stations for the SPO, and one stations for the MD. CONCLUSIONS: An MD rater is less likely to differ from a standard established by a consensus of MD ratings than are SP raters rating from recall. If an MD cannot be used, an SP observer is preferable to an SP rating from recall.

Analysis of Variance↗
PubMed1996

Inhibitory effect of ethanol on the Na(+)-ATPase activity of rat kidney proximal tubular cell plasma membranes.

The inhibitory effect of 2% ethanol (400 mM) in the incubation medium on several characteristics of the Na(+)-ATPase of basolateral plasma membranes from rat kidney proximal tubular cells was investigated. Ethanol did not change the Km of the enzyme for Mg2+, ATP or Na+; it did not change either the optimal pH or temperature values of the incubation medium for the enzyme to act and finally, it did not affect the apparent energy of activation of the enzyme. It was also found that 2% ethanol produced stronger inhibition of the ATPase when it is in an activated or stimulated state, than when it is working at its lower basal level. The presented results can be explained by assuming that 2% ethanol in the incubation medium inhibits Na(+)-ATPase activity by affecting the enzyme structure as well as its activating mechanism.

Adenosine Triphosphatases↗