Biomedical subjects
Publications and source records attributed to A Rothman.
Two patients with pulmonary atresia and intact ventricular septum each underwent early palliative surgery with a pulmonary valvotomy and an ascending aorta to pulmonary artery shunt. Adequate right ventricular growth and relief of pulmonary stenosis rendered the shunts unnecessary. The shunts were successfully occluded percutaneously with Gianturco coils.
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Mutations in PATCHED (PTC), the human homolog of the Drosophila patched gene, have been identified in most exons of the gene in patients with the basal cell nevus syndrome and in sporadic basal cell carcinomas. We have screened the 23 PTC exons for mutations using single strand conformation polymorphism analysis of DNA from 86 basal cell nevus syndrome probands, 26 sporadic basal cell carcinomas, and seven basal cell nevus syndrome-associated basal cell carcinomas. This screen identified mutations located in eight exons in 13 of the basal cell nevus syndrome patients and in three of the tumors. The most common mutations were frameshifts resulting in premature chain termination. These results provide further evidence for the crucial role of PTC as a tumor suppressor in human keratinocytes.
The optimal interventional management of coarctation of the aorta (CoA) remains controversial. Although some are strong advocates for surgery, the timing of operation in elective cases has been a matter of debate. Among those who advocate balloon angioplasty, some recommend dilation only of recurrent CoA. Others recommend dilation of native CoA, but only beyond a certain age. Finally, some are strong proponents of balloon angioplasty, even in the neonatal period, recognizing the need for reintervention (balloon dilation or surgery) in a significant proportion of patients. Recent reports suggest similar or slightly superior acute results of balloon dilation for native compared with recurrent CoA. Balloon-expandable stents are being used increasingly in complex CoAs and in failed dilations. A large multicenter study is required to determine the respective clinical roles of surgery and balloon dilation in the management of CoA.
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STUDY OBJECTIVES: The aims of this study were: to evaluate the performance of a novel arterial biopsy catheter in obtaining pulmonary endovascular samples in hypertensive dogs; to compare the results of pulmonary endoarterial biopsy in hypertensive vs normotensive dogs; and to assess the histologic changes in the hypertensive model. DESIGN AND INTERVENTIONS: Thirty-four dogs (27 with normal pulmonary arterial pressures and seven with pulmonary hypertension) were catheterized through an external jugular vein to obtain endovascular biopsy samples from distal pulmonary arteries 2 to 3 mm in luminal diameter. To induce pulmonary hypertension, seven dogs were given repeated infusions of 0.6- to 0.9-mm ceramic microspheres into the superior vena cava. Endoarterial samples were obtained at pulmonary systolic arterial pressures ranging from 10 to 110 mm Hg. MEASUREMENTS AND RESULTS: Sixty-two biopsy catheterization procedures were performed in the 34 dogs. After 12 initial procedures of technique refinement, endoarterial samples were obtained in each of the last 50 procedures (21 in normotensive dogs and 29 in hypertensive dogs). The average number of endovascular biopsy samples retrieved was 7.1 (range, 2 to 12) from a mean of 8.6 (range, 2 to 15) biopsy attempts per catheterization (success rate=83%). The average biopsy piece measured 1.13 mm in length, 0.33 mm in depth, and up to 1.0 mm in width. The biopsy success rates and endoarterial sample sizes were similar in normotensive and hypertensive dogs. Smooth muscle cells and endothelial cells were grown from the biopsy samples. There were no significant procedural complications, except for one self-limited hemorrhage. Histologically, samples obtained from dogs with pulmonary hypertension showed characteristic changes when compared with biopsies from normotensive dogs. CONCLUSION: This new endoarterial biopsy catheter was safe and effective when used to obtain pulmonary endoarterial samples in dogs with normal and experimentally elevated pulmonary arterial pressures. The quality and quantity of the biopsy samples allowed identification of pathologic changes.
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.
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.
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.
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.
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.
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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.
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.
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.
The presence of an accessory hepatic vein to the pulmonary venous atrium should be considered in the setting of progressive cyanosis following the Fontan procedure. An inferior vena caval angiogram with "levo-phase" should demonstrate it. Surgical intervention or transcatheter occlusion should lead to prompt resolution of cyanosis.
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.