Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80 degrees C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to approximately 150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.

The human p53 negative regulatory domain mediates inhibition of reporter gene transactivation in yeast lacking thioredoxin reductase.

Stimulation of target gene transcription by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. LexA/p53 fusion proteins were used to study the basis for thioredoxin reductase dependence. A fusion protein containing all 393 of the residues of p53 efficiently and specifically stimulated transcription of a LexOP-LacZ reporter gene in wild-type yeast but was several-fold less effective in delta trr1 yeast lacking the thioredoxin reductase gene. Thus, even when p53 was tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene transactivation remained dependent on thioredoxin reductase. A fusion protein containing only the activation domain of p53 stimulated reporter gene transcription equally in wild-type and delta trr1 cells, suggesting that p53 residues downstream from the activation domain created the requirement for thioredoxin reductase. Experiments using additional LexA/p53 truncation mutations indicated that the p53 negative regulatory domain, rather than the DNA-binding or oligomerization domains, created the requirement for thioredoxin reductase. The fusion protein results suggested that, under oxidative conditions, the negative regulatory domain inhibited the ability of DNA-bound p53 to stimulate transcription. However, deletion of the negative regulatory domain did not alleviate the requirement of non-LexA-containing p53 for thioredoxin reductase. The results, thus, suggest that oxidative conditions inhibit both DNA binding and transactivation by p53, and that inhibition of the latter requires the negative regulatory domain.

Cleavage of type 2 adenovirus DNA by HaeIII endonuclease. I. Catalog of HaeIII fragments.

Tye 2 adenovirus DNA was divided into 14 fragments by sequential use of BamI, HsuI, SmaI, anc EcoRI endonuclease. Each fragment was purified by gel electrophoresis and subsequently cleaved with HaeIII endonuclease. From the number of fragments produced, we could calculate the number of HaeIII cleavage sites: there are a total of 187 sites. HaeIII sites were not randomly distributed along the adenovirus chromosome. Most sites were clustered in the G + C-rich left half of the chromosome. The sum of the molecular weights of the HaeIII fragments is 22.4 . 10(6), within 2 % of the molecular weight of adenovirus DNA (22.9 . 10(6).

Cleavage of type 2 adenovirus DNA BY HaeIII endonuclease. II. Map of HaeIII sites in EcoRI fragments C and E.

Fourteen HaeIII cleavage sites have been located in EcoRI fragments C and E which together constitute the rightmost 16.6% of the type 2 adenovirus chromosome. In addition, nine sites recognized by SmaI, BalI, HsuI, HpaI, and HpaII endonucleases have also been mapped in this region.

Symmetrical adenovirus minichromosomes have hairpin replication intermediates.

A special class of panhandles (hairpin or foldback structures) arising from the replication of symmetrical adenovirus (Ad) minichromosome dimers and oligomers have been identified by two-dimensional gel electrophoresis. Hairpins provide evidence for replicative intermediates in the pathway for Ad complementary-strand synthesis. Furthermore, larger inverted sequences give Ad minichromosomes a replicative advantage.

In vitro replication directed by a cloned adenovirus origin.

A 5.7-kb recombinant plasmid, called XD-7, contains the terminal XbaI-E fragment from the left end of type 2 adenovirus cloned into the EcoRI site of pBR322. An average of 9% +/- 1% of input supercoiled, protein-free XD-7 DNA replicated as rolling circles with single-stranded tails ranging up to unit length and longer in reaction mixtures containing nuclear and cytoplasmic extracts from adenovirus-infected, but not uninfected, HeLa cells. The adenovirus origin was mapped on XD-7 by electron microscopy at the left boundary of the cloned adenovirus segment. Since replication proceeded rightwards, we conclude that the adenovirus l strand was displaced during replication. No origin was located at or near the EcoRI site on pBR322. Reversing the orientation of the adenovirus origin reversed the direction of replication, and deletion of the adenovirus origin abolished replication.

Mutational mapping of a cloned adenovirus origin.

We have developed a standardized, quantitative assay to study the function of a cloned adenovirus origin. We have shown that the adenovirus origin is located within the first 20 bp of the adenovirus inverted terminal repetition (ITR), a region containing a sequence conserved among human, simian, murine, and avian adenoviruses. Deletions removing or penetrating from either direction into the conserved sequence inactivated the cloned adenovirus origin. A point mutation within the conserved sequence impaired the adenovirus origin, but point mutations outside the conserved sequence had no effect. These results strongly suggest that the conserved sequence within the first 20 bp of the ITR alone constitutes the adenovirus origin (ori) signal.

Determinants of outcome in hospitalized infants with congenital heart disease.

To identify determinants of adverse outcome in this era of early, definitive treatment, retrospective data were analyzed for 1988 on infants aged less than 1 year with congenital cardiac disease hospitalized at The Johns Hopkins Hospital. In this cohort of 108 infants, 34% (37 of 108) had complex cardiac disease, 33% (36 of 108) had major extracardiac anomalies, 88 patients (81%) underwent 116 surgical procedures, 37% (40 of 108) were hospitalized for greater than 28 days and 29% (31 of 108) died during the first year. Univariate analysis showed that complex disease (i.e., severe ventricular hypoplasia, ventricular septal malalignment or outflow atresia), extracardiac anomalies, early initial presentation, and prolonged stay in the intensive care unit were significantly associated with infant death, whereas surgery was associated with a significantly increased rate of survival. The findings for complex disease and surgery persisted in multiple logistic regression analysis. It is concluded that outcome in most infants with congenital cardiac defects is now extremely favorable, and that major research and preventive efforts should focus on complex congenital cardiac defects.

Cervical aortic arch with aneurysm formation.

Two pediatric cases of cervical aortic arch with aneurysm formation are reported. Only female patients with cervical aortic arch have developed aneurysms, which may influence risk counseling of such patients.

Traumatic thoracic aortic rupture in the pediatric patient.

BACKGROUND: Traumatic thoracic aortic rupture is a rare injury in the pediatric patient. Experiences with thoracic aortic rupture in patients less than 17 years of age are needed to help identify factors that can influence injury occurrence, diagnosis, management, and outcome. METHODS: Between July 1989 and December 1995, 6 children were treated operatively for thoracic aortic rupture from blunt trauma at a level I pediatric trauma center. The average age was 13.2 years (range, 8 to 16 years). There were 4 females and 2 males. There were 5 motor vehicle accidents and 1 bicycle accident. Aortic injury was suspected based on the mechanism of injury and abnormal chest roentgenogram results, and was confirmed by aortography (3 cases) or chest computed tomography (2) and transesophageal echocardiography (3). Life-threatening central nervous system or gastrointestinal injuries were evaluated or treated first. Operative repair of the thoracic aorta was performed by cardiopulmonary bypass (2 patients) and clamp and sew technique (4). RESULTS: Aortic ruptures were complete transections at the ligamentum arteriosum in 5 of 6 (83%); the other case was a cervical arch pseudoaneurysm. Associated injuries included pulmonary contusion (100%), pelvic/long bone fractures (50%), visceral laceration/perforation (50%), central nervous system (33%), paraplegia (17%), and myocardial contusion (17%). There were no rib fractures. Four of 5 patients (80%) were not wearing seat belts, and 2 of these were ejected. The average time from injury to the operating room was 17.6 hours (range, 5 to 48 hours); the time from diagnosis to the operating room exceeded 5 hours with aortography and was less than 3 hours with chest computed tomography and transesophageal echocardiography. Each diagnostic modality accurately identified an aortic injury. The average time for cardiopulmonary bypass and for clamp and sew was 52 minutes (range, 49 to 55 minutes) and 34 minutes (range, 16 to 45 minutes), respectively. One patient with preoperative paraplegia regained partial function; there were no other patients with paraplegia. There were no deaths. All patients are alive 2 months to 7 years after repair. CONCLUSIONS: The multiply injured child with severe blunt trauma and an abnormal chest roentgenogram requires a search for aortic injury. We believe the most effective algorithm to follow for the diagnosis of traumatic thoracic aortic rupture in the child involves selective performance of chest computed tomography and transesophageal echocardiography. Our experience suggests that the mechanism of injury, the duration to diagnosis of an aortic injury, and failure to use seat belts may contribute to morbidity. A high index of suspicion and a systematic approach to the diagnosis and to the management strategy for injuries to the thoracic aorta can contribute to a good outcome in those few children who survive the injury.

A retrospective review of the role of transesophageal echocardiography in aortic and cardiac trauma in a level I Pediatric Trauma Center.

This study examined the role of transesophageal echocardiography in blunt aortic and cardiac trauma in a Pediatric Level I Trauma Center. In a > 5-year retrospective review, we identified 10 children with blunt cardiac (n = 4; tricuspid valve in two; mitral valve in one; aortic valve in one) and aortic (n = 6; aortic rupture in five, subintimal flap in one) trauma. Diagnosis of the cardiac injuries was made with transthoracic echocardiography, with transesophageal echocardiography providing additional anatomic detail and postoperative assessment in three of four children who required surgical intervention. Diagnosis of the aortic injuries was made with transesophageal echocardiography in five of six patients; one patient underwent aortography before transfer. Transesophageal echocardiography also identified depressed myocardial function in one child and aided in surgical management of the five aortic ruptures. In blunt chest trauma, transesophageal echocardiography provides accurate evaluation of cardiovascular structure and function and guides operative repair.

Criteria for extracorporeal membrane oxygenation in a population of infants with persistent pulmonary hypertension of the newborn.

Extracorporeal membrane oxygenation (ECMO) has been available since 1975 as a therapy of last resort to provide adequate oxygenation for term infants with acute lung disorders that do not respond to maximal medical therapy. Virtually all term infants with serious lung disease have persistent pulmonary hypertension of the newborn (PPHN) characterized by significant right-to-left shunting of blood and severe diffusion defects manifested as increased alveolar-arterial oxygen gradients (AaDO2). Criteria for initiation of ECMO therapy have been developed in several institutions but at the present time there are no universal criteria applicable to all infants with PPHN. We have attempted to establish entry criteria that may be used for different populations of infants with PPHN. Based on a retrospective review of 30 infants with PPHN in our institution, we have defined standards of maximal medical therapy. An alveolar-arterial oxygen difference (AaDO2) of greater than or equal to 610 for 8 hours has been shown to be associated with 79% mortality in this population. This AaDO2/time interval is established as a major criterion for institution of extracorporeal membrane oxygenation.

Emphysema predicts hospitalisation and incident airflow obstruction among older smokers: a prospective cohort study.

BACKGROUND: Emphysema on CT is common in older smokers. We hypothesised that emphysema on CT predicts acute episodes of care for chronic lower respiratory disease among older smokers. MATERIALS AND METHODS: Participants in a lung cancer screening study age ≥ 60 years were recruited into a prospective cohort study in 2001-02. Two radiologists independently visually assessed the severity of emphysema as absent, mild, moderate or severe. Percent emphysema was defined as the proportion of voxels ≤ -910 Hounsfield Units. Participants completed a median of 5 visits over a median of 6 years of follow-up. The primary outcome was hospitalization, emergency room or urgent office visit for chronic lower respiratory disease. Spirometry was performed following ATS/ERS guidelines. Airflow obstruction was defined as FEV1/FVC ratio <0.70 and FEV1<80% predicted. RESULTS: Of 521 participants, 4% had moderate or severe emphysema, which was associated with acute episodes of care (rate ratio 1.89; 95% CI: 1.01-3.52) adjusting for age, sex and race/ethnicity, as was percent emphysema, with similar associations for hospitalisation. Emphysema on visual assessment also predicted incident airflow obstruction (HR 5.14; 95% CI 2.19-21.1). CONCLUSION: Visually assessed emphysema and percent emphysema on CT predicted acute episodes of care for chronic lower respiratory disease, with the former predicting incident airflow obstruction among older smokers.

The Pediatric Heart Network: a primer for the conduct of multicenter studies in children with congenital and acquired heart disease.

Most contemporary diagnostic and treatment strategies for pediatric patients with cardiovascular disease are not supported by evidence from clinical trials but instead are based on expert opinion, single-institution observational studies, or extrapolated from adult cardiovascular medicine. In response to this concern, the National Heart, Lung, and Blood Institute established the Pediatric Heart Disease Clinical Research Network (PHN) in 2001. The purposes of this article are to describe the initiation, structure, and function of the PHN; to review the ongoing studies; and to address current and future challenges. To date, four randomized clinical trials and two observational studies have been launched. Design and conduct of complex, multicenter studies in children with congenital and acquired heart disease must address numerous challenges, including identification of an appropriate clinically relevant primary endpoint, lack of preliminary data on which to base sample size calculations, and recruitment of an adequate number of subjects. The infrastructure is now well developed and capable of implementing complex, multicenter protocols efficiently and recruiting subjects effectively. The PHN is uniquely positioned to contribute to providing evidence-based medicine for and improving the outcomes of pediatric patients with cardiovascular disease.

Intermediate in adenovirus type 2 replication.

Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.

Sequence conversion during postreplicative adenovirus overlap recombination.

Sequence conversion efficiently transfers genetic information in high yield during postreplicative adenovirus overlap recombination. This process is intrinsically nonreciprocal, depends on adenovirus-specific strand-displacement replication by both partner molecules, and requires that complementary sequences on displaced strands must exceed a minimal length to form a heteroduplex intermediate.

Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12.

Hybridization probes derived from the A and B subunit genes of the heat-labile enterotoxin (LT) of Escherichia coli were used to analyze DNA from Vibrio cholera strain 569B for cholera toxin gene sequences. Southern blot analysis indicated that the cholera toxin A and B subunit genes were each duplicated in the strain. One of the two toxin subunit gene pairs was cloned as a 5.1-kilobase DNA insert in plasmid pBR322. E. coli cells carrying the recombinant plasmid pJM17 were shown to produce cholera toxin, which was found to be largely cell associated. Protein chemical analysis indicated that the toxin was in its unnicked form and required additional proteolytic processing by trypsin to exhibit full toxicity in tissue culture. The alteration in E. coli of the secretion and proteolytic processing of cholera toxin parallels that previously observed for LT. An in vitro generated insertion mutation in the A subunit gene on pJM17 was shown to abolish production of the A chain but still allow production of the B chain. These observations, together with restriction mapping data, have demonstrated that the cholera toxin and LT genes are very similar in their genetic organization.