Genetic dissection of a mammalian replicator in the human beta-globin locus.

The timing and localization of DNA replication initiation in mammalian cells are heritable traits, but it is not known whether initiation requires specific DNA sequences. A site-specific recombination strategy was used to show that DNA sequences previously identified as replication initiation sites could initiate replication when transferred to new chromosomal locations. An 8-kilobase DNA sequence encompassing the origin of DNA replication in the human beta-globin locus initiated replication in the simian genome. Specific deletions within the globin origin did not initiate replication in these chromosomal sites. These data suggest that initiation of DNA replication in mammalian cells requires specific sequence information and extend the replicon hypothesis to higher eukaryotes.

Participation of the human beta-globin locus control region in initiation of DNA replication.

The human beta-globin locus control region (LCR) controls the transcription, chromatin structure, and replication timing of the entire locus. DNA replication was found to initiate in a transcription-independent manner within a region located 50 kilobases downstream of the LCR in human, mouse, and chicken cells containing the entire human beta-globin locus. However, DNA replication did not initiate within a deletion mutant locus lacking the sequences that encompass the LCR. This mutant locus replicated in the 3' to 5' direction. Thus, interactions between distantly separated sequences can be required for replication initiation, and factors mediating this interaction appear to be conserved in evolution.

p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine.

This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, gammaH2AX reverses after camptothecin removal. By contrast, gammaH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21(CDKN1A) (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced gammaH2AX formation in p21-/- cells, we conclude that enhanced gammaH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced gammaH2AX formation and inhibits camptothecin-induced p21(CDKN1A) upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that gammaH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21(CDKN1A) prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.

ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage.

BACKGROUND: Embryonic stem (ES) cells can contribute precursors to all adult cell lineages. Consequently, damage to ES cell genomes may cause serious developmental malfunctions. In somatic cells, cell-cycle checkpoints limit DNA damage by preventing DNA replication under conditions that may produce chromosomal aberrations. The tumor suppressor p53 is involved in such checkpoint controls and is also required to avoid a high rate of embryonic malformations. We characterized the cell-cycle and DNA-damage responses of ES cells to elucidate the mechanisms that prevent accumulation or transmission of damaged genomes during development. RESULTS: ES cells derived from wild-type mice did not undergo cell-cycle arrest in response to DNA damage or nucleotide depletion, although they synthesized abundant quantities of p53. The p53 protein in ES cells was cytoplasmic and translocated inefficiently to the nucleus upon nucleotide depletion. Expression of high levels of active p53 from an adenovirus vector could not trigger cell cycle arrest. Instead, ES cells that sustained DNA damage underwent p53-independent apoptosis. The antimetabolite-induced p53-dependent arrest response was restored in ES cells upon differentiation. CONCLUSIONS: Cell-cycle regulatory pathways in early embryos differ significantly from those in differentiated somatic cells. In undifferentiated ES cells, p53 checkpoint pathways are compromised by factors that affect the nuclear localization of p53 and by the loss of downstream factors that are necessary to induce cell-cycle arrest. A p53-independent programmed cell death pathway is effectively employed to prevent cells with damaged genomes from contributing to the developing organism. The p53-mediated checkpoint controls become important when differentiation occurs.

Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells.

Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites. Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP. The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites. This approach allows the detection of DNA excised by FLP-mediated recombination and facilitates the recovery of recombination products that would not be detected by available screening strategies. We used this system to show that the molecules excised by intrachromosomal recombination between tandem FLP recombinase target sites do not reintegrate into the host genome at detectable frequencies. We further applied the direct selection approach to recover a rare FLP-mediated recombination event displaying the characteristics of an unequal sister chromatid exchange between FLP target sites. Implications of this approach for the generation of duplications to assess their effect on gene dosage and chromosome stability are discussed.

Synthesis of complement proteins in amnion.

The amnion is a metabolically active tissue that has been identified as a site of synthesis of numerous products. We report that amnion tissue explants and amnion-derived epithelial cells synthesize and secrete six proteins of the complement system, C1r, C1s, C1 inhibitor, factor B, C3, and factor H. Synthesis of C2 was minimal and variable, and C5 was not detected. The six synthesized proteins had size and subunit composition characteristic of proteins synthesized in HEp2, a long term cell line derived from malignant epithelial cells. Constitutive and regulated synthesis of five of the six proteins was similar in amnion tissue and cells. However, synthesis of factor B was different in tissue and cells; constitutive synthesis was 12-fold higher in tissue than in cells, and interleukin-1 did not alter synthesis in tissue, but increased synthesis by 11.7-fold in cells. These results indicate that amnion may be a source of complement proteins present in the amnion fluid and may contribute to local host defense along with endometrial glandular epithelial cells, which synthesize C3. Furthermore, our results suggest that amnion tissue is stimulated in vivo to synthesize factor B and cannot respond to interleukin-1 with a further increase in the synthesis rate.

Variations in the apparent pH set point for activation of platelet Na-H antiport.

To explore the role of the Na-H antiport in essential hypertension, we studied the kinetics of cytosolic pH and external sodium activation of this transport system in platelets from 65 normotensive and essential hypertensive subjects on and off antihypertensive medications. Subjects included both blacks and whites, as well as men and women. The fluorescent dye 2'7-bis(carboxyethyl)-5,6-carboxyfluorescein was used to monitor the cytosolic pH in these cells. Platelets from black (hypertensive and normotensive) men and hypertensive white men demonstrated a highly significant alkaline shift in the apparent cytosolic pH set point for activation of the Na-H antiport. For the hypertensive subgroups, the cytosolic pH set point values (mean +/- SEM) were: white men, 7.45 +/- 0.052; white women, 7.04 +/- 0.089; black men, 7.66 +/- 0.148; and black women, 7.20 +/- 0.082. For the normotensive subgroups, the cytosolic pH set point values were: white men, 7.13 +/- 0.034; white women, 7.05 +/- 0.036; black men, 7.50 +/- 0.110; and black women, 7.20 +/- 0.176 (p = 0.0016 for race and p = 0.0001 for gender, using a three-way analysis of variance by race, gender, and hypertension). There were no race-, gender-, or blood pressure-related differences among the various cohorts in the kinetics of sodium activation of the Na-H antiport, the cellular buffering power, and basal pH. These results suggest that at basal pH the Na-H antiport is quiescent in platelets from both black and white women and normotensive white men.(ABSTRACT TRUNCATED AT 250 WORDS)

The ligand binding domain of the nicotinic acetylcholine receptor. Immunological analysis.

The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with alpha-bungarotoxin (alpha-BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR alpha-subunit binds alpha-BTX, whereas the mongoose-expressed domain is not recognized by alpha-BTX. On the other hand, both the mouse and mongoose domains bind to the site-specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of alpha-BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other.

Effects of essential hypertension and antihypertensive medications on sweat formation.

OBJECTIVE: Sweat volume and ionic composition depend to a large extent upon the cytosolic free calcium level in secretory sweat cells and sodium and potassium transport in the reabsorptive sweat duct. Since essential hypertension and its treatment with antihypertensive drugs is likely to be associated with altered cellular ionic regulation, the objective of this research was to explore sweat formation and sweat parameters in hypertensive and normotensive subjects. DESIGN: Black and white hypertensive and normotensive subjects of both genders were studied. Essential hypertensives were on or off antihypertensive medication. METHODS: Pilocarpine iontophoresis was used to induce sweat in a 5-cm2 area of the middle forearm. Sweat was analyzed for volume, sodium and potassium concentrations. RESULTS: Females demonstrated lower sweat volumes after pilocarpine stimulation than males. Untreated hypertensive white males exhibited a higher pilocarpine-induced sweat volume and sweat sodium excretion than normotensive white males, whilst hypertensive white males on antihypertensive medication showed a lower sweat volume and sweat sodium excretion than both normotensive white males and untreated essential hypertensive white males. Although untreated hypertensive white females did not show significant alterations in sweat parameters, treated hypertensive white females exhibited lower sweat volume and sweat sodium excretion than both the normotensive and untreated essential hypertensive white females. These hypertension and drug related alterations were not present in hypertensive black males and females. CONCLUSIONS: The results are consistent with the heterogeneous nature of essential hypertension and the diversity of the response to antihypertensive therapy. They suggest that the effect of antihypertensive medication on sweat formation is mediated through cytosolic free calcium.

Heredofamilial syndrome of mesodermal hamartomas, macrocephaly, and pseudopapilledema.

A 4 1/2-year-old boy with macrocephaly, pseudopapilledema, lipoangiomatosis, macropenia, and spotted pigmentations of the glans is reported. Lipoid masses were found in the subcutaneous tissue, tonsils, and probably the left lung. Some of these findings are consistent with features already reported by Riley and Smith, later by Bannayan, and recently by Ruvalcaba et al. We propose to unify the features of this syndrome and name it macrocephaly, hamartomas, and papilledema syndrome. The inheritance in our described case seems to be autosomal dominant.

Renal and absorptive hypercalciuria: a metabolic disturbance with varying and interchanging modes of expression.

BACKGROUND: In previous studies, the oral calcium loading and deprivation test has been used to distinguish between children with renal (fasting) hypercalciuria (RH) and absorptive hypercalciuria (AH). OBJECTIVE: We evaluated the long-term clinical course of 30 children with idiopathic hypercalciuria and investigated the influence of urinary sodium excretion, as a reflection of its intake, on urinary calcium excretion. METHODS: Thirty normocalcemic, normophosphatemic children (21 boys and 9 girls) with urinary calcium to creatinine ratios greater than 0.57 mmol/L/mmol/L ( > 0.21 mg/dL/mg/dL on the three consecutive examinations participated in this study. They were divided according to their responses to calcium deprivation and loading into AH (16 patients) and RH (14 patients). RESULTS: When restudied 3 to 7 years later, 6 of the 16 children with AH were normocalciuric and three demonstrated characteristics compatible with RH. The remaining seven patients maintained their initial AH pattern. Of the 14 children with RH, four were normocalciuric and four demonstrated AH. The remaining six children maintained their initial RH pattern. A significant positive correlation was observed between urine sodium and calcium excretion in children with AH or RH. Children who were normocalciuric at the second study had significantly lower values of urine sodium excretion when compared with those in whom hypercalciuria persisted. CONCLUSIONS: We suggest that AH and RH constitute a continuum. The change in characteristics observed during the second study suggests that any attempt to divide these patients into two physiologically distinct subtypes may be artificial. The main factor influencing urinary excretion of calcium in our patients seemed to be sodium intake.

The conservative management of vesicoureteric reflux: a review of 121 children.

A group of 121 children with vesicoureteric reflux (VUR) grades 1 to 3 managed conservatively were followed-up for a period of six to ten years. In the majority of patients VUR grades 1 and 2 disappeared spontaneously. Prognosis was less favorable in those who were seen initially with grade 3 VUR. A statistically significant higher incidence of reflux disappearance was observed in children who were seen before 4 years of age. In the majority of patients with paraureteric-vesicle diverticulum, reflux persisted. Reflux was more likely to disappear in children with lower incidence of urinary tract infection than in those with multiple infections.

Vesicoureteral reflux in boys with hypospadias.

Hypospadias in a congenital anomaly which in most cases has to be corrected surgically. We include micturition cystourethrography (MCU) in the preoperative workup of all these children as well as three months postoperatively. Meatal stenosis and müllerian duct remnants are known to occur in this condition, but vesicoureteral reflux (VUR) was an unexpected finding in many boys who were asymptomatic and had no evidence of urinary tract infection. Fifty-eight of 305 hypospadiacs, examined by MCU, proved to have reflux, most of them grade II. In 37, VUR was found before surgery and in the remaining 21 after surgical correction, many of them with meatal stenosis. In 10 patients reflux appeared in the postoperative period where there had been none in the preoperative study; 8 of the 10 had a postoperative stricture of the distal urethra, and VUR subsided in 4 after adequate dilatation. We conclude that vesicoureteral reflux is not uncommonly found in patients with hypospadias, even in those who are completely asymptomatic. We believe this is an additional incentive to include an MCU in the pre- and postoperative evaluation of patients with this anomaly.

Changes in renal enzyme activities following the administration of gentamicin to unilateral nephrectomized rats.

The effects of unilateral nephrectomy and the impact of gentamicin administration on renal tissue enzyme activities in adult Wistar rats were investigated. Gentamicin 200 mg/kg body wt. or an equivalent volume of saline to control rats was administered subcutaneously on three consecutive days, followed by unilateral nephrectomy. Rats were killed on day 3, 7 or 14 following nephrectomy. Alkaline phosphatase, predominantly a proximal tubular brush border enzyme, rose in both the experimental and control groups, however, significantly less in the gentamicin treated rats. Aspartate aminotransferase activity, an enzyme participating in renal glucogenesis, increased transiently in the control but remained unchanged in the experimental group. No difference in glucose-6-phosphate dehydrogenase activity between the two groups was observed, probably reflecting the localization of this enzyme to distal tubular segments, a site unaffected by gentamicin. Significant and similar increases in Mg2+ and Na+ K+ ATPase were observed on day 14 in both groups. The administration of the drug resulted in a marked reduction in oxygen consumption, with a higher oxidation to phosphorylation ratio (P/O). Serum creatinine concentration was significantly higher on days 3 and 7 in the experimental group reverting to control values on the 14th day. Urea concentration increased significantly on days 3 and 7, decreasing on the 14th day to values slightly, but significantly, higher than those of the controls.

Familial Mediterranean fever presenting with massive cardiac tamponade.

A 16-year-old girl, presenting initially with pericarditis and life threatening pericardial tamponade, developed clinical episodes characteristic of FMF few months later. This case report and several others reported previously, suggest that FMF should be considered in patients from certain ethnic groups presenting with pericardial effusion.

Sodium potassium adenosine triphosphatase activity in preterm and term infants and its possible role in sodium homeostasis during maturation.

AIM: To investigate sodium (NA(+)) potassium (K(+)) adenosine triphosphatase (ATPase) activity in newborn infants at different gestational ages, to elucidate the mechanism underlying poor renal sodium conservation in preterm infants. METHODS: Fifty three healthy newborn infants, gestational age 30-42 weeks, were studied. Umbilical cord red blood cell Na(+) K(+)ATPase activity, plasma renin activity, and plasma aldosterone activities were measured in all of them. Red blood cell Na(+) K(+)ATPase activity was re-examined in eight preterm infants, one and two weeks after birth. Total and ouabain sensitive ATPase activity was measured spectrophotometrically using a method that couples ATP hydrolysis with NADH oxidation. RESULTS: Red blood cell Na(+) K(+)ATPase activity was significantly lower (p<0.01) in preterm babies with a gestational age below 35 weeks, compared with those with aged 35 weeks and above: 2.3 (0.8) and 6.7 (1.3) nmol NADH/minute/mg protein, respectively. There was no correlation between gestational age, Na(+) K(+)ATPase, plasma renin activity and aldosterone values either in the preterm or term babies. Two weeks after birth, irrespective of gestational age, the enzyme activity of the preterm babies increased to values similar to those observed in the term neonates at birth. CONCLUSION: The differences in sodium homeostasis between term and preterm babies are modulated via changes in Na(+) K(+)ATPase activity.

The mechanism of carcinogen-induced DNA amplification: in vivo and in vitro studies.

Exposure to chemical carcinogens provides a means for the enhancement of the frequency of gene amplification and for the facilitation of research into its mechanism(s). Using carcinogen-induced SV40 amplification as a model system it was shown that amplification of the viral sequences occurs via a replication-dependent mode. This process involves overactivation of the origin region and the generation of inverted repeats. Carcinogen-induced enhancement of gene amplification is triggered by cellular factors that could act in trans. An in vitro amplification system, based on extracts from carcinogen-treated cells and SV40 template sequences, was used to further characterize the amplification intermediates. The major products of overreplication in this system consist of sequences derived from the origin region. Our studies suggest that the ability to overreplicate the origin region in vitro derives from the combined action of carcinogen-induced factors that trigger overinitiation, with the inherent inability of Chinese hamster cell extracts to fully replicate large plasmid templates. The newly replicated sequences are not associated with the parental molecule and contain hairpin or stem and loop structures. Based on these findings we propose a novel replicative mechanism for DNA amplification that allows the de novo formation of hairpin structures. According to this model, an obstruction of the replication fork may cause an overturning of the DNA polymerase, followed by a template switch that leads to the use of the newly replicated strand as a template. This mode of replication results in the generation of hairpin structures which can function as precursors for the duplicated inverted repeats which are commonly observed in amplified genomes. This model is supported by our in vitro and in vivo studies. The relevance of this model for the amplification of cellular sequences is discussed.

Functional upper airway obstruction in adolescents.

Functional upper airway obstruction is an uncommon manifestation of a conversive reaction characterized by recurrent stridor attacks caused by adduction of the vocal cords during inspiration. The oxygen saturation never drops to pathologic levels. The stridor is not accompanied by an appropriate degree of anxiety and is not associated with other symptoms. Patients benefit from verbal reassurance and speech therapy, but stridor attacks tend to recur and psychiatric consultation seems necessary in most cases. We present the case histories of three adolescent patients with nonorganic upper airway obstruction and describe the features that may facilitate the diagnosis of this condition. Early diagnosis and intervention may prevent unnecessary and potentially harmful investigations and therapy.

Midazolam in treatment of epileptic seizures.

Midazolam (Versed), the first water-soluble benzodiazepine, has had widespread acceptance as a parenteral anxiolitic agent. Its antiepileptic properties were studied in adult patients with good results. Midazolam was administered intramuscularly to 48 children, ages 4 months to 14 years, with 69 epileptic episodes of various types. In all but 5 epileptic episodes, seizures stopped 1-10 min after injection. These results suggest that midazolam administered intramuscularly may be useful in a variety of epileptic seizures during childhood, specifically when attempts to introduce an intravenous line in convulsing children are unsuccessful.

Rapidly growing head circumference as an isolated presenting symptom of brain abscesses in an infant.

A rapid and excessive enlargement in occipito frontal circumference (OFC) as the only manifestation of multiple cerebral abscesses in infancy is extremely rare. A 2-month-old asymptomatic infant presenting with increasing OFC is described. Surgical drainage of the cerebral abscesses followed by prolonged antibiotic therapy resulted in complete recovery.