A subfamily of alphoid repetitive DNA shared by the NOR-bearing human chromosomes 14 and 22

The nucleotide sequence of members of an alpha-repeat subfamily shared by human chromosomes 14 and 22 is presented. This subfamily is organized into a higher-order repeat unit composed of a tandem repetition of an ordered array of four related but distinct 340-bp repeat dimers. An analogous situation has been described for a related but distinct subfamily shared by chromosomes 13 and 21. These two subfamilies were further shown not to be present on the homologous chimpanzee chromosomes and therefore must have arisen by rearrangement of the human genome after separation of the two species. The sequence homology between the 13/21 and the 14/22 subfamilies is about 85%. The 14/22 subfamily represents the only major alphoid DNA species on these two chromosomes and is not present elsewhere in the human genome. Fluorescent in situ hybridizations show that sequences from the 13/21 and 14/22 subfamilies can be used as specific markers for their respective chromosomes.     

Seven polymorphic loci mapping to human chromosomal region 11q22-qter

Seven polymorphic loci that map to human chromosomal region 11q22-qter are revealed by DNA probes isolated from a chromosome-specific phage library constructed from a human X mouse somatic cell hybrid that has retained an 11q;16q translocation as the only human DNA. Three probes, each of which reveals a two-allele polymorphism, and four probes, each of which detects two linked RFLPs, have been characterized. Using a somatic cell hybrid mapping panel that divides 11q into four discrete sections, the seven clones have been localized to specific chromosomal regions. Localization of one of the clones has been confirmed and refined by in situ hybridization.     

Conversion of cyanide to formate and ammonia by a pseudomonad obtained from industrial wastewater

Summary A cyanide-degrading pseudomonad was isolated by selective enrichment in a chemostat inoculated with coke-plant activated sludge and maintained at a dilution rate of 0.042/h for 60 days with a feed of 10 mg/l cyanide. The isolate, a facultative methylotroph capable of growth on methanol and methylamine, degraded cyanide to formate and ammonia; it could utilize the released ammonia as a nitrogen source but did not further metabolize formate under the experimental conditions employed. Both cyanide-degrading enzyme activity and respiratory resistance to cyanide were inducible and were enhanced by repeated exposure to the compound. Cell-free extracts stoichiometrically converted cyanide to formate and ammonia in a reaction that did not require oxygen. Enzyme activity, lost upon dialysis, was restored by less than equimolar ratios of NAD(P)H or ascorbate to cyanide, indicating that the reductants did not function directly as co-enzymes.     

Higher rate of evolution of X chromosome alpha-repeat DNA in human than in the great apes.

The rate of introduction of neutral mutations is lower in man than in other primates, including the chimpanzee. This species is generally regarded as our closest relative among the great apes. We present here an analysis of sequences of X chromosomal alphoid repetitive DNA from man and the great apes, which supports the closer relationship between man and chimpanzee and indicates a considerably increased rate of recombination in the human repeat DNA. These results indicate that the 'molecular clock' is running more quickly in man.     

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.     

Brisk walking improves endurance fitness without changing body fatness in previously sedentary women.

The purpose of this study was to examine the influence of brisk walking on endurance fitness and the amount and distribution of body fat in previously sedentary women. Twenty eight women [mean age (SEM): 44.9 (1.5) years] followed the walking programme for 1 year, whilst 16 acted as controls [age 44.4 (2.3) years]. Changes in endurance fitness were evaluated by measuring the oxygen uptake (VO2) at a reference blood lactate concentration of 2 mmol.l-1. Two 1.61-km field tests of walking were completed, one at maximal speed and one at a "brisk" speed, as well as a 1.61-km walk on a motorised treadmill. The amount and distribution of body fat was determined by hydrostatic weighing and anthropometry and energy intake was evaluated using the 7-day weighed food intake method. Walkers completed an average of 157 min.week-1 of brisk walking over the year. The following were increased in walkers, relative to controls: brisk walking speed [walkers 1.73 (0.05) m.s-1 vs 1.88 (0.07) m.s-1; controls 1.69 (0.05) m.s-1 vs 1.70 (0.05) m.s-1 at baseline and 12 months respectively, P < 0.01], maximal walking speed and VO2 at 2 mmol.l-1. In addition, brisk walking reduced heart rate and blood lactate concentration during stepping as well as during standard, submaximal treadmill walking. It did not modify either the amount or the distribution of body fat, despite an unchanged energy intake.     

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.     

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus

Summary Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.     

Hemolymph concentrations of host ecdysteroids are strongly suppressed in precocious prepupae of Trichoplusia ni parasitized and pseudoparasitized by Chelonus near curvimaculatus.

Regulation of ecdysteroid production in lepidopteran prepupae was studied using a parasitic wasp (C. near curvimaculatus) which specifically suppresses host prepupal ecdysteroid production after the induction of precocious host metamorphosis. At the developmental stage at which the hemolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar "truly parasitized" hosts (hosts with a surviving endoparasite) had a strongly reduced ecdysteroid titer. However, during the photophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseudoparasitized prepupal hosts (in which the endoparasite was not present or died early in development) exhibited a sustained suppression in the hemolymph ecdysteroid titer. Small 5th instar pseudoparasitized hosts, which normally would molt to a 6th instar prior to metamorphosis, but which precociously attained the prepupal stage, also had a strongly reduced ecdysteroid titer. The late increase observed in truly parasitized hosts could be completely prevented by surgical removal of the parasite 24 h earlier, resulting in a titer similar to that in pseudoparasitized hosts. HPLC analysis of ecdysteroids in normal, truly parasitized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitized prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and those of Brown and Reed-Larsen (Biol Contr 1, 136 [1992]), showing endoparasite secretion of ecdysteroids just prior to its emergence from the host, strongly indicate that: (1) the prepupal peak in truly parasitized hosts originates from the endoparasite, and (2) the low level of ecdysteroids in pseudoparasitized hosts results from the host's intrinsic inability to express a normal level of prepupal ecdysteroid titer. While precocious 4th or 5th instar prepupae of similar size had similarly suppressed ecdysteroid titers, smaller 4th instar prepupae had a lower ecdysteroid titer than larger, precocious 5th instar prepupae. Rare 5th instar pseudoparasitized prepupae that were of nearly normal size showed a prepupal ecdysteroid titer distinctly greater than those of the usual smaller, precocious 5th instar prepupae. The data suggest that the competence of the host to express a normal hemolymph titer of prepupal ecdysteroids is more closely correlated with the size of the prepupae than with the instar attained.