Significant linkage disequilibrium between the Huntington disease gene and the loci D4S10 and D4S95 in the Dutch population.

Significant linkage disequilibrium has been found between the Huntington disease (HD) gene and DNA markers located around D4S95 and D4S98. The linkage-disequilibrium studies favor the proximal location of the HD gene, in contrast to the conflicting results of recombination analyses. We have analyzed 45 Dutch HD families with 19 DNA markers and have calculated the strength of linkage disequilibrium. Highly significant linkage disequilibrium has been detected with D4S95, consistent with the studies in other populations. In contrast with most other studies, however, the area of linkage disequilibrium extends from D4S10 proximally to D4S95, covering 1,100 kb. These results confirm that the HD gene most likely maps near D4S95.     

Origin heterogeneity of Hb Lepore-Boston gene in Italy

Forty-three hybrid delta-beta-globin genes were characterized by DNA sequence analysis and associated RFLP haplotypes in 40 families from Abruzzo and Campania, which are on the east and west coast of Italy, respectively. All the genes had the delta-globin sequence up to the exon 2 codon 87 and had the beta-globin sequence from IVS-2-8; between these two ends, they had 58 bp in common with the delta- and beta-globin genes. Thus, they were all of the Lepore-Boston type. A chromosomal background heterogeneity was present among the mutant genes. In fact, they were all associated with (+ - - - -) 5' subhaplotype, but 23/31 from Campania were associated with (+ +) 3' subhaplotype, whereas 12/12 genes from Abruzzo and 8/31 from Campania were associated with (+ -). DNA sequencing of homozygous subjects showed that (+ +) 3' subhaplotype was associated, at IVS-2-74, with G, while (+ -) was associated with T; that is they were associated with the beta-globin gene sequence of frameworks 1 and 2, respectively. The molecular characteristics of this heterogeneity, as well as its geographical patterns in the eastern and western regions of Italy, represent strong evidence for the recurrent and multicentric origins of the mutation.     

HPLC analysis of free amino acids and amino acids of total proteins in cultured cells: an application to the study of rat Sertoli cell protein metabolism.

A simple, rapid and, sensitive HPLC method, coupled with fluorometric detection, has been worked out and employed to determine the intracellular free amino acid concentrations and the amino acid composition of total proteins in rat Sertoli cell primary culture. Sertoli cells were isolated enzymatically from testes of 20- and 28-day-old rats and cultured at 32 degrees C in Eagle's minimum essential medium. On the second day of culture, cell monolayers were quickly rinsed with ice-cold saline, immediately frozen in liquid nitrogen, accurately harvested, and homogenized in 10% trichloroacetic acid. Tissue free amino acids were determined in the acidic soluble fraction following neutralization, while the precipitate was hydrolyzed for the evaluation of the fractional content of amino acids into total proteins. Amino acid samples were derivatized with o-phthaldialdehyde/3-mercaptopropionic acid and resolved by a linear one-step acetonitrile gradient in 12.5 mM sodium phosphate buffer, pH 7.2, employing a 5-microns particle size reversed-phase column. Fluorescence was monitored with excitation at 330 nm and emission at 450 nm. Under these conditions all major physiological amino acids could be satisfactory separated, identified, and subsequently quantified with the aid of standards. The run time was about 50 min; the linearity was excellent over a large range of concentrations (1-800 pmol) and the lower limit of sensitivity appeared to be 0.5 pmol. This method permits us to demonstrate age-dependent modifications in the intracellular amino acid pool and to adequately evaluate the process of protein synthesis in cultured Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of peroxisomes from frozen human liver samples.

This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen at -70 degrees C. Purification of these peroxisomes was obtained by a combination of two subcellular fractionation techniques: differential centrifugation and isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency measurements and by ultrastructural observation. The procedure described here may be useful for the isolation of other subcellular organelles from frozen human samples.     

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.     

Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]G.C adduct structure and comparison with the (+)-trans-anti-[BP]G.C enantiomer.

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)