Synthesis of oligoribonucleotides containing 4-thiouridine using the convertible nucleoside approach and the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group

Oligoribonucleotides containing 4-thiouridine were prepared using the Fpmp group for protection of the 2'-OH. Two uridine derivatives with the 1,2,4-triazolyl and the 2-nitrophenyl groups at position 4 were used to obtain 4-thiouridine by postsynthetic substitution with sodium hydrogen sulfide. Both uridine derivatives allow the preparation of the desired oligonucleotides in good yields.     

DNA damage response-mediated degradation of Ho endonuclease via the ubiquitin system involves its nuclear export

Yeast mating switch Ho endonuclease is rapidly degraded by the ubiquitin system and this depends on the DNA damage response functions, MEC1, RAD9, and CHK1. A PEST sequence marks Ho for degradation. Here we show that the novel F-box receptor, Ufo1, recruits phosphorylated Ho for degradation. Mutation of PEST residue threonine 225 stabilizes Ho, yet HoT225A still binds Ufo1 in vitro. Stable HoT225A accumulates within the nucleus, whereas HoT225E is degraded. Deletion of the nuclear exportin Msn5 traps native Ho in the nucleus and extends its half-life. These experiments suggest that Ho is degraded in the cytoplasm. In mec1 mutants stable Ho accumulates within the nucleus; Ho produced in mec1 cells does not bind Ufo1. Thus the MEC1 pathway has functions both in phosphorylation of Thr-225 for nuclear export and in additional phosphorylations for binding Ufo1. Cells with HO under its genomic promoter, but stabilized by deletion of the Msn5 exportin, proliferate, but are multibudded. These experiments elucidate some of the links between the DNA damage response and degradation of Ho by the ubiquitin system.     

A novel founder mutation in the RNASEL gene, 471delAAAG,is associated with prostate cancer in Ashkenazi Jews

HPC1/RNASEL was recently identified as a candidate gene for hereditary prostate cancer. We identified a novel founder frameshift mutation in RNASEL, 471delAAAG, in Ashkenazi Jews. The mutation frequency in the Ashkenazi population, estimated on the basis of the frequency in 150 healthy young women, was 4% (95% confidence interval [CI] 1.9%-8.4%). Among Ashkenazi Jews, the mutation frequency was higher in patients with prostate cancer (PRCA) than in elderly male control individuals (6.9% vs. 2.4%; odds ratio = 3.0; 95% CI 0.6-15.3; P=.17). 471delAAAG was not detected in the 134 non-Ashkenazi patients with PRCA and control individuals tested. The median age at PRCA diagnosis did not differ significantly between the Ashkenazi carriers and noncarriers included in our study. However, carriers received diagnoses at a significantly earlier age, compared with patients with PRCA who were registered in the Israeli National Cancer Registry (65 vs. 74.4 years, respectively; P<.001). When we examined two brothers with PRCA, we found a heterozygous 471delAAAG mutation in one and a homozygous mutation in the other. Loss of heterozygosity was demonstrated in the tumor of the heterozygous sib. Taken together, these data suggest that the 471delAAAG null mutation is associated with PRCA in Ashkenazi men. However, additional studies are required to determine whether this mutation confers increased risk for PRCA in this population.     

Genetic variation in the 6p22.3 gene DTNBP1, the human ortholog of the mouse dysbindin gene,is associated with schizophrenia

Prior evidence has supported the existence of multiple susceptibility genes for schizophrenia. Multipoint linkage analysis of the 270 Irish high-density pedigrees that we have studied, as well as results from several other samples, suggest that at least one such gene is located in region 6p24-21. In the present study, family-based association analysis of 36 simple sequence-length-polymorphism markers and of 17 SNP markers implicated two regions, separated by approximately 7 Mb. The first region, and the focus of this report, is 6p22.3. In this region, single-nucleotide polymorphisms within the 140-kb gene DTNBP1 (dystrobrevin-binding protein 1, or dysbindin) are strongly associated with schizophrenia. Uncorrected, empirical P values produced by the program TRANSMIT were significant (P<.01) for a number of individual SNP markers, and most remained significant when the data were restricted to include only one affected offspring per nuclear family per extended pedigree; multiple three-marker haplotypes were highly significant (P=.008-.0001) under the restricted conditions. The pattern of linkage disequilibrium is consistent with the presence of more than one susceptibility allele, but this important issue is unresolved. The number of markers tested in the adjacent genes, all of which are negative, is not sufficient to rule out the possibility that the dysbindin gene is not the actual susceptibility gene, but this possibility appears to be very unlikely. We conclude that further investigation of dysbindin is warranted.     

Disruption of neurogenesis by amyloid beta-peptide,and perturbed neural progenitor cell homeostasis,in models of Alzheimer's disease

Neurogenesis occurs in the adult mammalian brain and may play roles in learning and memory processes and recovery from injury, suggesting that abnormalities in neural progenitor cells (NPC) might contribute to the pathogenesis of disorders of learning and memory in humans. The objectives of this study were to determine whether NPC proliferation, survival and neuronal differentiation are impaired in a transgenic mouse model of Alzheimer's disease (AD), and to determine the effects of the pathogenic form of amyloid beta-peptide (Abeta) on the survival and neuronal differentiation of cultured NPC. The proliferation and survival of NPC in the dentate gyrus of the hippocampus was reduced in mice transgenic for a mutated form of amyloid precursor protein that causes early onset familial AD. Abeta impaired the proliferation and neuronal differentiation of cultured human and rodent NPC, and promoted apoptosis of neuron-restricted NPC by a mechanism involving dysregulation of cellular calcium homeostasis and the activation of calpains and caspases. Adverse effects of Abeta on NPC may contribute to the depletion of neurons and cognitive impairment in AD.     

Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae)

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, P<0.01), dry rubber (r=0.81, P<0.01)] together with flow time (r=0.85, P<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Properties of triple helices formed by parallel-stranded hairpins containing 8-aminopurines

Parallel-stranded hairpins with a polypyrimidine sequence linked to a complementary purine carrying 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine bind polypyrimidine sequences complementary (in an antiparallel sense) to the purine part by a triple helix. The relative stabilities of triplexes were assessed by UV-absorption melting experiments as a function of pH and salt concentration. Hairpins carrying 8-aminopurines give very stable triple helical structures even at neutral pH, as confirmed by gel-shift experiments, circular dichroism and nuclear magnetic resonance spectroscopy. The modified hairpins may be redesigned to cope with small interruptions in the polypyrimidine target sequence.     

Resolution of parallel and antiparallel oligonucleotide triple helices formation and melting processes by multivariate curve resolution

A procedure is described for the complete resolution of concentration profiles of oligonucleotide triplexes as a function of pH and temperature. The pH and temperature ranges at which triplexes are present and the relative concentrations of all the species involved in acid-base and conformational equilibria are successfully estimated from Multivariate Curve Resolution analysis of UV absorbance spectra recorded along acid-base titrations and melting experiments of single stranded, hairpin and their mixtures. The dependence of formation constants upon pH was successfully estimated. The hairpin h26 (5'-GAAGGAGGAGA-TTTT-TCTCCTCCTTC-3'), and the single stranded oligonucleotides s11CT (5'-CTTCCTCCTCT-3'), s11AG (5'-AGAGGAGGAAG-3') and s11TG (5'-TGTGGTGGTTG-3') were synthesized and their protonation and conformational equilibria were studied in detail. The procedure was shown to be especially useful for the study of triplexes with a low hypochromism upon formation.     

Chickens fed with biomass of the red microalga Porphyridium sp. have reduced blood cholesterol level and modified fatty acid composition in egg yolk

The biomass of the red alga Porphyridium sp.constitutes a unique combination of soluble sulfatedpolysaccharide that accounts for about 70% of thealgal dry weight, and various polyunsaturatedfatty acids (PUFA) such as arachidonic andeicosapentaenoic acid (AA, 20:4 6 and EPA,20:5 3). In view of earlier results in ourlaboratory showing a reduction in serum cholesteroland triglyceride levels in rodents fed with red algalbiomass, we set out to examine the influence of algalbiomass as a feed additive on the metabolism ofchickens, with an emphasis on blood and eggcholesterol levels. For that purpose, lyophilizedalgal biomass was fed to 12–13, 30-week-old, WhiteLeghorn chickens for 10 days at a proportion of 5% or10% of the standard chicken diet. Twelve chickensfed with unsupplemented diet served as the control. No differences in body weight, egg number, and eggweight were found between the algal-fed chickens (atboth concentrations) and the control. However,chickens fed with algal biomass consumed 10% lessfood for both groups, and their serum cholesterollevels were significantly lower (by 11% and 28% forthe groups fed with 5% and 10% supplement,respectively) as compared with the respective valuesof the control group. Egg yolk of chickens fed withalgae tended to have reduced cholesterol levels (by10%) and increased linoleic acid and arachidonic acidlevels (by 29% and 24%, respectively). In addition,the color of the egg yolk was darker as a result ofthe higher carotenoid levels (2.4 fold higher) forchickens that fed with 5% supplement. Theseresults encourage the development of an improvedchicken feed having dietary fibers and polyunsaturatedfatty acids.