Synthesis and Some Properties of Modified Oligonucleotides. II. Oligonucleotides Containing 2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.     

Synthesis of 2′-Substituted Inosine Analogs via Unusual Masking of the 6-Hydroxyl Group

2′-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate was obtained via an unusually facile dimethylamine transfer from dimethylformamide.

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Use of a Base-Labile Protected Derivative of 6-Mercaptohexanol for the Preparation of Oligonucleotides Containing a Thiol Group at the 5′-End

Abstract

The preparation of a base-labile (Dnpe) protected derivative of 6-mercaptohexanol is described. The use of the phosphoramidite derivative of this compound improves both yields and the time needed for the preparation of oligonucleotides containing a thiol group at the 5′-end.     

Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10–17 yr and 18–50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization–MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer''s disease, which is frequently associated with Down syndrome.Down syndrome (DS)¹ is a frequent genetic disorder in humans characterized by premature aging (1). A clinical feature of people with DS is the increased prevalence and severity of periodontal disease compared with age-matched subjects of similar levels of intellectual impairment and compared with the general population (2). Common conditions observed in DS are marginal gingivitis, acute and subacute necrotizing gingivitis, advanced periodontitis, gingival recession, and pocket formation (3, 4). It is known that saliva plays an important role in maintaining oral and dental health, because of the presence of a variety of antimicrobial peptides mainly derived from gland secretion, oral epithelial cells, and neutrophils (5). Several papers reported that neutrophils and T-lymphocyte function is impaired in people with DS (6–9). However, the salivary secretion of the antimicrobial LL-37 in young individuals with DS was found normal (10). A review of the literature (11, 12) reveals only sporadic and contradictory reports that attempt to explain the role of saliva in the oral health of subjects with DS, and on the whole, information on the biochemical composition of their saliva is scarce. On the basis of the above information, in the present study, we proposed to investigate the salivary proteome of DS subjects by an intact protein-based “top-down” approach. The spectrum of salivary peptides of DS subjects was compared with that of sex and age-matched healthy control groups to determine qualitative and quantitative differences. Interestingly, the results showed that several members of the S100A family, which possess different biological functions, and also described as potential markers of the Alzheimer Disease, were significantly increased in saliva of Down syndrome subjects with respect to controls.     

Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD⁺ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.     

Hypolipidemic and antiatherosclerotic potential of Pleurotus ostreatus, cultived by submerged fermentation in the high-fat diet fed rats

The ability of Pleurotus ostreatus biomass, cultived by submerged fermentation, to produce beneficial effect on lipid profile and macrophages activity during a high-fat diet (HFD) for a long-term intake was investigated. Blood samples were collected through cardiac puncture to measure the plasma cholesterol, triglycerides, low-density protein (LDL), high-density protein (HDL), aspartate aminotransferase (AST) activity, urea-blood urea nitrogen (BUN)/creatinine ratio of rats fed on an HFD for 4 months. Dosage of lipid hydroperoxides was carried out on methanolic extract of liver tissue. Peritoneal macrophages activity was evaluated in relation to the superoxide anion, hydrogen peroxide and nitric oxide production, phagocytosis and lysosomal volume. The administration of P. ostreatus significantly altered the lipid profile and oxidative stress as related to the LDL and triglycerides decrease and inhibitory effects on superoxide anion and hydrogen peroxide production. All findings of this study lead us to suggest that the P. ostreatus maybe a beneficial agent in the hyperlipidemia and atherosclerosis treatments.     

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

Mutational analysis in podocin-associated hereditary nephrotic syndrome in Polish patients: founder effect in the Kashubian population

Hereditary nephrotic syndrome is caused by mutations in a number of different genes, the most common being NPHS2. The aim of the study was to identify the spectrum of NPHS2 mutations in Polish patients with the disease. A total of 141 children with steroid-resistant nephrotic syndrome (SRNS) were enrolled in the study. Mutational analysis included the entire coding sequence and intron boundaries of the NPHS2 gene. Restriction fragment length polymorphism (RFLP) and TaqMan genotyping assay were applied to detect selected NPHS2 sequence variants in 575 population-matched controls. Twenty patients (14 %) had homozygous or compound heterozygous NPHS2 mutations, the most frequent being c.1032delT found in 11 children and p.R138Q found in four patients. Carriers of the c.1032delT allele were exclusively found in the Pomeranian (Kashubian) region, suggesting a founder effect origin. The 14 % NPHS2 gene mutation detection rate is similar to that observed in other populations. The heterogeneity of mutations detected in the studied group confirms the requirement of genetic testing the entire NPHS2 coding sequence in Polish patients, with the exception of Kashubs, who should be initially screened for the c.1032delT deletion.     

Ross Sea Mollusca from the Latitudinal Gradient Program: R/V Italica 2004 Rauschert dredge samples

Information regarding the molluscs in this dataset is based on the Rauschert dredge samples collected during the Latitudinal Gradient Program (LGP) on board the R/V “Italica” in the Ross Sea (Antarctica) in the austral summer 2004. A total of 18 epibenthic dredge deployments/samplings have been performed at four different locations at depths ranging from 84 to 515m by using a Rauschert dredge with a mesh size of 500μm. In total 8,359 specimens have been collected belonging to a total of 161 species. Considering this dataset in terms of occurrences, it corresponds to 505 discrete distributional records (incidence data). Of these, in order of abundance, 5,965 specimens were Gastropoda (accounting for 113 species), 1,323 were Bivalvia (accounting for 36 species), 949 were Aplacophora (accounting for 7 species), 74 specimens were Scaphopoda (3 species), 38 were Monoplacophora (1 species) and, finally, 10 specimens were Polyplacophora (1 species). This data set represents the first large-scale survey of benthic micro-molluscs for the area and provides important information about the distribution of several species, which have been seldom or never recorded before in the Ross Sea. All vouchers are permanently stored at the Italian National Antarctic Museum (MNA), Section of Genoa, enabling future comparison and crosschecking. This material is also currently under study, from a molecular point of view, by the barcoding project “BAMBi” (PNRA 2010/A1.10).