Novel synthesis of 2'-O-methylguanosine

An efficient and chemoselective synthesis of 2'-O-methylguanosine (6) has been accomplished in high yield without protection of the guanine base. The salient feature of the synthesis of 6 lies in the application of methylene-bis-(diisopropylsilyl chloride), (MDPSCl(2), 2) as a new 3',5'-O-protecting group for nucleosides. Use of CH(3)Cl as a weak electrophile and NaHMDS as a mild base was crucial to the success of the 2'-O-methylation of 3',5'-O-protected guanosine.     

CFTR gene mutations – including three novel nucleotide substitutions – and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.     

Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato

We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.     

Vibrational spectroscopic characterization of new calcium phosphate bioactive coatings

In this work calcium phosphate (CaP) compounds with different PO(3-)(4)/HPO(2-)(4) R molar ratios in the 0.65-149 range were synthesized. In fact, all these CaPs contain different amounts of HPO(2-)(4) and PO(3-)(4) ions as well as the amorphous precursors (tricalcium phosphate and octacalcium phosphate) of hydroxyapatite deposition, which was shown by in vitro and in vivo measurements. Spectroscopical IR and Raman results showed the presence of bands whose intensity ratio can be related to the molar ratio R; in particular, the Raman I(962)/I(987) and the IR I(1035)/I(1125) intensity ratios were characterized as markers of the molar ratio. For these CaP compounds a nucleation model, which was based on the ability of HPO(2-)(4) ions to form strong H bonds with PO(3-)(4) ions, was proposed.     

Disturbance and Seasonal Dynamics of Mycorrhizae in a Tropical Deciduous Forest in Mexico1

Mycorrhizal fungi were sampled in a deciduous tropical forest on the Pacific coast of Mexico during different seasons and in natural treefall gaps and pastures. All 12 plant species sampled in the forest were arbuscular mycorrhizal. The percent root infection and spore production were closely related to the phenology of the plants. Most tree species and all herbaceous species had the highest infection in the summer rainy season, but two species, Opuntia excelsa and Jacquinia pungens, had highest infection in the dry season. Unusually high rainfall during the dry season was associated with increased infection but not increased spore production. Spore density was low for all species at all sample times, except at the beginning of the July 1993 rainy season in, when we observed up to 28 spores/g soil. The percent cover of shrubs or herbs did not increase in gaps after two years, and we observed no colonizing seedlings. No plant species with cover higher than 2.7 percent occurred exclusively in gaps or forest. The percent mycorrhizal infection did not differ significantly between gaps and forest. Spore counts were as high in the gaps as in the forest in two of the three gaps but lower in the third gap. The lack of significant response of plants in these gaps after two years differed from the rapid response in tropical rainforests. It is likely related to the small size of the gaps and to light infiltration to the forest floor. Pastures were dominated by two species of exotic grasses and one species of mycorrhizal fungus, whereas forests had 15 fungal species. The slow regrowth of vegetation in gaps was not limited by mycorrhizal fungi, since they were still abundant after the treefalls, but recovery in pastures could be affected by low fungal diversity and dominance of grasses.     

Dislocation of Type I Membrane Proteins from the ER to the Cytosol Is Sensitive to Changes in Redox Potential

The human cytomegalovirus (HCMV) gene products US2 and US11 dislocate major histocompatibility class I heavy chains from the ER and target them for proteasomal degradation in the cytosol. The dislocation reaction is inhibited by agents that affect intracellular redox potential and/or free thiol status, such as diamide and N-ethylmaleimide. Subcellular fractionation experiments indicate that this inhibition occurs at the stage of discharge from the ER into the cytosol. The T cell receptor α (TCR α) chain is also degraded by a similar set of reactions, yet in a manner independent of virally encoded gene products. Diamide and N-ethylmaleimide likewise inhibit the dislocation of the full-length TCR α chain from the ER, as well as a truncated, mutant version of TCR α chain that lacks cysteine residues. Cytosolic destruction of glycosylated, ER-resident type I membrane proteins, therefore, requires maintenance of a proper redox potential for the initial step of removal of the substrate from the ER environment.     

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Résumé

À l'inverse de la situation des vertébrés et de la majorité des insectes, chez qui un gène unique code pour l'acétylcholinestérase (AChE), quatre gènes d'AChE ont été clones et caractérisés chez Caenorhabditis elegans. Le gène ace-1 (localisé sur le chromosome X) et le gène ace-2 (chromosome I) assurent respectivement l'expression de l'AChE dans les tissus musculaire (ace-1) et nerveux (ace-2). Les gènes ace-x et ace-y ne sont séparés que de quelques centaines de paires de bases sur le chromosome II et leur rôle est pour l'instant inconnu.     

A Laboratory Study of Sleep and Dreaming in a Case of Asperger's Syndrome

Asperger's Syndrome (AS) is a pervasive developmental disorder whose continuity with High-Functioning Autism is still a matter of debate. Clinical observations suggest that patients with AS may present the same sleep disorders as autistic patients, including difficulties in initiating and maintaining sleep as well as poor dream recall. We recorded the sleep of a 25-year-old male patient with AS for two nights using a full EEG montage and compared the second night to that of a group of normal participants. We found low levels of slow wave sleep (SWS: stages 3 + 4), high levels of stage 1, and a large number of awakenings. The organization of REM sleep was unremarkable, including normal REM density. Analyses of phasic EEG events revealed a very low incidence of sleep spindles and a normal number of K-complexes over bilateral frontal and central EEG leads. In order to collect dream reports, the patient was awakened three times over two nights following at least 15 minutes of REM sleep in each case. On each occasion the patient was not aware of any mental activity happening just prior to awakening. These observations are discussed with regards to the connections that may exist between EEG sleep spindle activity, selective attention, and the capacity to generate a dream report.