Biosynthetic experiments with tall plants under field conditions. 18O2 incorporation into humulone from Humulus lupulus

Five segments of a large hop plant (Humulus lupulus var. Hallertauer Magnum) carrying several cones were enclosed in sealed glass vessels that were gassed with (18)O(2). After 14 days, the segments were harvested and humulone and cohumulone were analysed by NMR spectroscopy and mass spectroscopy. The oxygen atoms in position 6 of humulone and cohumulone showed 9% (18)O enrichment, respectively. It follows that the C-6 hydroxy groups were introduced by oxygenase catalysis.     

Cryptoendolithic actinomycetes from antarctic sandstone rock samples: Micromonospora endolithica sp. nov. and two isolates related to Micromonospora coerulea Jensen 1932

Three cryptoendolithic, aerobic actinomycetes (AA-459T, AA-319 and AA-321) from antarctic sandstone were characterised phenotypically and by molecular taxonomic methods. The isolates had single spores on substrate mycelium, meso-diaminopimelic acid (m-DAP) and glycine (cell wall type II), a whole cell sugar pattern D (galactose, xylose, arabinose, glucose or rhamnose) and phospholipids of type PII (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol). Their predominant fatty acids were iso-16:0 and iso-15:0 or 17:1omega8c, the menaquinone profile was complex with mainly MK10 (H4) and MK10 (H6). A wide variety of sugars and several acids were utilised for growth. The isolates were sensitive to a few antibiotics, but formation and excretion of antibiotics was not observed. Phenotypically, isolates AA-319 and AA-321 were similar. Phylogenetic analysis of 16S rRNA gene sequences revealed close relationship of strains AA-319 and AA-321 with each other (99.5%) and clustering (98.5%) with Micromonospora coerulea DSM 43143T. DNA-DNA hybridisation showed both strains to be genomically highly similar to strain DSM 43143T. Phenotypically they could be viewed as separate taxa, but presently they will be considered as strains of Micromonospora coerulea. Strain AA-459T was phylogenetically close to Micromonospora chersina DSM 44151T (99.1%) and to Micromonospora rosaria DSM 803T, but DNA-DNA similarity with M. chersina DSM 44151T was low with 28.9/33.5 %, indicating the presence of a different and new species. Consequently, isolate AA-459T (DSM 44398T NRRL B-24248T) is described as the type strain of Micromonospora endolithica sp. nov.     

Caries and periodontal disease of the elderly in Pomerania, Germany: results of the Study of Health in Pomerania

Objective: The aim of this study was to describe the oral health status of older adults living in north‐eastern Germany. Materials and Methods: Representative samples of adults aged 60 years or older were examined as part of Study of the Health in Pomerania, a cross‐sectional, population‐based study. Data on 1446 subjects aged 60–79 years were evaluated for coronal caries using the decayed/missing/filled teeth (DMFT) index, root caries using the root caries index (RCI), calculus, plaque, bleeding on probing, pocket depth and attachment loss. Results: The prevalence of edentulousness varied from 16% in the 60–65‐year‐old group to 30% in the 75–79‐year‐old group, whereas the median number of remaining natural teeth per subject varied from 14 in the youngest age group (60–65 years) to one in the oldest (75–79 years). Among subjects aged 60–69 years, a quarter (26%) of the teeth examined had coronal restoration against 17% in the oldest age group (70–79 years). Coronal caries was found in 2% of the teeth in both age groups. Among teeth with gingival recession, 6% had fillings on root surfaces and 2% had root caries, irrespective of age. In all, 11% of the subjects had at least one untreated coronal lesion and 27% had at least one untreated root caries lesion. Plaque score, calculus score and bleeding on probing were higher in the oldest age group (70–79 years). The prevalence of periodontal disease expressed as the presence of at least one periodontal pocket of 4 mm and more, was higher in men and among the younger subjects (men aged 60–69 years: 85% vs. 71% in 70–79‐year‐old men; women aged 60–69 years: 71% vs. 62% in 70–79‐year‐olds). The prevalence of attachment loss of 3 mm or more followed a similar pattern. Conclusions: It seems therefore that in this population, the major oral health concern is related to caries and the small number of teeth retained among the dentate subjects.     

Efficiency of microsatellite enrichment in<Emphasis Type="Italic">Prosopis chilensis</Emphasis> using magnetic capture

Microsatellites (i.e., simple sequence repeats [SSRs]) are highly variable genetic markers that are widely used at an intraspecific level in population genetic studies. Here we employed an enrichment strategy for microsatellite isolation by using microsatellite oligoprobes and magnetic capture of the fragments (Fischer and Bachmann, 1998) inProsopis chilensis (Mol.) Stuntz (Fabaceae). We analyzed the obtained level of enrichment by sequencing 120 enriched genomic fragments. A total of 521 SSR motives were detected. According to specific search criteria (SSR motifs ≥3 repeat units and ≥6 bp length), 95.8% of the clones contained SSR motifs. Of these, 7.8% showed homology to chloroplast sequences and 92.2% to nuclear sequences. When regarding only nuclear SSRs with 5 or more repeat units and a minimum length of 10 bp, the level of enrichment was 30.8%. A FASTA search against the European Molecular Biology Laboratory (EMBL) database univocally revealed 4 clones in transcribed regions, 102 clones in genomic regions with unknown function, and 9 clones in chloroplast regions. Among the loci with longer repeat units (≥10 bp, ≥5 repeat units), 3 were in transcribed regions and 65 were in other genomic regions. We discuss the applicability of these markers for population genetic studies.     

Secretion and molecular forms of NESP55, a novel genomically imprinted neuroendocrine-specific protein from AtT-20 cells

NESP55 (neuroendocrine secretory protein of M(r) 55,000) is a paternally imprinted proteoglycan, expressed specifically in endocrine cells and the nervous system. We investigated the subcellular localization and secretion of NESP55 in AtT-20 cells. NESP55 accumulated in the medium linearly over 24 h exceeding its intracellular content 3.7-fold by that time. Incubation of cells at 16 degrees C, to block protein export, inhibited basal secretion by 79%. Stimulation of AtT-20 cells with 8-Br-cAMP increased secretion of NESP55 by only 45%. The NESP55 secretory vesicles sedimented at a density of 1.2-1.4 M, which is slightly lighter than that of the large dense core vesicles. Immunofluorescence studies revealed immunoreactivity in the Golgi apparatus and a punctuate staining of processes or neurites. Our data demonstrate that NESP55 is mainly sorted to and released from a population of constitutive secretory vesicles, which are transported out of the perikarya into processes or axons. In addition, some NESP55 is also routed to the regulated pathway. The signal peptide of NESP55, as determined with peptide antisera, is 46 amino acids long and represents the best conserved region of this molecule suggesting that the signal peptide may have a function of its own. The subcellular localization and export of NESP55 from cells are reminiscent of neuronal proteoglycans forming the extracellular matrix, which are implicated in the development and maintenance of neuronal circuits and mechanisms of axonal guidance.     

Small molecule biaryl FSH receptor agonists. Part 1: Lead discovery via encoded combinatorial synthesis

High-throughput screening of two million compounds in 37 distinct encoded combinatorial libraries using FSH receptor transfected cells provided small molecule agonists such as 1 (EC(50)=3 microM) and 2 (EC(50)=3.9 microM), based on which a focused combinatorial library with a total of 31372 compounds was designed, synthesized, and screened to reveal 72 novel biaryl FSH receptor agonists such as 8a-c as well as a unique combinatorial SAR.     

Amino acids runs and genomic compositional biases in vertebrates

A compositional analysis of a sample of 50 zebrafish proteins containing at least one alanine run and of their open reading frames (ORFs) has been performed. The sample of poly(Ala) proteins showed a tendency to have runs of other amino acids (His/H, Gln/Q, Ser/S, Pro/P). Their ORFs and the first and second codon positions had higher GC contents than a reference gene set. The "universal" correlation between the GC content of the first+second and third codon positions (GC1+2 vs GC3) does not hold, but I provide an explanation in terms of genomic heterogeneity. Significant correlation between AHQS content and GC3 was obtained, reflecting codon bias favoring G/C at the third codon position of these amino acids. A correspondence analysis (COA) of relative synonymous codon usage showed that the poly(Ala) proteins have a biased distribution according to the second axis of the COA, which correlates with gene expression in zebrafish. A comparison with human is undertaken.     

Evaluation of diagnostic relevance of mRNA levels in peripheral blood: predictive value for mortality in hemodialysis patients

In clinical practice, diagnosis and risk prediction are usually based on the analysis of serum or plasma proteins whereas gene expression analysis is not used on a routine basis. In order to compare the diagnostic and predictive relevance of serum protein and peripheral blood mRNA levels, we determined cytokine levels of end-stage renal failure patients undergoing hemodialysis. These patients face a high mortality mainly due to acceleration of atherosclerosis and subsequent severe vascular events. mRNA expression of the pro-inflammatory cytokine TNF alpha was significantly elevated in hemodialysis patients and further increased after 2 h of dialysis treatment. In contrast, gene expression of the anti-inflammatory cytokine TGF beta was significantly decreased. Patients who died during the observation period of 36 months had significantly increased mRNA levels of TNF alpha and decreased TGF beta mRNA expression at baseline. Survival analysis indicated that increased TNF alpha mRNA levels (P < 0.02) and TNF alpha/TGF beta mRNA ratios (P < 0.001) predict mortality. The corresponding cytokines in serum showed some association with disease, but serum concentrations neither changed during hemodialysis nor predicted mortality. This study shows that gene expression patterns of circulating leukocytes may present an important new diagnostic tool to predict clinical outcome in patients with inflammatory vascular diseases.     

beta-Amyloid efflux mediated by p-glycoprotein

A large body of evidence suggests that an increase in the brain beta-amyloid (Abeta) burden contributes to the etiology of Alzheimer's disease (AD). Much is now known about the intracellular processes regulating the production of Abeta, however, less is known regarding its secretion from cells. We now report that p-glycoprotein (p-gp), an ATP-binding cassette (ABC) transporter, is an Abeta efflux pump. Pharmacological blockade of p-gp rapidly decrease extracellular levels of Abeta secretion. In vitro binding studies showed that addition of synthetic human Abeta1-40 and Abeta1-42 peptides to hamster mdr1-enriched vesicles labeled with the fluorophore MIANS resulted in saturable quenching, suggesting that both peptides interact directly with the transporter. Finally, we were able to directly measure transport of Abeta peptides across the plasma membranes of p-gp enriched vesicles, and showed that this phenomenon was both ATP- and p-gp-dependent. Taken together, our study suggests a novel mechanism of Abeta detachment from cellular membranes, and represents an obvious route towards identification of such a mechanism in the brain.     

Structural analysis of CTLA-4 function in vivo

CTLA-4-mediated inhibition of T cell activation may be accomplished by competition for ligands and/or by signals mediated through the intracellular domain. Studies have implicated Tyr201 in the cytoplasmic domain of CTLA-4 in regulating CTLA-4 signal transduction and intracellular trafficking. To investigate the mechanism of CTLA-4 function in vivo, transgenes encoding wild-type CTLA-4 (FL), a mutant lacking the cytoplasmic domain of CTLA-4 (DeltaCTLA-4 tail), or a CTLA-4 Tyr201 mutant (Y201V) were introduced into CTLA-4-deficient mice. CTLA-4-/- mice display an autoimmune lymphoproliferative disorder resulting in tissue destruction and early death. When either the FL or the Y201V transgene was bred into CTLA-4-/- animals, a complete rescue from lymphoproliferation and autoimmunity was observed. In contrast, CTLA-4-/- mice expressing the DeltaCTLA-4 tail transgene were long lived with no evidence of multiorgan lymphocytic infiltration, but exhibited lymphadenopathy and accumulated large numbers of activated T cells. Furthermore, these animals displayed a Th2-biased phenotype which conferred susceptibility to Leishmania infection. These results indicate that the inhibitory effect of CTLA-4 is mediated in part through the ability of the extracellular domain to compete for ligands. The cytoplasmic domain of CTLA-4, however, is required for complete inhibitory function of the receptor and for regulation of Th cell differentiation in vivo.