Detection of allergen specific immunoglobulins by microarrays coupled to microfluidics

Allergen microarrays are under development for a component‐resolved diagnosis of Type I (IgE‐mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA‐co‐NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software‐controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion‐limited to a kinetic‐limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.     

Characterisation of thermotolerant Saccharomyces cerevisiae hybrids

Thermotolerant Saccharomyces strains were crossed with mesophilic Saccharomyces cerevisiae and with cryotolerant Saccharomyces bayanus. The former hybrid is fertile confirming thermotolerant strains as S. cerevisiae. The spores from this hybrid, though, possess a low germinability and give cultures that grow poorly. The hybrid cryotolerant x thermotolerant is sterile and show a new combination of the parental strains' traits improving their technological application. © Rapid Science Ltd. 1998     

DAU 6285: a novel antagonist at the putative 5-HT4 receptor.

The antagonistic properties of DAU 6285, an azabicycloalkyl benzimidazolone derivative, at putative 5-hydroxytryptamine4 (5-HT4) receptors were investigated in in vitro preparations of guinea-pig ileum and human atrium, in comparison to ICS 205-930. DAU 6285 behaved as a competitive antagonist in all the preparations examined. Its affinity (pA2) ranged between 6.50 and 7.12 in the test models considered. The affinity of ICS 205-930 was 2-3 fold lower. At variance with ICS 205-930, DAU 6285 displayed a weak affinity for 5-HT3 receptors (pKi = 6.1, rat cortex; pA2 less than 5, guinea-pig ileum). In the guinea-pig ileum, DAU 6285 (10 microM) did not exert antimuscarinic, antihistaminic, antinicotinic or myolytic activity. Moreover, it did not bind to other 5-HT receptor subtypes, or to adrenergic, dopaminergic, benzodiazepine, nicotine, GABA receptors. DAU 6285 may represent a suitable tool for studies in the field of 5-HT4 receptors.     

Novel aromatase and 5α-reductase inhibitors

Inhibitors of aromatase and 5α-reductase may be of use for the therapy of postmenopausal breast cancer and benign prostatic hyperplasia, respectively. FCE 27993 is a novel steroidal irreversible aromatase inhibitor structurally related to exemestane (FCE 24304). The compound was found to be a very potent competitive inhibitor of human placental aromatase, with a K_i of 7.2 nM (4.3 nM for exemestane). In preincubation studies with placental aromatase FCE 27993, like exemestane, was found to cause time-dependent inhibition with a higher rate of inactivation ( ) and a similar K_i(inact) (56 vs 66 nM). The compound was found to have a very low binding affinity to the androgen receptor (RBA 0.09% of dihydrotestosterone) and, in contrast to exemestane, no androgenic activity up to 100 mg/kg/day s.c. in immature castrated rats. Among a series of novel 4-azasteroids with fluoro-substituted-17β-amidic side chains, three compounds, namely FCE 28260, FCE 28175 and FCE 27837, were identified as potent in vitro and in vivo inhibitors of prostatic 5α-reductase. Their IC₅₀ values were found to be 16, 38 and 51 nM for the inhibition of the human enzyme, and 15, 20 and 60 nM for the inhibition of the rat enzyme, respectively. When given orally for 7 days in castrated and testosterone (Silastic implants) supplemented rats, the new compounds were very effective in reducing prostate growth. At a dose of 0.3 mg/kg/day inhibitions of 42, 36 and 41% were caused by FCE 28260, FCE 28175 and FCE 27837, respectively.     

Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France.

Summary Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c₅₅₂ biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.Abbreviations BV Benzyl-Viologen - NTG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - NIR nitrite reductase - FR fumarate reductase - HYD hydrogenase - CYT c₅₅₂ cytochrome c₅₅₂     

Limitations on the use of polymerase chain reaction--restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the species Saccharomyces cerevisiae

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.     

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.