Immunodetection and immunopurification of aflatoxins using a high affinity monoclonal antibody to aflatoxin B1

A monoclonal antibody was obtained from BALB/c mice immunized with aflatoxin Bl (AFB1) conjugated to bovine serum albumin. This IgG2a antibody, ASCI, with K light chain has a high specificity for AFB1. In an indirect enzyme-linked immunosorbent assay the antibody litre in ascites fluid was 1: 6000 for 50% binding to plates coated with aflatoxin-poly-L-lysine. The assay is sensitive to 2.5 pg aflatoxin/assay. ASCI cross-reacts with closely related aflatoxin metabolites such as AFB2, AFM1 and AFG1. However, ASCI displays negligible cross-reactivity with other related aflatoxin analogues such as AFM2, AFP1, AFQ1 and aflatoxicol. An immunoabsorbent was prepared by coupling ASCI antibody to Ultrogel AcA 22. This immunomatrix was used to purify aflatoxins at 0–1 ng/ml levels from contaminated body fluids such as bovine milk. The antibody affinity column was regenerated and re-used several times. Owing to its high specificity for AFB1 and AFM1, ASCI will be of value in immunodetection and immunopurification of these toxins in various foodstuffs.     

Pulsed-field gel electrophoresis analysis of the genome of Streptomyces ambofaciens strains

The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.     

Melatonin Biosynthesis in Drosophila: Its Nature and Its Effects

Drosophila melanogaster homogenates incubated with tritiated 5-hydroxytryptophan, 5-hydroxytryptamine, or N-acetylserotonin have the ability of converting them into labelled indolamines, including melatonin. All these compounds were characterized by two-dimensional thin-layer chromatography by comparison with nonradiolabelled standards, and melatonin by mass spectrometry and radioimmunoassay. On the other hand, the injection of pharmacological doses of melatonin into 2-day-old female flies diminishes the mating speed and the oviposition rate.     

A Go-like protein in Drosophila melanogaster and its expression in memory mutants.

G proteins couple receptors for extracellular signals to several intracellular effector systems and play a key role in signalling transduction mechanisms. In particulate preparations of Drosophila melanogaster heads, only one substrate for pertussis toxin at 39-40 kd was detected. This substrate, which showed only one isoform when analysed by isoelectric focusing, was recognized by immunoblotting and immunoprecipitation techniques using a polyclonal antibody against the alpha subunit of the Go protein purified from bovine brain and can be thus considered as a Go-like protein. Antibodies obtained against a carboxy-terminal sequence of the alpha subunit of Go (but not of Gi1 or Gi2) and against an internal sequence shared by all the alpha subunits, were also able to cross-react with the alpha subunit of this protein in insects. We have also studied the Go-like protein in several D.melanogaster mutants, primarily in memory and learning mutants. In these mutants there was a sex-dependent enhancement in pertussis toxin-catalysed ADP-ribosylation with respect to the wild-type. This increase could be attributed in part to an increase in the alpha subunit of the Go-like protein, as revealed by immunoblotting with anti-Go alpha polyclonal antibody. This report constitutes the first evidence for the participation of a Go protein in learning and memory.     

Calamintha cretica (Lamiaceae), a Cretan endemic: Distribution and essential oil composition

The distribution of Calamintha cretica , a taxon restricted on the massif of Levka Ori (White Mountains W. Crete, Greece), is presented. The essential oils of three populations were examined by means of GC and GC-MS. The essential oil yield varied from 0.5% to 1.9%, whereas the major compounds were in all cases piperitenone oxide (26.4–41.3%) and piperitone oxide (33.8–59.9%). Like all other Calamintha taxa examined to date, it is a species rich in p -menthane compounds. The results are further discussed in relation to their chemotaxonomic value.     

Analyse des procédures du test de Hühner ou test postcoïtal tel qu'il est pratiqué en région Alsace

The Huhner test is an easy, unpainful, unexpensive test which must be done first at the time of unfertility exploration. His clinical and prognosticated interest is much debated because many imprecisions in its realisation and interpretation occur. Our multicentric study proves that this test is quite standardized in his realisation but a loss of its efficacity appears by the fact of a inadequate collaboration between attending physicians and biologists.     

Pulse fluorimetry study of energy transfers between tryptophan residues and NADPH in beef liver glutamate dehydrogenase complexes

A method is proposed to determine the rates of singlet energy transfers in an array of chromophores containing a finite number of donors and fluorescent acceptors. This method is based on measurements of transfer efficiency coupled with pulse fluorimetry. Three classes of donors can be distinguished which differ in their energy transfer rate. The rates of the first, the second and the third class are respectively greater than, of the order of, and smaller than the emission rate. The method is applied to the study of the energy transfers from tryptophan residues to NADPH, in ternary and quaternary glutamate dehydrogenase complexes. Practically, all these tryptophan residues belong to the first class. They can be divided into two subclasses having different transfer rate values. The distances between these residues and the NADPH site are of the order of 2.5 nm. In addition, the ligand binding induces a protein conformational change, leading to a fluorescence quenching of the tryptophanyl emission.     

The Muridae glyceraldehyde-3-phosphate dehydrogenase family

Summary Although only one gene is known to be functional, numerous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences are scattered throughoutMus musculus andRattus rattus genomes. In this report we show that: (1) GAPDH pseudogenes are repeated to comparable extents, at least 400 copies, in 12 other Muridae species; (2) the complete, or nearly so, sequence of GAPDH messenger RNA is amplified, and a high proportion, if not all of these copies, are intronless; (3) GAPDH pseudogenes are preferentially located in heavily methylated and DNAse I-insensitive regions of chromatin; and (4) the presence of atypical GAPDH-related mRNAs in different cellular contexts raises the possibility that more than one GAPDH gene is transcribed.     

The chemical basis for sex recognition in Drosophila melanogaster

Chemical cues were recognized to play a predominant role in initiating male courtship behaviour in Drosophila melanogaster as measured by the number and duration of wing-vibration responses elicited in test males. The effect was associated with compounds specific to the female cuticle, for which we describe a simple extraction procedure. Female active extracts were compared with behaviourally non-active extracts from males, using gas-liquid and thin-layer chromatography. Using these preparative methods, long-chain hydrocarbons were isolated and activity was found only among unsaturated molecules. One, heptacosadiene, inducing the highest level of courtship, appears to be the main aphrodisiac pheromone of the female D. melanogaster. This compound is specific to females of the species and is the most abundant of their cuticular hydrocarbons.     

Purification of adult Drosophila melanogaster lipophorin and its role in hydrocarbon transport

Lipophorin was isolated from homogenized adult Drosophila melanogaster. It is stained by Sudan Black and has a native molecular mass of 640 kD and a density of 1.12 g/ml. It consists of two glycosylated apoproteins of 240 and 75 kDa. Gas chromatography and mass spectrometry showed that lipophorins isolated separately from virgin 3-day-old male and female flies were associated with specific hydrocarbons, and that these were the same hydrocarbons found in male and female cuticles, respectively. Moreover, a pool of internal hydrocarbons was demonstrated for the first time, with chain lengths similar to those of the cuticular pool. Studies on the fate of the hydrocarbons synthesized de novo after topical applications of radiolabelled fatty acid precursors showed a decrease of the internal pool of hydrocarbons with time, concomitant with an increase of the cuticular pool. These results suggest that hydrocarbons synthesized at an internal site, possibly in oenocytes, may be transported to the cuticle of the flies by lipophorin. © 1996 Wiley-Liss, Inc.