Androgen synthesis and aromatization by equine corpus luteum microsomes

Whereas mare corpus luteum does not produce androgens or estrogens in vivo, the incubation of mare corpus luteum microsomes with progesterone and NADPH resulted in 17 alpha-hydroxyprogesterone and estrogen production with a small yield of androstenedione. In the presence of an aromatase inhibitor (4-hydroxyandrostenedione), 17 alpha-hydroxyprogesterone and androstenedione were accumulated. Aromatization of testosterone and androstenedione occurred via stereospecific loss of the 1 beta, 2 beta hydrogen atoms and was inhibited by MgCl2, KCl, and EDTA. The Km of estrogen synthetase from equine corpus luteum for testosterone was 18.5 +/- 2.7 nM and for androstenedione was 11.5 +/- 1.5 nM. 19-Norandrogens were aromatized with a slightly higher efficiency than were androgens, but the affinity of the aromatase was lower for 19-norandrogens than for androgens. Our results suggest that aromatases from equine testis and corpus luteum are closely related enzymes. On the other hand, the question arises as to the relationship among the cell origin, the synthetizing abilities, and in vivo production of the corpus luteum in different mammalian species.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

Sequence-specific assignments of the 1H nuclear magnetic resonance spectra of reduced high-potential ferredoxin (HiPIP) from Chromatium vinosum.

The 1H resonances of the high-potential [4Fe-4S]2+ ferredoxin from Chromatium vinosum have been assigned through conventional sequential methodology applied to 2D NMR spectra. Almost 80% of the residues were identified using standard 2D COSY, HOHAHA, and NOESY pulse sequences. These residues correspond to four segments of the primary structure that do not interact strongly with the iron-sulfur cluster. A minor correction to the amino acid sequence is strongly suggested by these NMR data. Additional protons more sensitive to the proximity of the cluster were assigned by a combination of NOESY experiments with fast repetition rates and short mixing times and of HOHAHA spectra recorded with reduced spin-lock duration aimed at compensating for the short relaxation rates. Hence, the contributions of 79 residues out of 85 were identified in NMR spectra, among which the assignments of 64 residues were completed. Even the fastest relaxing protons, like those of the cysteine ligands, could be correlated, partly because the strong hyperfine shifts isolate them from the crowded diamagnetic region. However, other protons, in particular those involved in NH-S hydrogen bonds with the iron-sulfur cluster, were more difficult to identify, most probably because their relatively broad signals overlapped with those of protons not or less perturbed by the active site. The availability of the major part of the 1H NMR assignments has enabled the detection and identification of many interresidue NOESY cross peaks. These data are in full agreement with the elements of secondary structure previously revealed by X-ray crystallographic analysis of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identity of the RNA-binding protein K of hnRNP particles with protein H16, a sequence-specific single strand DNA-binding protein.

Protein H16, which we have identified previously in mammalian cell lines, binds in vitro to two single stranded DNA sites on the late strand of the early promoter of SV40. It has no other single strand binding site in the SV40 genome and does not bind to double stranded DNA. In vitro, H16 can be shown to stimulate strongly the activity of purified RNA polymerase II. Here we have purified this 70 kDa protein from cultured monkey cells and have sequenced three of its tryptic peptides. The analysis indicates that H16 is the simian homolog of human protein K, a nuclear RNA-binding protein found in heterogeneous nuclear ribonucleoprotein (hnRNP) particles, which contains a KH domain present in several proteins including the fragile X mental retardation gene product (FMR1). The binding affinities of protein K/H16 for RNA and DNA were subsequently compared in detail. They showed that under conditions where K/H16 binds strongly to its single stranded DNA site, it binds very weakly to the corresponding RNA sequence. This result suggests a possible shuttling of the protein from RNA to DNA during processes which involve opening of the DNA double helix.     

Linkage analysis of schizophrenia with five dopamine receptor genes in nine pedigrees

Alterations in dopamine neurotransmission have been strongly implicated in the pathogenesis of schizophrenia for nearly 2 decades. Recently, the genes for five dopamine receptors have been cloned and characterized, and genetic and physical map information has become available. Using these five loci as candidate genes, we have tested for genetic linkage to schizophrenia in nine multigenerational families which include multiple affected individuals. In addition to testing conservative disease models, we have used a neurophysiological indicator variable, the P50 auditory evoked response. Deficits in gating of the P50 response have been shown to segregate with schizophrenia in this sample and may identify carriers of gene(s) predisposing for schizophrenia. Linkage results were consistently negative, indicating that a defect at any of the actual receptor sites is unlikely to be a major contributor to schizophrenia in the nine families studied.     

Transient sex-related changes in the mice hypothalamo-pituitary-adrenal (HPA) axis during the acute phase of the inflammatory process

The potential role of endogenous sex hormones in regulating hypothalamo-pituitary-adrenal (HPA) axis function was investigated after a single injection of endotoxin in adult (8 week old) BALB/c mice of both sexes. The effect of LPS on plasma ACTH, corticosterone (B), testosterone and oestradiol (E) levels and on anterior pituitary (AP) ACTH and adrenal B contents at different times after treatment was studied. The results indicate that: (a) basal B but not ACTH plasma levels were significantly higher in female than in male mice; (b) LPS significantly increased both ACTH and B plasma levels over the baseline 2 h after injection, both hormone levels being higher in female than in male mice; (c) although plasma ACTH concentrations recovered the basal value at 72 h after LPS in animals of both sexes, plasma B levels returned to the baseline only at 120 h after treatment; (d) E plasma levels significantly increased 2 h after LPS and returned to the baseline at 72 h post-treatment, in both sexes; (e) at 2 h after LPS, testosterone plasma levels significantly decreased in male mice and increased in female mice, recovering the baseline level at 120 and 72 h after LPS, respectively; (f) AP ACTH content was similar in both sexes in basal condition and it was significantly diminished 72 h post-treatment without sex difference; whereas AP ACTH returned to basal content 120 h after LPS in males, it remained significantly decreased in females; (g) basal adrenal B content was higher in female than in male mice, and it significantly increased in both sexes 2 h post-LPS, maintaining this sex difference. Whereas adrenal B returned to basal content 72 h after treatment in male mice, it remained significantly enhanced up to 120 h post-LPS in female animals. The data demonstrate the existence of a clear sexual dimorphism in basal condition and during the acute phase response as well as in the recovery of the HPA axis function shortly after infection.     

A rapid assay for chloroplast-encoded triazine resistance in higher plants

We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through time and space and can serve as a model system applicable to other gene monitoring needs.     

Mating Call Discrimination in Female European (Coturnix c. coturnix) and Japanese Quail (Coturnix c. japonica)

Each year, thousands of domestic Japanese and hybrid quails are released within the breeding range of the European quail. We showed recently that no post‐zygotic isolating mechanisms have yet been established between these subspecies. The aim of this study was to investigate whether pre‐zygotic mechanisms are strong enough to prevent hybridization. We tested the level of subspecies selectivity in females of European and Japanese quail respectively using playbacks of European, hybrid and Japanese male mating calls. European quail females emitted the greatest number of rally calls in response to mating calls by conspecific males. Their responses were the weakest to mating calls produced by males of the other subspecies and intermediate to mating calls by hybrid males. In contrast, Japanese quails produced similar responses to all types of mating calls. These results suggest that mixed pairs could form in the wild. The European quail could thus become one of the most endangered galliforms of the Western Palearctic.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.