Analysis of human V H gene repertoire expression in peripheral CD19+ B cells

Using CD19 B-cell selection and polymerase chain reaction-amplified cDNA libraries, we analyzed the peripheral immunoglobulin heavy chain variable repertoire of three healthy adult donors. Here we report that most of the CD19+ circulating B cells expressed unmutated V _H-D-J_H rearrangements. By specific V _H family hybridization, we show that V _H gene family utilization in the periphery roughly corresponds to the complexity of these families in the germline and appears to be relatively constant among the analyzed subjects. However, sequence data of clones picked at random from one IgM cDNA library reveals that in spite of this random utilization, the V _H gene expression in naive circulating B cells is highly biased towards the expression of a limited set of V _H genes. As previously reported by others, this restricted mechanism is also found for the D and J _H segments.The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL nucleotide sequence database and have been assigned the accession numbers Z47213-Z47243 and Z47349     

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABA_A receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [³H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [³H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [³H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABA_A receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABA_A receptors. The presence of α-subunit(s), as revealed by [³H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABA_A receptors are extrasynaptic and expressed in astroglia.     

A review of issues related to the quantification of bacteria from the phyllosphere

Abstract: Quantification of the size of epiphytic bacterial populations and characterization of their composition involves definition of a sampling strategy in time and space, the choice of methods for liberating the bacteria from the leaf surface and for recovering them for subsequent determination of the number of viable or culturable cells. This literature review focuses on some of the issues related to these choices. After briefly reviewing the different types of epiphytic colonizers we consider the biological, methodological, and statistical consequences of the choice of the sampling unit and of the spatial and temporal variability of population size and composition for epiphytic bacteria. The different methods available for the detection and enumeration of naturally occurring microorganisms in the phyllosphere are discussed. Advantages and drawbacks of each are described in this review designed as a 'hands-on' guide.     

Studies of mercurated derivatives of acetylacetone and ethyl acetoacetate by solid state nuclear magnetic resonance and vibrational spectroscopies

Seven new mono- and/or dimercurated compounds involving acetylacetone (2,4-pentanedione) or ethyl acetoacetate (3-ethyl ketobutanoate) were synthesized in aqueous medium. In all cases, mercuration occurred at methylene carbon atoms. All compounds were carefully analyzed by solid state carbon-13 nuclear magnetic resonance. Assignments were confirmed by using selective sequences, which allowed a total editing of the spectra. It was shown that deshielding of ¹³C mercurated sites occurred when the rate of mercuration increased. It was also possible to measure direct ¹J(¹⁹⁹Hg¹³C) coupling constants. The main bands of the vibrational spectra (infrared and Raman) were assigned. It was proved that v(C = O) and δ(C(γ)-H) could be directly related to the mercuration rate of molecules.     

Endothelin Stimulates Phospholipase D in Striatal Astrocytes

Abstract: In primary cultures of mouse striatal astrocytes prelabeled with [³H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [³H]phosphatidic acid and [³H]diacylglycerol. In the presence of ethanol, a production of [³H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC₅₀ = 2–5 n M ). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a G_i/G_o protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [³H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally depended on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.     

Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl₂ with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl₂, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.     

Structure-function relationships and molecular genetics of the 3β-hydroxysteroid dehydrogenase gene family

The isoenzymes of the 3β-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3β-HSD) gene family catalyse the transformation of all 5-ene-3β-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3β-hydroxy- and 3-keto-5-androstane steroids. The two human 3β-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3β-HSD isoenzymes prefer NAD⁺ to NADP⁺ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr³⁶ in the rat type III and of Phe³⁶ in mouse type IV enzymes instead of Asp³⁶ found in other 3β-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3β-HSD proteins possess an intrinsic 17β-HSD activity psecific to 5-androstane 17β-ol steroids, thus suggesting that such “secondary” activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3β-HSD deficiency, the structures of the types I and II 3β-HSD genes in 12 male pseudohermaphrodite 3β-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3β-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3β-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3β-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (1%). Mutations found in nonsalt-loser patients have some residual activity ranging from 1 to 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3β-HSD superfamily.