Production of Guanine from NH4CN Polymerizations

The synthesis of adenine from the polymerization of concentrated ammonium cyanide solutions is well known. We show here that guanine is also produced by this reaction but at yields ranging from 10 to 40 times less than that of adenine. This synthesis is effective at both +80 and −20°C. Since high concentrations of NH₄CN are obtainable only by freezing, this prebiotic synthesis would be applicable to frozen regions of the primitive Earth, the Jovian satellite Europa and other icy satellites, and the parent body of the Murchison meteorite. Received: 18 September 1998 / Accepted: 31 March 1999     

Apoptose au cours de la spermatogenèse et dans le sperme éjaculé

It has become clear in recent years that programmed cell death occurs spontaneously in the cycle of the seminiferous epithelium. Induced germ cell apoptosis occurs at specific stages of the spermatogenic cycle and the existence of supracellular control of germ cell death during spermatogenesis has been documented. If apoptosis is a key phenomenon in the control of sperm production, the existence and role of apoptosis in ejaculated sperm cells remain controversial. Apoptosis — as determined by DNA fragmentation (TUNEL) and ultrastructural analysis — is abnormally frequent in the sperm cells of the ejaculate of sterile men with classical biochemical and ultrastructural pattern. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa and the consequences of DNA damage if the apoptotic spermatozoa is used for ICSI. Percentages of DNA fragmentation in human ejaculated sperm are correlated with fertilization rates both after FIV and ICSI. Detection of DNA fragmentation in human sperm could provide additional information about the biochemical integrity of sperm and may be used in future studies for fertilization failures not explained by conventional sperm parameters. However, the analysis of other molecular markers of apoptosis (Fas, Annexine V ...) is necessary to assess the role of apoptosis in human ejaculated sperm cells.     

Size distribution and buoyant density of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis>

The size and density of microbial cells determine the time that pathogens can remain airborne and thus, their potential to infect by the respiratory route. We determined the density and size distribution of Burkholderia pseudomallei cells in comparison with other Burkholderia species, including B. mallei and B. thailandensis, all prepared and analyzed under similar conditions. The observed size distribution and densities of several bacterial strains indicates that aerosolized particles consisting of one or of a few B. pseudomallei cells should be efficiently retained in the lungs, highlighting the risk of transmission of melioidosis by the respiratory route when the pathogen is present in fluids from infected patients or aerosolized from the environment.     

Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA

In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E. coli. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.     

An integrative approach combining ion mobility mass spectrometry,X-ray crystallography,and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α1-antitrypsin upon ligand binding

Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α₁-antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α₁-antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α₁-antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α₁-antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.PDB Code(s): 4PYW     

A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.     

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 μm from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.     

Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D3

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.     

Identification of the hepatocyte Na+-dependent bile acid transport protein using monoclonal antibodies

Monoclonal antibodies have been utilized to characterize the hepatocyte Na+-dependent bile acid transport system. Sinusoidal plasma membrane proteins in the 49-54-kDa range, which are thought to be components of this transport system, based on photo-affinity labeling and reconstitution studies, have been partially purified by affinity chromatography and utilized as an immunogen for the production of a panel of monoclonal antibodies (mAb). One of these mAbs, 25A-3, recognized both a 49- and a 54-kDa protein as assessed by immunoprecipitation. In addition, it was shown to protect the bile acid transport system from inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a dose-dependent manner. DIDS covalently labeled membrane proteins of 49 and 54 kDa, and this process could be significantly inhibited when performed in the presence of mAb 25A-3. Furthermore, the DIDS-labeled membrane proteins were immunoprecipitated by 25A-3. These results establish that one of these membrane components is the bile acid carrier protein. Another mAb (25D-1) which immunoprecipitated only a 49-kDa protein was shown to block the protective effect of 25A-3 on DIDS inhibition of bile acid transport. In addition both antibodies effected each other's binding capacity to hepatocytes and reacted with the same 49-kDa protein as established by sequential immunoprecipitation. Binding studies indicated that there are approximately 3.3 X 10(6) 49-kDa transport molecules/hepatocyte. These results firmly establish that the 49-kDa protein is the Na+-dependent hepatocyte bile acid transporter.