Structural investigation of the ligand binding domain of the zebrafish VDR in complexes with 1alpha,25(OH)2D3 and Gemini: purification, crystallization and preliminary X-ray diffraction analysis

The nuclear receptor of Vitamin D can be activated by a large number of agonist molecules with a wide spectrum in their stereochemical framework. Up to now most of our structural information related to the protein-ligand complex formation is based on an engineered ligand binding domain (LBD) of the human receptor. We now have extended our database, using a wild-type LBD from zebrafish that confirms the previously reported results and allows to investigate the binding of ligands that induce significant conformational changes at the protein level.     

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V_H families/segments was observed showing that ONCL did not introduce cloning biases for or against any V_H family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 10¹⁰ and by selecting five unique Fabs against GAPDH antigen.     

Carnosine Release from Olfactory Bulb Synaptosomes Is Calcium-Dependent and Depolarization-Stimulated

Abstract: The dipeptide carnosine (β-alanyl-L-histidine) has been proposed as a neurotransmitter in the mammalian olfactory pathway. Therefore, the efflux of in vivo -synthesized [¹⁴C]carnosine from mouse olfactory bulb synaptosomes was investigated. Carnosine was found to be released from the olfactory bulb synaptosomes by two mechanisms. The first is a slow spontaneous process that is independent of depolarization. The rate of this release was doubled in the presence of 1 m M external carnosine. Release by the second mechanism was markedly stimulated in the presence of calcium by depolarization with either 60 m M K⁺ or 300 μ M veratridine. Omission of calcium abolished the stimulatory effect of both of these agents. Further, blockage of the veratridine-induced depolarization by tetrodotoxin also inhibited carnosine release. These results are consistent with the hypothesis that carnosine acts as a neurotransmitter in the mouse olfactory pathway.     

Antepartum Membrane Stripping in GBS Carriers,Is It Safe? (The STRIP-G Study)

Objective

Stripping of the membranes is an established and widely utilized obstetric procedure associated with higher spontaneous vaginal delivery rates, reduced need for formal induction of labor and a lower likelihood of post-term pregnancy. Due to the theoretical concern of bacterial seeding during the procedure many practitioners choose not to sweep the membranes in Group B Streptococcus (GBS) colonized patients. We conducted ‘the STRIP-G study’ in order to determine whether maternal and neonatal outcomes are affected by GBS carrier status in women undergoing membrane stripping.

Study design

We conducted a prospective study in a tertiary referral center, comparing maternal and neonatal outcomes following membrane stripping among GBS-positive, GBS-negative, and GBS-unknown patients. We compared the incidence of composite adverse neonatal outcomes (primary outcome) among the three study groups, while secondary outcome measure was composite adverse maternal outcomes.

Results

A total of 542 women were included in the study, of which 135 were GBS-positive, 361 GBS-negative, and 46 GBS-unknown status. Demographic, obstetric, and intra-partum characteristics were similar for all groups. Adverse neonatal outcomes were observed in 8 (5.9%), 31 (8.6%), and 2 (4.3%) in the GBS-positive, GBS-negative, and unknown groups, respectively (P = 0.530), (Odds Ratio between GBS-Positive vs. GBS-Negative groups 0.67 (95%, CI = 0.30–1.50)); while composite adverse maternal outcomes occurred in 9 (6.66%), 31 (8.59%), and 5 (10.87%) in the GBS-positive, GBS-negative, and unknown groups, respectively (P = 0.617).

Conclusions

Antepartum membrane stripping in GBS carriers appears to be a safe obstetrical procedure that does not adversely affect maternal or neonatal outcomes.     

Strigolactones interact with ethylene and auxin in regulating root-hair elongation in Arabidopsis

Strigolactones (SLs) or derivatives thereof have been identified as phytohormones, and shown to act as long-distance shoot-branching inhibitors. In Arabidopsis roots, SLs have been suggested to have a positive effect on root-hair (RH) elongation, mediated via the MAX2 F-box. Two other phytohormones, auxin and ethylene, have been shown to have positive effects on RH elongation. Hence, in the present work, Arabidopsis RH elongation was used as a bioassay to determine epistatic relations between SLs, auxin, and ethylene. Analysis of the effect of hormonal treatments on RH elongation in the wild type and hormone-signalling mutants suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs, whereas the effect of SLs on RH elongation requires ethylene synthesis. SL signalling was not needed for the auxin response, whereas auxin signalling was not necessary, but enhanced RH response to SLs, suggesting that the SL and auxin hormonal pathways converge for regulation of RH elongation. The ethylene pathway requirement for the RH response to SLs suggests that ethylene forms a cross-talk junction between the SL and auxin pathways.     

Correction method for null alleles in species with variable microsatellite flanking regions, a case study of the black-lipped pearl oyster Pinctada margaritifera

In bivalves, heterozygote deficiencies and departures from Hardy-Weinberg equilibrium (HWE) in microsatellite analysis are common and mainly attributed to inbreeding, genetic patchiness (Walhund effect), or null alleles. We checked for the occurrence of null alleles at 3 microsatellite loci in 3 populations of black-lipped pearl oyster, Pinctada margaritifera, using a step-by-step method to re-amplify homozygotes and null individuals with redesigned primer pair combinations. After amplification with original primer pairs, the 3 populations exhibited null alleles, absence of structure, and significant departure from HWE for all 3 loci due to heterozygote deficiencies. After 3 re-amplification steps, with modified primer sets, all loci were corrected for null alleles. Once corrected, all populations appeared at HWE, demonstrating that null alleles were responsible for the initial disequilibrium of the populations. Furthermore, analysis from corrected genotypes demonstrates significant genetic differentiation for one population from the other 2.     

Strigolactones affect lateral root formation and root-hair elongation in Arabidopsis

Strigolactones (SLs) have been proposed as a new group of plant hormones, inhibiting shoot branching, and as signaling molecules for plant interactions. Here, we present evidence for effects of SLs on root development. The analysis of mutants flawed in SLs synthesis or signaling suggested that the absence of SLs enhances lateral root formation. In accordance, roots grown in the presence of GR24, a synthetic bioactive SL, showed reduced number of lateral roots in WT and in max3-11 and max4-1 mutants, deficient in SL synthesis. The GR24-induced reduction in lateral roots was not apparent in the SL signaling mutant max2-1. Moreover, GR24 led to increased root-hair length in WT and in max3-11 and max4-1 mutants, but not in max2-1. SLs effect on lateral root formation and root-hair elongation may suggest a role for SLs in the regulation of root development; perhaps, as a response to growth conditions.