The role of fluorinated pyrimidine analogues in the induction of the in vitro expression of the fragile X chromosome

Summary The modes of action of 5-fluoro-2-deoxyuridine (FdUrd) and 5-fluoro-2-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3x10^-8 M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect. Second, inhibition of the in situ thymidylate synthetase activity measured as an increase in the level of [³H]-dThd incorporation coincided with the inhibition of cell proliferation. Third, the inhibition of the thymidylate synthetase-dependent incorporation of [³H]-dUrd into newly synthesized DNA coincided with the inhibition of cell proliferation. The effects of FdUrd and FdCyd on the in vitro expression of fragile site Xq27 of fragile X chromosomes was shown to be based on the depletion of the intracellular pool of thymidine-5-monophosphate (dTMP), as fudged from the following observations: First, both the FdUrd- and FdCyd-dependent induction of site Xq27 coincided with the antiproliferative effects of the respective fluoropyrimidines. Second, addition of thymidine (dThd) to the culture medium both prevented the expression of site Xq27 and neutralized the cytotoxicity of FdUrd and of FdCyd. On the basis of these findings, we provide further evidence for the concept that the fragile X site is located in an AT-rich region.     

Influence of the phosphorus and nitrogen source on nisin production in Lactococcus lactis subsp. lactis batch fermentations using a complex medium

The influence of different phosphorus and nitrogen sources on Lactococcus lactis subsp. lactis NIZO 22186 growth and nisin production was studied in batch fermentations using a complex medium. KH₂PO₄ was found to be the best phosphorus source for nisin production. Increasing initial phosphate concentrations from 0 to 5% KH₂PO₄ exerted a double effect, creating favourable pH conditions and particularly stimulating the nisin production levels, which were highest at 5% KH₂PO₄. Up to now, no such high initial phosphate concentrations have been reported for the production of other antibiotics or bacteriocins. Nisin, a lanthionine-containing peptide antibiotic with bacteriocin properties, clearly behaved as a primary metabolite, since its formation was linked with active growth and was not suppressed by phosphate concentrations up to 5%. A complex medium supplemented with cotton seed meal as nitrogen source also gave very high nisin yields. Correspondence to: L. De Vuyst     

Cyclitol metabolism is a central feature of Burkholderia leaf symbionts

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant–bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled 17 new endophyte genomes representing endophytes from 13 plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.     

La procréation médicalement assistée avec spermatozoïdes immatures: Impératifs cliniques et techniques chirurgicales

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.     

Construction and characterization of a functional chimeric murine-human antibody directed against human fibrin fragment-D dimer

Fibrin-directed monoclonal antibodies may be clinically useful for in vitro thrombus imaging and for the targeting of fibrinolytic agents to blood clots. One such murine monoclonal antibody, (mAb-15C5), raised against the fragment-D dimer epitope of cross-linked human fibrin, was previously characterized [Holvoet, P., Stassen, J. M., Hashimoto, Y., Spriggs, D., Devos, P. & Collen, D. (1989) Thromb. Haemostasis 61, 307-313] has recently been cloned and expressed [Vandamme, A.-M., Bulens, F., Bernar, H., Nelles, L., Lijnen, H. R. & Collen, D. (1990) Eur. J. Biochem. 192, 767-775]. In order to reduce the immunogenicity of the murine mAb-15C5 in man, we have now constructed a murine--human chimera of mAb-15C5, by substituting the cDNA sequences encoding the constant regions of the murine kappa light chain and gamma 1 heavy chain by the corresponding human genomic sequences. Both chimeric murine--human Ig chains were cloned into two separately selectable expression vectors, which were contransfected into Chinese hamster ovary (CHO) cells. Murine--human chimeric mAb-15C5 (mAb-15C5Hu) was purified from the conditioned medium of selected cell lines by chromatography on Zn-chelating Sepharose, protein-A-Sepharose and on insolubilized antigen (fragment-D dimer), with a final yield of 29 micrograms/l and a recovery of 33%. SDS/PAGE without reduction revealed a homogeneous band with a mobility similar to that of natural mAb-15C5, whereas after reduction, both the heavy and the light chains had slightly slower mobilities than their natural counterparts. Expression in the presence of tunicamycin suggested that the differences in gamma 1-chain mobility were due to different N-glycosylation patterns. Immunoblotting of proteins from SDS gels showed immunological reactivity of recombinant mAb-15C5Hu with goat anti-(human IgG) IgG and of recombinant and natural murine mAb-15C5 with goat anti-(mouse IgG) IgG. Competitive binding revealed a comparable affinity of recombinant murine mAb-15C5, recombinant mAb-15C5Hu and natural mAb-15C5, for fragment-D dimer, indicating that recombinant mAb-15C5Hu was obtained in a functionally intact form. Thus, mAb-15C5Hu may constitute a useful alternative to mAb-15C5 for in vivo use in man.     

Micro‐dose placement of phosphorus induces deep rooting of upland rice

Plant and Soil - Upland rice production is often constrained by phosphorus deficiency (P) and drought events. Methods are needed to maximize P use efficiency, while promoting deep root development...     

Clinical Evaluation of Rega 8: An Updated Genotypic Interpretation System That Significantly Predicts HIV-Therapy Response

Introduction

Clinically evaluating genotypic interpretation systems is essential to provide optimal guidance in designing potent individualized HIV-regimens. This study aimed at investigating the ability of the latest Rega algorithm to predict virological response on a short and longer period.

Materials & Methods

9231 treatment changes episodes were extracted from an integrated patient database. The virological response after 8, 24 and 48 weeks was dichotomized to success and failure. Success was defined as a viral load below 50 copies/ml or alternatively, a 2 log decrease from the baseline viral load at 8 weeks. The predictive ability of Rega version 8 was analysed in comparison with that of previous evaluated version Rega 5 and two other algorithms (ANRS v2011.05 and Stanford HIVdb v6.0.11). A logistic model based on the genotypic susceptibility score was used to predict virological response, and additionally, confounding factors were added to the model. Performance of the models was compared using the area under the ROC curve (AUC) and a Wilcoxon signed-rank test.

Results

Per unit increase of the GSS reported by Rega 8, the odds on having a successful therapy response on week 8 increased significantly by 81% (OR = 1.81, CI = [1.76–1.86]), on week 24 by 73% (OR = 1.73, CI = [1.69–1.78]) and on week 48 by 85% (OR = 1.85, CI = [1.80–1.91]). No significant differences in AUC were found between the performance of Rega 8 and Rega 5, ANRS v2011.05 and Stanford HIVdb v6.0.11, however Rega 8 had the highest sensitivity: 76.9%, 76.5% and 77.2% on 8, 24 and 48 weeks respectively. Inclusion of additional factors increased the performance significantly.

Conclusion

Rega 8 is a significant predictor for virological response with a better sensitivity than previously, and with rules for recently approved drugs. Additional variables should be taken into account to ensure an effective regimen.