Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED₉₉ of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.     

CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening

AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.     

A novel Mr 77,000 protein of the XY body of mammalian spermatocytes: its localization in normal animals and in Searle’s translocation carriers

We describe a novel XY body protein of rat and mice pachytene spermatocytes called XY77. Biochemical characterization showed that protein XY77 (Mr 77,000; pH value 8.3) is present in meiotic but absent in postmeiotic stages of spermatogenesis. With the aid of an antibody against protein XY77 together with another specific for XY body-associated protein XY40 we also investigated the localization of these proteins in mice carrying Searle’s translocation, a reciprocal X-autosomal translocation. We show here that in these mice the distribution of both XY77 and XY40 is abnormal. Our results indicate that in Searle’s translocation alterations are not restricted to the translocated autosome, but also involve chromatin segments corresponding originally to the sex chromosomes X and Y. Received: 21 December 1996; in revised form: 1 February 1997 / Accepted: 15 February 1997     

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure^? and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure^? and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure^?, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure^? method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure^? treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure^? method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure^? could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.     

Caloric Restriction Decreases Orthostatic Tolerance Independently from 6° Head-Down Bedrest

Astronauts consume fewer calories during spaceflight and return to earth with an increased risk of orthostatic intolerance. Whether a caloric deficiency modifies orthostatic responses is not understood. Thus, we determined the effects of a hypocaloric diet (25% caloric restriction) during 6° head down bedrest (an analog of spaceflight) on autonomic neural control during lower body negative pressure (LBNP). Nine healthy young men completed a randomized crossover bedrest study, consisting of four (2 weeks each) interventions (normocaloric bedrest, normocaloric ambulatory, hypocaloric bedrest, hypocaloric ambulatory), each separated by 5 months. Muscle sympathetic nerve activity (MSNA) was recorded at baseline following normocaloric and hypocaloric interventions. Heart rate (HR) and arterial pressure were recorded before, during, and after 3 consecutive stages (7 min each) of LBNP (-15, -30, -45 mmHg). Caloric and posture effects during LBNP were compared using two-way ANOVA with repeated measures. There was a strong trend toward reduced basal MSNA following caloric restriction alone (normcaloric vs. hypocaloric: 22±3 vs. 14±4 burst/min, p = 0.06). Compared to the normocaloric ambulatory, both bedrest and caloric restriction were associated with lower systolic blood pressure during LBNP (p<0.01); however, HR responses were directionally opposite (i.e., increase with bedrest, decrease with caloric restriction). Survival analysis revealed a significant reduction in orthostatic tolerance following caloric restriction (normocaloric finishers: 12/16; hypocaloric finishers: 6/16; χ2, p = 0.03). Caloric restriction modifies autonomic responses to LBNP, which may decrease orthostatic tolerance after spaceflight.     

Fast identification of folded human protein domains expressed in <Emphasis Type="Italic">E. coli</Emphasis> suitable for structural analysis

Background  

High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.     

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.     

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.