Ciliary neurotrophic factor prevents unweighting-induced functional changes in rat soleus muscle.

The purpose of the present work was to see whether changes in rat soleus characteristics due to 3 wk of hindlimb suspension could be modified by ciliary neurotrophic factor (CNTF) treatment. Throughout the tail suspension period, the cytokine was delivered by means of an osmotic pump (flow rate 16 microg. kg(-1). h(-1)) implanted under the hindlimb skin. In contrast to extensor digitorum longus, CNTF treatment was able to reduce unweighting-induced atrophy in the soleus. Twitch and 146 mM potassium (K) tensions, measured in small bundles of unloaded soleus, decreased by 48 and 40%, respectively. Moreover, the time to peak tension and the time constant of relaxation of the twitch were 48 and 54% faster, respectively, in unloaded soleus than in normal muscle. On the contrary, twitch and 146 mM K contracture generated in CNTF-treated unloaded and normal soleus were not different. CNTF receptor-alpha mRNA expression increased in extensor digitorum longus and soleus unloaded nontreated muscles but was similar in CNTF-treated unloaded muscles. The present results demonstrate that exogenously provided CNTF could prevent functional changes occurring in soleus innervated muscle subject to unweighting.     

A new MspI restriction fragment length polymorphism in the hemophilia B locus

Summary Using a partial cDNA probe for human coagulation factor IX, we have detected a new restriction fragment length polymorphism in human DNA digested with MspI. The frequency of the minor allele is 0.20±0.05 and average heterozygosity is about 0.32. The MspI RELP is in strong linkage disequilibrium with the TaqI RFLP previously described, but should nevertheless be useful in segregation analysis in case of homozygosity for the TaqI minor allelc.     

Beta amino acid‐modified and fluorescently labelled kisspeptin analogues with potent KISS1R activity

Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a dual luciferase reporter assay in KISS1R‐transfected HEK293T cells. In general terms the results reflect the outcomes of other assay formats and a number of potent agonists were identified among the analogues, including β²‐hTyr‐modified and fluorescently labelled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation

The signal for somatic sex determination in mammals, Caenorhabditis elegans and Drosophila melanogaster is chromosomal, but the overall mechanisms do not appear to be conserved between the phyla. However it has been found quite recently that the C. elegans sex-determining gene Mab-3 contains a domain highly homologous to the Drosophila sex-determining gene doublesex (dsx) and shares a similar role. These data suggest that at least some aspects of the regulation of sex determination might be conserved. In humans, a doublesex-related gene (DMRT1) was identified at less than 30 kb from the critical region for sex reversal on chromosome 9p24 (TD9). In order to get insights into the role of DMRT1 in sex determination/differentiation, we have isolated DMRT1 mouse homologue (Dmrt1) and analysed its expression pattern. The gene is expressed in the genital ridges of both sexes during the sex-determining switch and it shows male/female dimorphism at late stages of sex differentiation.     

A DNA fragment from the human X chromosome short arm which detects a partially homologous sequence on the Y chromosomes long arm.

An X linked human DNA fragment (named DXS31 ) which detects partially homologous sequences on the Y chromosome has been isolated. Regional localisation of the two sex linked sequences was determined using a panel of rodent-human somatic cell hybrids. The X specific sequence is located at the tip of the short arm ( Xp22 .3-pter), i.e. within or close to the region which pairs with the Y chromosome short arm at meiosis. However the Y specific sequence is located in the heterochromatic region of the long arm ( Yq11 -qter) and lies outside from the pairing region. DNAs from several XX male subjects were probed with DXS31 and in all cases a double dose of the X linked fragment was found, and the Y specific fragment was absent. DXS31 detects in chimpanzee a male-female differential pattern identical to that found in man. However results obtained in a more distantly related species, the brown lemur, suggest that the sequences detected by DXS31 in this species might be autosomally coded. The features observed with these X-Y related sequences do not fit with that expected from current hypotheses of homology between the pairing regions of the two sex chromosomes, nor with the pattern observed with other X-Y homologous sequences recently characterized. Our results suggest also that the rule of conservation of X linkage in mammals might not apply to sequences present on the tip of the X chromosome short arm, in bearing with the controversial issue of steroid sulfatase localisation in mouse.     

Isolation and characterization of a family of sequences dispersed on the human X chromosome

During a systematic search for X-specific sequences we isolated a DNA fragment (called G1.3) that hybridizes to six further homologous X-specific genomic fragments that map to at least four different regions of the human X chromosome. Genomic segments of 11-30 kb (called G1.3 a, b, c, d, and e or DNF22S1 to DNF22S5) have been subsequently cloned for five of the seven repetitions and characterized by restriction mapping. Single-copy sequences have been used to analyze homology between cloned repetitions, to confirm X specificity, and to regionally localize the repetitions. Sequence homology between members of this family seems to be very high (80-90%) and to extend over at least 5 to 12 kb. In situ hybridization and Southern blotting experiments with a panel of human-rodent hybrid cell lines demonstrated that four of the cloned sequences map to three different regions within Xp21.2-pter and the fifth one (G1.3c) maps to Xq28. The family is present with the same complexity and X specificity in macaques (20-30 x 10(6) years divergence with man), whereas no related sequences were detected in the mouse. To our knowledge small families of dispersed chromosome-specific sequences have been described only for the human Y chromosome. The possible functional or evolutionary significance of this family is discussed.     

Functional disomy of Xp22-pter in three males carrying a portion of Xp translocated to Yq

A number of Xp22;Yq11 translocations involving the transposition of Yq material to the distal short arm of the X chromosome have been described. The reciprocal product, i.e. the derivative Y chromosome resulting from the translocation of a portion of Xp to Yq, has never been recovered. We searched for this reciprocal product by performing dosage analysis of Xp22-pter loci in 9 individuals carrying a non-fluorescent Y chromosome. In three mentally retarded and dysmorphic patients, dosage analysis indicated the duplication of Xp22 loci. Use of the highly polymorphic probe CRI-S232 demonstrated the inheritance of paternal Xp-specific alleles in the probands. In situ hybridization, performed in one case, confirmed that 29CL pseudoautosomal sequences were present, in addition to Xpter and Ypter, in the telomeric portion of Yq. To our knowledge, these are the first cases in which the translocation of Xp material to Yq has been demonstrated. The X and Y breakpoints were mapped in the three patients by dosage and deletion analysis. The X breakpoint falls, in the three cases, in a region of Xp22 that is not recognized as sharing sequence similarities with the Y chromosome, thus suggesting that these translocations are not the result of a homologous recombination event.     

Solid Phase Synthesis and Circular Dichroism Analysis of (i → i + 4) Cyclic Lactam Analogues of Kisspeptin

The α-helix is one of the most common secondary structure elements adopted by proteins and is commonly stabilized in synthetic peptides via the formation of a covalent side-chain to side-chain lactam bridge. In this study, we explored the application of various side-chain to side-chain lactam bridges to helix stabilization of kisspeptin analogues, an interesting candidate for ligand-based drug discovery with potential as anti-metastatic agents. We successfully synthesised a series of Asp/Lys, Lys/Asp, Glu/Lys and Lys/Glu lactams, finding peptide (1) cyclo(4,8)Tyr-Asn-Trp-Glu-Ala-Phe-Gly-Lys-Arg-Phe-NH₂, to exhibit characteristic α-helical activity in aqueous buffer, in comparison to the linear native peptide, which showed no helical character.     

Targeting kidney CLC-K channels: Pharmacological profile in a human cell line versus Xenopus oocytes

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca²⁺ is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks.