Solution conformation of Monensin free acid,a typical representative of the polyetherin antibiotics

The solution conformation of the ionophore Monensin in its free-acid form bears a close resemblance to that of its Na⁺ salt. The backbone is folded into a closed loop, and the pseudocyclic structure is shut by head-to-tail H bonding between the carboxylic function and the alcoholic functions of the last six-membered ring with the mediation of a water molecule. A mode of trapping is proposed and compared to features observed in some other membrane-active complexones.     

A model for the enhancement of fitness in cyanobacteria based on resonance of a circadian oscillator with the external light-dark cycle

Fitness enhancement based on resonating circadian clocks has recently been demonstrated in cyanobacteria [Ouyang et al. (1998). Proc. Natl Acad. Sci. U.S.A.95, 8660-8664]. Thus, the competition between two cyanobacterial strains differing by the free-running period (FRP) of their circadian oscillations leads to the dominance of one or the other of the two strains, depending on the period of the external light-dark (LD) cycle. The successful strain is generally that which has an FRP closest to the period of the LD cycle. Of key importance for the resonance phenomenon are observations which indicate that the phase angle between the circadian oscillator and the LD cycle depends both on the latter cycle's length and on the FRP. We account for these experimental observations by means of a theoretical model which takes into account (i) cell growth, (ii) secretion of a putative cell growth inhibitor, and (iii) the existence of a cellular, light-sensitive circadian oscillator controlling growth as well as inhibitor secretion. Building on a previous analysis in which the phase angle was considered as a freely adjustable parameter [Roussel et al. (2000). J. theor. Biol.205, 321-340], we incorporate into the model a light-sensitive version of the van der Pol oscillator to represent explicitly the cellular circadian oscillator. In this way, the model automatically generates a phase angle between the circadian oscillator and the LD cycle which depends on the characteristic FRP of the strain and varies continuously with the period of the LD cycle. The model provides an explanation for the results of competition experiments between strains of different FRPs subjected to entrainment by LD cycles of different periods. The model further shows how the dominance of one strain over another in LD cycles can be reconciled with the observation that two strains characterized by different FRPs nevertheless display the same growth kinetics in continuous light or in LD cycles when present alone in the medium. Theoretical predictions are made as to how the outcome of competition depends on the initial proportions and on the FRPs of the different strains. We also determine the effect of the photoperiod and extend the analysis to the case of a competition between three cyanobacterial strains.     

Association of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Elements with Specific Serotypes and Virulence Potential of Shiga Toxin-Producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) strains (n = 194) representing 43 serotypes and E. coli K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (n = 81) was further analyzed for subtype I-E cas and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E cas and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (P < 0.05). Fewer spacers were found in strains that had a greater probability of causing outbreaks and disease than in those with lower virulence potential (P < 0.05). The relationship between the CRISPR-cas system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.     

Polyamine flux analysis by determination of heavy isotope incorporation from 13C, 15N-enriched amino acids into polyamines by LC-MS/MS

The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the (13)C, (15)N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC-MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine-N (1)-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC-MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux.     

Carbonic Anhydrase Inhibitors: Synthesis of Sulfonamides Incorporating 2, 4, 6–Trisubstituted-Pyridinium-Ethylcarboxamido Moieties Possessing Membrane-Impermeability and in Vivo Selectivity for the Membrane-Bound (CA IV) Versus the Cytosolic (CA I and CA II) Isozymes

A new approach is proposed for the selective in vivo inhibition of membrane-bound versus cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes with a class of positively-charged, membrane-impermeant sulfonamides. Aromatic/heterocyclic sulfonamides acting as strong (but unselective) inhibitors of this zinc enzyme were derivatized by the attachment of trisub-stituted-pyridinium-ethylcarboxy moieties (obtained from 2, 4, 6–trisubstituted-pyrylium salts and β-alanine) to the amino, imino, hydrazino or hydroxyl groups present in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes, i.e., hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound isozyme; h = human; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, ethoxzolamide), are unable to penetrate biological membranes, as shown by CJ vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors suggest that: (i) when using the new type of positively-charged sulfonamides. only the membrane-bound enzyme (CA IV) was inhibited. whereas the cytosolic isozymes (CA I and II) were not affected, (ii) in the experiments in which the classical compounds (acetazolamide, bcn-zolamíde. etc.) were used. unselective inhibition of all CA isozymes (I. II and IV) occurred.     

Focal adhesion kinase protein levels in gut epithelial motility

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.     

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.     

Novel receptor partners and function of receptor activity-modifying proteins

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.