Cross-adaptation and molecular modeling study of receptor mechanisms common to four taste stimuli in humans

Psychophysical cross-adaptation experiments were performed with two carbohydrates, sucrose (SUC) and fructose (FRU), and two sweeteners, acesulfame-K (MOD) and dulcin (DUL). Seven subjects were asked to match concentrations that elicited the same intensity as a sucrose reference (30 g/l). Cross-adaptation levels were calculated as the ratio of isointense concentrations measured for a given stimulus before and under adaptation. On average, cross-adaptation between SUC and FRU is low and apparently reciprocal. By contrast, cross-adaptation between SUC and MOD is clearly non-reciprocal: SUC adapts MOD significantly (24%, P < 0.005), but MOD fails to adapt SUC (2%, P < 0.79). Significant and reciprocal cross-enhancement is observed between DUL and MOD (approximately -20%, P < 0.03), and also between SUC and DUL (approximately -15%, P < 0.08). In parallel, molecular modeling of the four tastants was performed in order to look for the 12 common binding motifs that were isolated on 14 other tastants in a previous study. SUC and FRU each display 10 out of the 12 binding motifs, whereas DUL and MOD only display four and five distinct motifs respectively and do not have any motif in common. Experimental cross-adaptation levels seem to correlate well with the number of motifs that molecules have in common. FRU and SUC share a majority of binding motifs and correlatively show mutual cross-adaptation. Four motifs of MOD are found among the 10 motifs of SUC, which may explain why SUC cross-adapts MOD but not vice versa. By contrast, DUL and MOD do not share any motif and do not cross- adapt. The various molecular mechanisms that may be responsible for cross-adaptation and/or cross-enhancement are discussed in light of our results.      

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

A top-down proteomics approach for differentiating thermal resistant strains of Enterobacter sakazakii

Thermal tolerance has been identified as an important factor relevant to the pathogenicity of Enterobacter sakazakii in human neonates. To identify a biomarker specific for this phenotypic trait, intact protein expression profiles of 12 strains of E. sakazakii were obtained using liquid chromatography mass spectrometry. Proteins were extracted from the bacterial cells, separated by reversed-phase liquid chromatography and mass analyzed. At the end of the chromatography run, the uncharged masses of the multiply charged proteins were determined via automated software routines. The resulting data provided an accurate mass expression profile of the proteins found in the individual strains. From the individual expression profiles, it was possible to identify unique proteins corresponding to strains with thermal resistance. One protein found only in the thermal tolerant strains was sequenced and identified as homologous to a hypothetical protein found in the thermal tolerant bacteria, Methylobacillus flagellatus KT. The protein sequence of this protein was then used to reverse-engineer PCR primers for the gene sequence associated with the protein. In all cases, only thermal tolerant strains of E. sakazakii produced amplified PCR products, demonstrating the specificity of this biomarker.     

Proton transfer limits protein translocation rate by the thylakoid DeltapH/Tat machinery

The thylakoid transmembrane DeltapH is the sole energy source driving translocation of precursor proteins by the DeltapH/Tat machinery. Consequently, proton translocation must be coupled to precursor translocation. For the precursor of the 17 kDa protein of the oxygen-evolving complex (pOE17), the protein translocation process is characterized by a steep drop in efficiency at an external pH below 7.0 and above 8.7. As the membrane DeltapH is virtually unaffected from pH 6.5 to 9.2, the loss in import efficiency is a consequence of the titration of multiple residues within the translocation machinery. Transport is retarded by a factor of 2-3 in deuterium oxide (D(2)O) relative to water, strongly suggesting that proton-transfer reactions limit translocation rate. The solvent isotope effect manifests itself after the precursor binds to the membrane, indicating that the rate-limiting step is a later event in the transport process.     

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice

Background  

Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process.     

In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence

Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (rho = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.     

The anti-cyclic citrullinated peptide response in tuberculosis patients is not citrulline-dependent and sensitive to treatment

Introduction  

Patients with tuberculosis (TB) frequently produce anti-citrullinated protein antibodies (ACPA). The objective of this study is to characterize the citrulline-dependence of the ACPA reactivity in sera of patients with mycobacterium infections.