Hox2 Genes Are Required for Tonotopic Map Precision and Sound Discrimination in the Mouse Auditory Brainstem

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The interaction of arginine- and tryptophan-rich cyclic hexapeptides with Escherichia coli membranes.

Cyclization of R- and W-rich hexapeptides has been found to enhance specifically the antimicrobial activity against Gram-negative Escherichia coli. To gain insight into the role of the bacterial outer membrane in mediating selectivity, we assayed the activity of cyclic hexapeptides derived from the parent sequence c-(RRWWRF) against several E. coli strains and Bacillus subtilis, L-form bacteria, and E. coli lipopolysaccharide (LPS) mutant strains, and we also investigated the peptide-induced permeabilization of the outer and inner membrane of E. coli. Wall-deficient L-form bacteria were distinctly less susceptible than the wild type strain. The patterns of peptide-induced permeabilization of the outer and inner E. coli membranes correlated well with the antimicrobial activity, confirming that membrane permeabilization is a detrimental effect of the peptides upon bacteria. Truncation of LPS had no influence on the activity of the cyclic parent peptide, but the highly active c-(RRWFWR), with three adjacent aromatic residues, required the complete LPS for maximal activity. Furthermore, differences in the activity of the parent peptide and its all-D sequence indicated stereospecific interactions with the LPS mutant strains. We suggest that, depending on the primary sequence of the peptides, either hydrophobic interactions with the fatty acid chains of lipid A, or electrostatic interactions disturbing the polar core region and interference with saccharide-saccharide interactions prevail in the barrier-disturbing effect upon the outer membrane and thereby provide peptide accessibility to the inner membrane. The results underline the importance of tryptophan and arginine residues and their relative location for a high antimicrobial effect, and the activity-modulating function of the outer membrane of E. coli. In addition to membrane permeabilization, the data provided evidence for the involvement of other mechanisms in growth inhibition and killing of bacteria.     

Isolation of lactoferrin from milk of different species: calorimetric and antimicrobial studies

Lactoferrin (LF) is an iron-binding glycoprotein found in different biological fluids of mammals and in neutrophils. It has been proposed to be involved in many functions, including protection from pathogens. In this work, purification of lactoferrin using an ion-exchange chromatography (SP-Sepharose) was attempted for the milk of the following animals: sheep (Ovis aries), goat (Capra hircus), camel (Camelus bactrianus), alpaca (Lama pacos), elephant (Elephas maximus) and grey seal (Halichoerus grypus), as well as human (Homo sapiens). Lactoferrin was identified in all the milks apart from that from grey seal. The thermal stability of the purified lactoferrins, in their native and iron-saturated forms, was studied by differential scanning calorimetry (DSC). Maximum temperature, onset temperature and enthalpy change of denaturation were higher when lactoferrins were saturated with iron than in their native form, indicating an increase in the stability of the protein structure upon iron-binding. Human lactoferrin was found to be the most heat-resistant and the other lactoferrins presented different degrees of thermoresistance, that of elephant being the least resistant. The antimicrobial activity of the different isolated lactoferrins was investigated against Escherichia coli 0157:H7. The minimal inhibitory concentrations (MICs) were determined by measuring the absorbance at 620 nm. The minimum bactericidal concentrations (MBCs) were also measured and it was found that camel lactoferrin was the most active lactoferrin against E. coli 0157:H7, whereas alpaca and human lactoferrins were the least active.     

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.     

High Functional Diversity in Mycobacterium tuberculosis Driven by Genetic Drift and Human Demography

Mycobacterium tuberculosis infects one third of the human world population and kills someone every 15 seconds. For more than a century, scientists and clinicians have been distinguishing between the human- and animal-adapted members of the M. tuberculosis complex (MTBC). However, all human-adapted strains of MTBC have traditionally been considered to be essentially identical. We surveyed sequence diversity within a global collection of strains belonging to MTBC using seven megabase pairs of DNA sequence data. We show that the members of MTBC affecting humans are more genetically diverse than generally assumed, and that this diversity can be linked to human demographic and migratory events. We further demonstrate that these organisms are under extremely reduced purifying selection and that, as a result of increased genetic drift, much of this genetic diversity is likely to have functional consequences. Our findings suggest that the current increases in human population, urbanization, and global travel, combined with the population genetic characteristics of M. tuberculosis described here, could contribute to the emergence and spread of drug-resistant tuberculosis.     

CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells

The aim of this study was to characterize the role of CFTR during Cd²⁺-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr^+/+ and cftr^?/? mice were used. In cftr^+/+ cells, the application of Cd²⁺ (5 μM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl^? conductance sensitive to CFTR_inh-172. Thereafter Cd²⁺ induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl^? and TASK2 K⁺ conductances. By contrast, cftr^?/? cells exposed to Cd²⁺ were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr^+/+ cells, Cd²⁺ enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR_inh-172 or in cftr^?/? cells. Additionally, Cd²⁺ exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr^+/+ cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd²⁺ exposure by modulating the intracellular content of glutathione.     

Poly(methyl metacrylate) conductive fiber optic transducers as dual biosensor platforms

Herein the development of an alternative optic-conductive fiber configuration applied for the construction of biosensing platforms. This new approach is based on applying the chemical polymerization of pyrrole onto the surface of polymethyl metacrylate (PMMA) fibers to create a polymer—a conductive surface, onto which an additional photoactive polypyrrole-benzophenone (PpyBz) film is electrochemically generated upon the fiber surface. Irradiation of the benzophenone groups embedded in the Ppy films with UV radiation (350 nm) formed active radicals that allowed the covalent attachment of the desired bioreceptors. Characterization of the amperometric biosensing matrix was accomplished by using a model Urease (Urs) through electrochemical impedance spectroscopy (EIS) and amperometry. Both techniques have shown a low charge transfer resistance (340 kΩ) and a high sensitivity (12.3 μA mM⁻¹ cm⁻²). Thereafter, the construction of an optical biosensing matrix based on horseradish peroxidase (HRP) production of photons was carried out. The high signal to noise (S/N) ratio (1600) indicated clearly that this approach can serve as a new platform to replace glass optical fibers based on biosensors.     

Identification of Cardiac Myosin-binding Protein C as a Candidate Biomarker of Myocardial Infarction by Proteomics Analysis

Acute myocardial infarction (AMI) is a common cause of death for which effective treatments are available provided that diagnosis is rapid. The current diagnostic gold standards are circulating cardiac troponins I and T. However, their slow release delays diagnosis, and their persistence limits their utility in the identification of reinfarction. The aim was to identify candidate biomarkers of AMI. Isolated mouse hearts were perfused with oxygenated protein-free buffer, and coronary effluent was collected after ischemia or during matched normoxic perfusion. Effluents were analyzed using proteomics approaches based on one- or two-dimensional initial separation. Of the 459 proteins identified after ischemia with one-dimensional separation, 320 were not detected in the control coronary effluent. Among these were all classic existing biomarkers of AMI. We also identified the cardiac isoform of myosin-binding protein C in its full-length form and as a 40-kDa degradation product. This protein was not detected in the other murine organs examined, increased markedly with even trivial myocardial infarction, and could be detected in the plasma after myocardial infarction in vivo, a profile compatible with a biomarker of AMI. Two-dimensional fluorescence DIGE of ischemic and control coronary effluents identified more than 200 asymmetric spots verified by swapping dyes. Once again existing biomarkers of injury were confirmed as well as posttranslational modifications of antioxidant proteins such as peroxiredoxins. Perfusing hearts with protein-free buffers provides a platform of graded ischemic injury that allows detailed analysis of protein release and identification of candidate cardiac biomarkers like myosin-binding protein C.Acute myocardial infarction (AMI)1 is a common cause of death for which effective treatments are available provided that the condition is rapidly diagnosed. The modern diagnosis of AMI relies on the rise and fall of a specific serum biomarker accompanied by an appropriate circumstance such as chest pain or revascularization. In this accepted paradigm, the diagnosis cannot be ruled in or ruled out without the definite presence or definite absence of a serum biomarker. The ideal biomarker of cardiac injury should be cardiac specific and released rapidly after myocardial injury in direct proportion to the extent of damage. Furthermore, the biomarker should have a high sensitivity and specificity (1). Several biomarkers of AMI have been described in the literature, but only a few, none of which are ideal, have found their way into routine clinical practice. For example, CK-MB starts to increase 4–8 h after coronary artery occlusion and returns to base line within 2–3 days (2). However, its use is limited by its presence in skeletal muscle and normal serum and by sensitivity of the assay to interference, causing some to question its utility (3). Myoglobin is another cytoplasmic protein found in cardiac and skeletal, but not smooth, muscle. It is released even earlier within 1–2 h of AMI and peaks within 5–6 h (2). Unfortunately, any injury to skeletal muscle also causes elevated levels of myoglobin, reducing specificity. Fatty acid-binding proteins (FABPs) are small (15-kDa) cytoplasmic proteins expressed in all tissues with active fatty acid metabolism. Among the nine proteins, heart-specific FABP (H-FABP) is found in heart but also kidney, brain, skeletal muscle, and placenta (4). Following acute myocardial infarction, H-FABP can be detected within 20 min and peaks at 4 h, considerably faster even than CK/CK-MB in the same patient cohort. Although H-FABP concentrations in normal plasma are low, they are known to rise nonspecifically during physical exertion (without a troponin rise), kidney injury, and stroke (5).The most specific and sensitive cardiac proteins released after acute myocardial infarction are cardiac troponins I and T. Both troponins I and T are released slowly, peaking ∼18 h after myocardial infarction, and remain elevated for 7–10 days (2). This slow release is likely the result of their relatively inaccessible cellular location compared with CK-MB, myoglobin, and H-FABP. Troponins regulate the physical interaction of actin and myosin and thus are found almost entirely associated within the crystalline structure of the sarcomere of striated muscle cells (6). The troponin complex is composed of three forms: I, T, and C. Troponins I and T exist as cardiac specific isoforms with epitopes that differ from the corresponding skeletal isoforms. In addition, the absent or extremely low normal circulating levels of troponin provide the greatest dynamic range of any of the currently available biomarkers (7). Although there is no doubt troponins have revolutionized the detection and management of patients with AMI (8), they do have disadvantages. The slow release of troponin delays diagnosis and the initiation of specific treatments that could salvage heart tissue in those in whom it is raised. Similarly, patients in whom it is absent and who are ultimately reassured and discharged are admitted to the hospital unnecessarily. Furthermore, the persistence of troponins limits their utility in the diagnosis of reinfarction.It is therefore widely accepted that there is a need for new biomarkers that can diagnose AMI earlier during its natural history and/or that have a short plasma half-life, allowing use in diagnosis and quantification of reinfarction. The purpose of this study was to use the platform of the crystalloid perfused mouse hearts to perform a systematic proteomics analysis of the coronary effluent after minimal AMI to identify new potential biomarkers (9).