Discovery of a potent phenolic N1-benzylidene-pyridinecarboxamidrazone selective against Gram-positive bacteria

As part of a study into antimycobacterial compounds a set of phenolic N1-benzylidene-pyridinecarboxamidrazones was prepared and evaluated. This report describes the unexpected discovery of a potent compound with a pronounced selectivity for Gram-positive bacteria over Gram-negative micro-organisms. In addition, this compound is active against various drug-resistant Gram-positive bacteria.     

Exercise training improves myocardial tolerance to ischemia in male but not in female rats

Acute exercise increases myocardial tolerance to ischemia-reperfusion (I-R) injury in male but not in female rat hearts, possibly due to a decreased heat shock protein 70 (Hsp70) response in the female hearts. This study examined whether repetitive exercise training would increase Hsp70 and myocardial tolerance to I-R injury in female rat hearts. Adaptations in myocardial manganese superoxide dismutase (MnSOD) and endothelial nitric oxide synthase (eNOS) were also assessed. Ten-week old male (M) and female (F) Sprague-Dawley rats (n = 40 total) exercise-trained for 14 wk; the last 8 wk consisted of running 1 h at 30 m/min (2% incline), 5 days/wk. Following training, left ventricle mechanical function (LVMF) was monitored for 30 min of reperfusion following 30 min of global ischemia (Langendorff procedure). Myocardial Hsp70 content was not different in M and F control groups, while increases were observed in both trained groups (M greater than F; P < 0.05). Although MnSOD content did not differ between groups, endothelial nitric oxide synthase (eNOS) levels were decreased in F, with no change in M, following training (P < 0.05). Hearts from control F demonstrated a greater recuperation of all indices of LVMF following I-R compared with control M hearts (P < 0.05). Hearts of trained M exhibited improved recovery of LVMF (left ventricular diastolic pressure, left ventricular end-diastolic pressure, +dP/dt, -dP/dt) during reperfusion compared with control M hearts (P < 0.05). In contrast, hearts of trained F did not show any change in recovery from I-R. Hence, exercise training is more beneficial to M than F in improving myocardial function following I-R injury.     

Red white and blue – bright light effects in a diurnal rodent model for seasonal affective disorder

Despite the common use of bright light exposure for treatment of seasonal affective disorder (SAD), the underlying biology of the therapeutic effect is not clear. Moreover, there is a debate regarding the most efficacious wavelength of light for treatment. Whereas according to the traditional approach full-spectrum light is used, recent studies suggest that the critical wavelengths are within the range of blue light (460 and 484 nm). Our previous work shows that when diurnal rodents are maintained under short photoperiod they develop depression- and anxiety-like behavioral phenotype that is ameliorated by treatment with wide-spectrum bright light exposure (2500 lux at the cage, 5000 K). Our current study compares the effect of bright wide-spectrum (3,000 lux, wavelength 420- 780 nm, 5487 K), blue (1,300 lux, wavelength 420-530 nm) and red light (1,300 lux, wavelength range 600-780 nm) exposure in the fat sand rat (Psammomys Obesus) model of SAD. We report results of experiments with six groups of sand rats that were kept under various photoperiods and light treatments, and subjected to behavioral tests related to emotions: forced swim test, elevated plus maze and social interactions. Exposure to either intense wide-spectrum white light or to blue light equally ameliorated depression-like behavior whereas red light had no effect. Bright wide-spectrum white light treatment had no effect on animals maintained under neutral photoperiod, meaning that light exposure was only effective in the pathological-like state. The resemblance between the effects of bright white light and blue light suggests that intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in the underlying biology of SAD and light therapy.     

Babysitting behavior by age/sex classification in squirrel monkeys (Saimiri sciureus)

The aunting behavior in a captive group of 22 squirrel monkeys containing three infants was done in terms of the age/sex classification of those animals involved. The time course of the aunting phenomena and the type and intensity of the interactions between the mothers and the aunts were recorded. Males as well as females were observed to ascertain if the babysitters were sex specific. Observations were gathered before, during, and after a particular threat to any monkey who was carrying an infant. Three categories of protective behavior (protect, retreat, and nothing) were tabulated. The results indicated that most aunting and protection occurred between infant ages 2-1/2-5 weeks when the infants were growing rapidly but not as yet socially self-sufficient. Mothers protected infants the most against juveniles, then subadult males, and least against other adult females. Subadult males were occasionally observed to carry and protect older infants. Aunting behavior was discussed in terms of the selective pressures by which it may have evolved.     

A truncated RHAMM protein for discovering novel therapeutic peptides

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7?kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7?kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7?kDa RHAMM. Therefore, in terms of its key binding properties, the 7?kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.     

Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope-labeled concatenated peptide standards

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.     

Immunodominant Francisella tularensis antigens identified using proteome microarray

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.