Reaction of phenylglyoxal with arginine. The effect of buffers and pH.

Phenylglyoxal reacts much more rapidly with N²-acetylarginine than with either N²-acetyllysine or N-acetylcysteine. The rate of the reaction of phenylglyoxal with either N-acetylarginine or arginine increases with increasing pH from 7.5 to 11.5. The model reaction with arginine is much faster in bicarbonate, diethylamine, or triethylamine buffer than in N-ethylmorpholine, borate, phosphate, or Tris buffer. This activation by various buffers should be taken into consideration when glyoxal derivatives are used to modify arginyl residues.     

Purification and properties of the hemagglutinin from Wistaria floribunda seeds.

Extracts of Wistaria floribunda seeds contain separable erythroagglutinating and lymphocyte mitogenic activities. We wish to report the purification and characterization of the erythroagglutinating lectin of these seeds. A phosphate-buffered saline (PBS) extract of the ground seeds was made to 50% ethanol and the precipitate, which contained both the agglutinin and mitogen, was dissolved in PBS. The erythroagglutinating activity was adsorbed onto insoluble polyleycyl derivatized A + H active hog gastric mucin. After desorption with 0.2 M D-galactose and removal of the sugar by dialysis, the eluate displayed three protein bands on polyacrylamide gel electrophoresis. The major component represented 85% of the mixture. Immunoelectrophoresis of the mixture demonstrated immunochemical identity among the proteins. Gel filtration through Sephadex G-200 resulted in purification of the major component. Based upon the composition and subunit molecular weight, it was concluded that the three components represent a dimer, tetramer, and octamer of a single glycopolypeptide chain of 28 000. The erythroagglutinin has a pI at pH 5.4 and one cystine per dimeric unit.     

Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.     

What is hidden in the pannexin treasure trove: the sneak peek and the guesswork

Connexins had been considered to be the only class of the vertebrate proteins capable of gap junction formation; however, new candidates for this function with no homology to connexins, termed pannexins were discovered. So far three pannexins were described in rodent and human genomes: Panx1, Panx2 and Panx3. Expressions of pannexins can be detected in numerous brain structures, and now found both in neuronal and glial cells. Hypothetical roles of pannexins in the nervous system include participating in sensory processing, hippocampal plasticity, synchronization between hippocampus and cortex, and propagation of the calcium waves supported by glial cells, which help maintain and modulate neuronal metabolism. Pannexin also may participate in pathological reactions of the neural cells, including their damage after ischemia and subsequent cell death. Recent study revealed non-gap junction function of Panx1 hemichannels in erythrocytes, where they serve as the conduits for the ATP release in response to the osmotic stress. High-throughput studies produced some evidences of the pannexin involvement in the process of tumorigenesis. According to brain cancer gene expression database REMBRANDT, PANX2 expression levels can predict post diagnosis survival for patients with glial tumors. Further investigations are needed to verify or reject hypotheses listed.     

Discovery of 1-amino-4-phenylcyclohexane-1-carboxylic acid and its influence on agonist selectivity between human melanocortin-4 and -1 receptors in linear pentapeptides

Linear pentapeptides (Penta-cis-Apc-DPhe-Arg-Trp-Gly-NH2) containing 1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc) and substituted Apc are potent hMC4R agonists and they are inactive or weakly active in hMC1R, hMC3R, and hMC5R agonist assays. This study, together with our earlier report on 5-BrAtc, demonstrated the importance of replacing His6 with phenyl-containing rigid templates in achieving good hMC4R agonist potency and selectivity against hMC1R in linear pentapeptides.     

Developability studies before initiation of process development

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2−8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.     

Single Cell Genomics of the Brain: Focus on Neuronal Diversity and Neuropsychiatric Diseases

Single cell genomics has made increasingly significant contributions to our understanding of the role that somatic genome variations play in human neuronal diversity and brain diseases. Studying intercellular genome and epigenome variations has provided new clues to the delineation of molecular mechanisms that regulate development, function and plasticity of the human central nervous system (CNS). It has been shown that changes of genomic content and epigenetic profiling at single cell level are involved in the pathogenesis of neuropsychiatric diseases (schizophrenia, mental retardation (intellectual/leaning disability), autism, Alzheimer’s disease etc.). Additionally, several brain diseases were found to be associated with genome and chromosome instability (copy number variations, aneuploidy) variably affecting cell populations of the human CNS. The present review focuses on the latest advances of single cell genomics, which have led to a better understanding of molecular mechanisms of neuronal diversity and neuropsychiatric diseases, in the light of dynamically developing fields of systems biology and “omics”.     

Ground-foraging ants (Hymenoptera: Formicidae) and rainfall effect on pitfall trapping in a deciduous thorn woodland (caatinga), Northeastern Brazil

The semi-arid Caatinga is the fourth largest biome of Brazil, which biota still remains one of the most poorly known, especially with regard to invertebrate groups. In this study, a ground-foraging ant assemblage was surveyed during one year and the effect of rainfall on pitfall trapping was assessed. The study was performed in an area located in the municipality of Pentecoste (3 degrees 48' S - 39 degrees 20' W), in the State of Ceará. A 200m transect with 20 equidistant sampling points was established. Transect sampling was performed once a month during 12 months, over the period August 2008-August 2009. At each sampling point, a pitfall trap partially filled with a mixture of ethanol and monoethylene glycol was placed at the beginning of each month and remained in the field for seven days. 39 species belonging to six subfamilies and 19 genera, plus two unidentified species, were collected, with Pheidole (10 spp.) and Camponotus (8 spp.) being the taxa with the most species. 23 species were frequent, being found in more than 50% of the 12 transect samplings. Five species had an intermediate frequency (25 to 50%), while 13 were relatively infrequent (less than 25%). Most of the species (22) showed low occurrence, being found in less than 10% of the 240 samples (20 samples each month, during 12 months). Only five species were collected in more than 50% of the samples, those species being also responsible for most of the total abundance (number of captured individuals of all species) observed each month. The species-accumulation curves (observed and estimated) indicated that sampling sufficiency was attained, and that about 92% of the estimated ground-foraging ant fauna had been collected. 40 and 29 species were collected in the dry and rainy season, respectively, with monthly species richness ranging from 13 to 28. The total ant abundance showed a drastic decrease during the rainy season, and a negative linear correlation was found between rainfall and total ant abundance (R2 = 0.68). A similar negative linear correlation was found for species occurrences against rainfall (R2 = 0.71), and for mean number of species per pitfall trap against rainfall (R2 = 0.71). However, some species showed equal abundance, occurrence and mean number of individuals per pitfall trap in both seasons, while others showed a much higher abundance and occurrence during the rainy season. Pitfall trapping as a method to sample ground-foraging ant assemblage of the Caatinga biome and potential factors responsible for lower pitfall trap performance during rainy season are discussed.