Ca2+ clearance mechanisms in neurohypophysial terminals of the rat

The importance of intracellular calcium ([Ca2+]i) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+]i transients, cytoplasmic Ca2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+]i and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+]i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+]i or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, La3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+]i, and also slowed down the recovery phase of both [Ca2+]i and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca2+ clearance or in the maintenance of basal [Ca2+]i or release of AVP.     

Structural insights into humanization of anti-tissue factor antibody 10H10

Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.     

Forage Crop <Emphasis Type="Italic">Lolium multiflorum</Emphasis> Assisted Synthesis of AgNPs and Their Bioactivities Against Poultry Pathogenic Bacteria in In Vitro

Italian ryegrass is one of main feed for livestock animals/birds. It has potential antioxidant metabolites that can improve their health and protect them against various infectious diseases. In this work, we studied synthesis of silver nanoparticles assisted by forage crop Lolium multiflorum as a green synthesis way. Potential antibacterial efficacy of these synthesized nanosized silver nanoparticles against poultry pathogenic bacteria was then studied. Aqueous extract of IRG was used as reducing agent for bio-reduction of silver salt to convert Ag⁺ to Ag⁰ metallic nano-silver. Size, shape, metallic composition, functional group, and crystalline nature of these synthesized silver nanoparticles were then characterized using UV–Vis spectrophotometer, FESEM, EDX, FT-IT, and XRD, respectively. In addition, antibacterial effects of these synthesized AgNPs against poultry pathogenic bacteria were evaluated by agar well diffusion method. UV–Vis spectra showed strong absorption peak of 440–450 nm with differ reaction time ranging from 30 min to 24 h. FESEM measurements revealed particles sizes of around 20–100 nm, majority of which were spherical in shape while a few were irregular. These biosynthesized silver nanoparticles using IRG extract exhibited strong antibacterial activities against poultry pathogenic microorganisms, including Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, and Bacillus subtilis. Overall results confirmed that IRG plant extract possessed potential bioactive compounds for converting silver ions into nanosized silver at room temperature without needing any external chemical for redox reaction. In addition, such synthesized AgNPs showed strong antibacterial activities against pathogenic bacteria responsible for infectious diseases in poultry.     

Models of delta-hemolysin membrane channels and crystal structures

Molecular modeling and energy calculations have been used to study how delta-hemolysin and melittin helices may aggregate on membrane surfaces and insert through membranes to form channels. In these models adjacent antiparallel amphipathic helices form planar "raft" structures, in which one surface is hydrophobic and the other hydrophilic. Models of delta-hemolysin crystal structure were developed using these "rafts." These models are based on the unit cell constants and the crystal symmetry obtained from the preliminary crystal data. Energy calculations favor channel models of delta-hemolysin with six or eight monomers per channel.     

Do Emotions Expressed Online Correlate with Actual Changes in Decision-Making?: The Case of Stock Day Traders

Emotions are increasingly inferred linguistically from online data with a goal of predicting off-line behavior. Yet, it is unknown whether emotions inferred linguistically from online communications correlate with actual changes in off-line activity. We analyzed all 886,000 trading decisions and 1,234,822 instant messages of 30 professional day traders over a continuous 2 year period. Linguistically inferring the traders’ emotional states from instant messages, we find that emotions expressed in online communications reflect the same distributions of emotions found in controlled experiments done on traders. Further, we find that expressed online emotions predict the profitability of actual trading behavior. Relative to their baselines, traders who expressed little emotion or traders that expressed high levels of emotion made relatively unprofitable trades. Conversely, traders expressing moderate levels of emotional activation made relatively profitable trades.     

Recruitment of cAMP-response Element-binding Protein and Histone Deacetylase Has Opposite Effects on Glucocorticoid Receptor Gene Transcription

Glucocorticoids control the synthesis of the glucocorticoid receptor (GR) in various tissues through a negative feedback regulation of the mRNA. In this study, we have identified feedback regulatory domains in the human GR gene promoter and examined the roles of GR, the cAMP-response element-binding protein (CREB), and HDAC-6 in association with promoter elements of the human GR gene. Using breast cancer T47D and HeLa-GR cells, we identify specific negative glucocorticoid-response elements in the GR gene. The feedback regulatory domains were also involved in interactions with CREB. GR-bound negative glucocorticoid-response elements recruited HDAC-6, and this was dependent on treatment with dexamethasone. Both CREB and HDAC-6 formed complexes with GR-dexamethasone. The HDAC-6 LXXLL motif between amino acids 313 and 418 made direct contact with the GR AF-1 domain. Interestingly enough, although the level of GR decreased in CREB knockdown cells, it was elevated in HDAC-6 knockdown cells. Our results suggest that CREB-P is dephosphorylated and that HDAC-6 is recruited by the GR, and they play opposite roles in the negative feedback regulation of the GR gene.