Comparative studies of somatostatin-14 and some of its fragments on passive avoidance behavior, open field activity and on barrel rotation phenomenon in rats

Behavioral effects of somatostatin-14, and some of its fragments [somatostatin(3–8), somatostatin(9–14), somatostatin(7–10)] after intracerebroventricular (ICV) administration have been investigated in male rats. In a passive avoidance learning test, somatostatin-14 (0.6 nM) given immediately after the learning session increased the avoidance latency at 24 hr after the injection, when compared to a somatostatin(3–8) (0.6 nM)-treated group. However, compared to a saline-treated group, the peptides did not significantly influence the avoidance latency. Somatostatin-14 administered in higher dose (6.0 nM) decreased the avoidance latency compared to the saline-treated group, while its fragments did not influence it. In an open field behavioral test, immediately after the 24-hr passive avoidance test, 6 nM of somatostatin-14 decreased the rearing activity, while the fragments did not influence this behavior. Somatostatin-14 produced barrel rotation in a dose-related manner, but after the injection of a high dose of the peptide (12 nM) all of the animals died in cardiorespiratory failure (apnea, pulmonary oedema). The fragments did not produce barrel rotation.     

Two novel proteins activate superoxide generation by the NADPH oxidase NOX1

NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47(phox) and p67(phox) subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47(phox) and p67(phox) are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47(phox) homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67(phox) homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47(phox) was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).     

New antifungal peptides. Synthesis,bioassays and initial structure prediction by CD spectroscopy

The synthesis, in vitro evaluation and conformational study of KKWKMRRNQFWIKIQR-NH₂, HFRWRQIKIWFQNRRMKWKK-NH₂ and RQPKIWFPNRRKPWKK-NH₂ acting as antifungal agents are reported. These peptides displayed a moderate but significant antifungal effect against both pathogenic fungi Candida albicans and Cryptococcus neoformans. The conformational analysis of these peptides was carried out using both theoretical and experimental methods.     

Spike-Timing Theory of Working Memory

Working memory (WM) is the part of the brain''s memory system that provides temporary storage and manipulation of information necessary for cognition. Although WM has limited capacity at any given time, it has vast memory content in the sense that it acts on the brain''s nearly infinite repertoire of lifetime long-term memories. Using simulations, we show that large memory content and WM functionality emerge spontaneously if we take the spike-timing nature of neuronal processing into account. Here, memories are represented by extensively overlapping groups of neurons that exhibit stereotypical time-locked spatiotemporal spike-timing patterns, called polychronous patterns; and synapses forming such polychronous neuronal groups (PNGs) are subject to associative synaptic plasticity in the form of both long-term and short-term spike-timing dependent plasticity. While long-term potentiation is essential in PNG formation, we show how short-term plasticity can temporarily strengthen the synapses of selected PNGs and lead to an increase in the spontaneous reactivation rate of these PNGs. This increased reactivation rate, consistent with in vivo recordings during WM tasks, results in high interspike interval variability and irregular, yet systematically changing, elevated firing rate profiles within the neurons of the selected PNGs. Additionally, our theory explains the relationship between such slowly changing firing rates and precisely timed spikes, and it reveals a novel relationship between WM and the perception of time on the order of seconds.     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

NOX5 variants are functionally active in endothelial cells

NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.     

Complementary identification of multiple flux distributions and multiple metabolic pathways

Cell robustness and complexity have been recognized as unique features of biological systems. Such robustness and complexity of metabolic-reaction systems can be explored by discovering, or identifying, the multiple flux distributions (MFD) and redundant pathways that lead to a given external state; however, this is exceedingly cumbersome to accomplish. It is, therefore, highly desirable to establish an effective computational method for their identification, which, in turn, gives rise to a novel insight into the cellular function. An effective approach is proposed for complementarily identifying MFD in metabolic flux analysis and multiple metabolic pathways (MMP) in structural pathway analysis. This approach judiciously integrates flux balance analysis (FBA) based on linear programming and the graph-theoretic method for determining reaction pathways. A single metabolic pathway, with the concomitant flux distribution and the overall reaction manifesting itself as the desired phenotype under some environmental conditions, is determined by FBA from the initial candidate sequence of metabolic reactions. Subsequently, the graph-theoretic method recovers all feasible MMP and the corresponding MFD. The approach's efficacy is demonstrated by applying it to the in silico Escherichia coli model under various culture conditions. The resultant MMP and MFD attaining a unique external state reveal the surprising adaptability and robustness of the intricate cellular network as a key to cell survival against environmental or genetic changes. These results indicate that the proposed approach would be useful in facilitating drug discovery.     

Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5)

NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.     

Elevated levels of inflammatory biomarkers in the cerebrospinal fluid after coronary artery bypass surgery are predictors of cognitive decline

Recovery from cardiac surgery is marred for many patients by the development of neurological, psychological or cognitive dysfunction. An uncontrolled inflammatory reaction, in response to surgical stress, may be responsible. To confirm this hypothesis, the present study evaluated changes in the levels of cytokines in cerebrospinal fluid after coronary artery bypass grafting. One week post-operatively, the concentration of the pro-inflammatory cytokine interleukin-6 markedly increased; 6 months after surgery, however, its level normalized with an increased concentration of the anti-inflammatory interleukin-4. This suggests that a regulated immune response may participate in developing adverse neurologic events and complications following cardiac interventions, and cytokines in the cerebrospinal fluid may serve as specific biomarkers and predictors of developing cognitive decline after coronary surgery.