Vascular smooth muscle influences the release of endothelium-derived relaxing factor

Conditioned medium was collected from vascular smooth-muscle cells grown in culture to determine if these cells synthesize vasoactive substances. The medium caused a short-acting endothelium-independent constriction of rat aorta, followed by a prolonged, endothelium-dependent relaxation. This relaxation was mediated through the release of endothelium-derived relaxing factor (EDRF) as it was abolished by the addition of methylene blue (5 x 10(-6) M), haemoglobin (10(-6) M) or methyl arginine, but was not affected by indomethacin (10(-5) M). Smooth-muscle medium stimulated the production of EDRF from both rat and rabbit thoracic aortic rings as well as from cultured bovine pulmonary artery endothelial cells. The prolonged stimulation of EDRF by smooth-muscle medium was not mimicked by known physiological stimuli to EDRF release; EDRF-stimulating activity was not affected when smooth-muscle cells were grown in the presence of indomethacin (10(-5) M), although serum in the medium was required. The EDRF-stimulating substance(s) in the smooth-muscle medium was heat stable and associated with a high molecular mass (30,000 greater than Mr greater than 3500) water-soluble species that is as yet unidentified.     

MicroRNA-146a Is Upregulated by and Negatively Regulates TLR2 Signaling

TLR signaling is a crucial component of the innate immune response to infection. MicroRNAs (miRNAs) have been shown to be upregulated during TLR signaling. Specifically, microRNA-146a (miR-146a) plays a key role in endotoxin tolerance by downregulating interleukin-1 receptor-associated kinase 1 (IRAK-1). The aim of this study was to assess the role of miR-146a in the TLR2 signaling and development of bacterial lipoprotein (BLP) self-tolerance and cross-tolerance to bacteria. Expression of miR-146a increased in a dose- and time-dependent manner in BLP-stimulated human THP-1 promonocytic cells. In BLP-tolerised cells miR-146a was even further upregulated in response to BLP re-stimulation (p<0.001). Re-stimulation of BLP-tolerised cells with heat-killed gram-negative Salmonella typhimurium (S. typhimurium), but not gram-positive Staphylococcus aureus (S. aureus), led to significant overexpression of miR-146a (p<0.05). Transfection of naive cells with a miR-146a mimic substantially suppressed TNF-α production (p<0.05). Furthermore, overexpression of miR-146a resulted in strong reduction in IRAK-1 and phosphorylated IκBα expression in naive and S. typhimurium-stimulated THP-1 cells. Collectively, miR-146a is upregulated in response to BLP and bacterial stimulation in both naive and BLP-tolerised cells. Overexpression of miR-146a induces a state analogous to tolerance in BLP-stimulated cells and therefore may represent a future target for exogenous modulation of tolerance during microbial infection and sepsis.     

Interdiffusion of PCBM and P3HT Reveals Miscibility in a Photovoltaically Active Blend

Developing a better understanding of the evolution of morphology in plastic solar cells is the key to designing new materials and structures that achieve photoconversion efficiencies greater than 10%. In the most extensively characterized system, the poly(3‐hexyl thiophene) (P3HT):[6,6]‐phenyl‐C₆₁‐butyric‐acid‐methyl‐ester (PCBM) bulk heterojunction, the origins and evolution of the blend morphology during processes such as thermal annealing are not well understood. In this work, we use a model system, a bilayer of P3HT and PCBM, to develop a more complete understanding of the miscibility and diffusion of PCBM within P3HT during thermal annealing. We find that PCBM aggregates and/or molecular species are miscible and mobile in disordered P3HT, without disrupting the ordered lamellar stacking of P3HT chains. The fast diffusion of PCBM into the amorphous regions of P3HT suggests the favorability of mixing in this system, opposing the belief that phase‐pure domains form in BHJs due to immiscibility of these two components.     

Genetic variation in DNMT3B and increased global DNA methylation is associated with suicide attempts in psychiatric patients

Recently, a significant epigenetic component in the pathology of suicide has been realized. Here we investigate candidate functional SNPs in epigenetic‐regulatory genes, DNMT1 and DNMT3B, for association with suicide attempt (SA) among patients with co‐existing psychiatric illness. In addition, global DNA methylation levels [5‐methyl cytosine (5‐mC%)] between SA and psychiatric controls were quantified using the Methylflash Methylated DNA Quantification Kit. DNA was obtained from blood of 79 suicide attempters and 80 non‐attempters, assessed for DSM‐IV Axis I disorders. Functional SNPs were selected for each gene (DNMT1; n = 7, DNMT3B; n = 10), and genotyped. A SNP (rs2424932) residing in the 3′ UTR of the DNMT3B gene was associated with SA compared with a non‐attempter control group (P = 0.001; Chi‐squared test, Bonferroni adjusted P value = 0.02). Moreover, haplotype analysis identified a DNMT3B haplotype which differed between cases and controls, however this association did not hold after Bonferroni correction (P = 0.01, Bonferroni adjusted P value = 0.56). Global methylation analysis showed that psychiatric patients with a history of SA had significantly higher levels of global DNA methylation compared with controls (P = 0.018, Student's t‐test). In conclusion, this is the first report investigating polymorphisms in DNMT genes and global DNA methylation quantification in SA risk. Preliminary findings suggest that allelic variability in DNMT3B may be relevant to the underlying diathesis for suicidal acts and our findings support the hypothesis that aberrant DNA methylation profiles may contribute to the biology of suicidal acts. Thus, analysis of global DNA hypermethylation in blood may represent a biomarker for increased SA risk in psychiatric patients.     

Diversity in the C3b [corrected] contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family

To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B^18–85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B^18–85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg⁴⁴ and Tyr⁵¹ as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.     

A Perspective on Neuronal Cell Death Signaling and Neurodegeneration

Although neuronal cell death through apoptotic pathways represents a common feature of dysferopathies, the canonical apoptotic changes familiar from nonneuronal cells are late events. Loss of neuronal function occurs at a much early time, when synaptic-based neuronal connectivity fails. In this context, apoptotic pathways may normally serve a cleanup role, rather than a pathogenic one. Reframing the consideration of cell death in the nervous system to include the early stages of axonal degeneration provides a better understanding of the roles played by various apoptotic signaling pathways in neurodegenerative diseases. Focusing on disease-specific mechanisms that initiate the sequence that eventually leads to neuronal loss should facilitate development of therapies that preserve neuronal function and neuronal numbers.     

Stability and folding rates of domains spanning the large A-band super-repeat of titin

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.     

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.KEY WORDS: Brain, Intravital microscopy, Leukocytes, Microglia, Neurodegeneration, Zebrafish     

Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.