Saturated and trans-unsaturated fatty acids elicit high levels of superoxide generation in intact and cell-free preparations of neutrophils

Saturated and trans-unsaturated fatty acids, such as laurate and elaidate, elicited O2- generation in intact porcine and human neutrophils and also in a cell-free preparation of porcine neutrophils. The activities thus induced were comparable to those induced by cis-unsaturated fatty acids. However, the activation by saturated or trans-unsaturated fatty acids was depressed almost completely in the presence of Ca2+ at around 1 mM, which is usually contained in the media for phagocytes. In contrast, the activation by cis-unsaturated fatty acids such as arachidonate was scarcely affected by Ca2+. These findings appear to demand reevaluation of the effects of long chain fatty acids on the respiratory burst system in phagocytes.     

Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.     

Role of luteinizing hormone-releasing factor (LH-RF) and LH on maintenance of early gestation in rats.

The role of luteinizing hormone (LH) and LH-releasing hormone (LH-RH) in the maintenance of early pregnancy in rats was studied. Serum levels of progesterone (P) and LH were measured daily in untreated pregnant rats from Day 4 through parturition. Serum levels of P and LH were determined on Days 11 and 15 of pregnancy in animals treated with antisera to LH (LH-A/S) and to LH-RH (LH-RH-A/S) on Days 8-10. Serum levels of P peaked on Days 7 and 16 in untreated animals, after which they declined sharply just before delivery. Serum LH fluctuated between 30-160 ng/ml during pregnancy but did not exhibit any distinctive peaks. Treatment with .2 ml LH-A/S on Days 8-10 reduced serum P to virtually undetectable levels on Day 11, and only a slight recovery was evident on Day 15. Lower doses of LH-A/S had no effect. Administration of 1.3 ml LH-RH-A/S had no effect on serum levels of P or LH, and did not impede fetal development. The results indicate that LH is essential to the luteotropic complex of early pregnancy in the rat, and also that LH-RH-A/S can maintain to some extent basal levels of P and LH during early pregnancy.     

Deletions of any single residues in Glu40-Ser48 loop connecting a domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase result in almost complete inhibition of conformational transition and hydrolysis of phosphoenzyme intermediate

Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca(2+)-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca(2+)-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.     

Community-wide plasmid gene mobilization and selection

Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host''s chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions.     

Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells

The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155–7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling.     

Retinal Vascular Caliber Is Associated with Cardiovascular Biomarkers of Oxidative Stress and Inflammation: The POLA Study

Purpose

Retinal vascular caliber has been linked with increased cardiovascular risk and is predictive of cardiovascular pathology, including stroke and coronary heart disease. Oxidative stress, as well as inflammatory mechanisms, plays a major role in the pathogenesis and progression of atherosclerosis, plaque rupture and vascular thrombotic propensity. The purpose of this study is to explore the relationship between retinal vascular calibers and biomarkers of oxidative stress and inflammation, in subjects free of cardiovascular pathology.

Patients and Methods

Cross-sectional analysis from a community-dwelling cohort comprising 1224 individuals aged 60 years and over, without a history of coronary or peripheral artery disease or stroke. Retinal vascular caliber was measured from fundus photographs using semi-automated standardized imaging software. Oxidative stress was evaluated using plasma superoxide dismutase 2 and glutathione peroxidase (GPx-3) activities, and inflammatory state was assessed using plasma high sensitivity C-reactive protein (hsCRP) and orosomucoid.

Results

In a multivariate model controlling for cardiovascular risk factors, larger retinal arteriolar caliber was independently related to higher level of GPx-3 activity (p = 0.003) whereas larger venular caliber was associated with higher levels of hsCRP (p = 0.0001) and orosomucoid (p = 0.01).

Conclusion

In the present study, biomarkers of oxidative stress regulation and inflammation were independently associated with retinal vascular calibers. This suggests that an assessment of retinal vessels may offer early and non-invasive detection of subclinical vascular pathology.     

Single-Molecule Observation of the Ligand-Induced Population Shift of Rhodopsin,A G-Protein-Coupled Receptor

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.     

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1 fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.     

TCR‐β repertoire analysis of antigen‐specific single T cells using a high‐density microcavity array

The antigen specificity of cytotoxic T cells, provided by T‐cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single‐cell level is required. In this study, we applied a high‐density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR‐β gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent‐labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T‐cell population. The isolated single cells were then directly utilized for RT‐PCR. By sequencing the amplified PCR products, antigen‐specific TCR‐β repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single‐cell level. This simple, accurate, and cost‐effective technique for single‐cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells. Biotechnol. Bioeng. 2010;106: 311–318. © 2010 Wiley Periodicals, Inc.