Role of activated macrophages in Acanthamoeba keratitis

The purpose of this study was to determine whether activating the conjunctival macrophages would affect the course of Acanthamoeba spp. keratitis in a Chinese hamster model of this disease. Chinese hamster spleen cells were stimulated with concanavalin A (Con A), and interferon gamma (IFN-gamma) -containing supernatants were collected 24 hr later. The IFN-gamma-containing supernatants were loaded into liposomes, which were fed to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for production of nitric oxide (NO) with the use of Griess reagent. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A-activated spleen cell culture supernatants. Control liposomes were loaded with phosphate-buffered saline (PBS). Macrophages exposed to supernatants from Con A-stimulated spleen cells produced 4-fold-higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A-stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with PBS-containing liposomes demonstrated evidence of corneal disease at day 14 compared to 10% incidence of infection in the Con A-treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatant was significantly reduced compared to the group treated with liposomes containing PBS (P < 0.05). Macrophages stimulated with IFN-gamma-containing supernatants killed significant numbers of the trophozoites in vitro (P < 0.05). Killing was inhibited by cytochalasin D, but not by L-N6-1-iminoethyl-L-lysine dihydrochloride (L-NIL), which is a selective inhibitor of inducible NO synthase (INOS).     

Ion pairs in non-redundant protein structures

Ion pairs contribute to several functions including the activity of catalytic triads, fusion of viral membranes, stability in thermophilic proteins and solvent-protein interactions. Furthermore, they have the ability to affect the stability of protein structures and are also a part of the forces that act to hold monomers together. This paper deals with the possible ion pair combinations and networks in 25% and 90% non-redundant protein chains. Different types of ion pairs present in various secondary structural elements are analysed. The ion pairs existing between different subunits of multisubunit protein structures are also computed and the results of various analyses are presented in detail. The protein structures used in the analysis are solved using X-ray crystallography, whose resolution is better than or equal to 1.5 A and R-factor better than or equal to 20%. This study can, therefore, be useful for analyses of many protein functions. It also provides insights into the better understanding of the architecture of protein structure.     

Investigations on unconventional hydrogen bonds in RNA binding proteins: the role of CH...O=C interactions

We have investigated the roles played by C-H...O=C interactions in RNA binding proteins. There was an average of 78 CH...O=C interactions per protein and also there was an average of one significant CH...O=C interactions for every 6 residues in the 59 RNA binding proteins studied. Main chain-Main chain (MM) CH...O=C interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH...O=C interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH...O=C interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH...O=C interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH...O=C interacting polar amino acid residues were solvent exposed while, majority of the CH...O=C interacting non polar residues were excluded from the solvent. Long and medium-range CH...O=C interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH...O=C interacting residues had a higher conservation score. Significant percentage of CH...O=C interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH...O=C interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Linkage and association analysis of candidate genes for TB and TNFα cytokine expression: evidence for association with IFNGR1, IL-10, and TNF receptor 1 genes

Tuberculosis (TB) is a growing public health threat globally and several studies suggest a role of host genetic susceptibility in increased TB risk. As part of a household contact study in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB by developing an intermediate phenotype model for TB susceptibility, analyzing levels of tumor necrosis factor-α (TNFα) in response to culture filtrate as the phenotype. In the present study, we analyzed candidate genes related to TNFα regulation and found that interleukin (IL)-10, interferon-gamma receptor 1 (IFNGR1), and TNFα receptor 1 (TNFR1) genes were linked and associated to both TB and TNFα. We also show that these associations are with progression to active disease and not susceptibility to latent infection. This is the first report of an association between TB and TNFR1 in a human population and our findings for IL-10 and IFNGR1 replicate previous findings. By observing pleiotropic effects on both phenotypes, we show construct validity of our intermediate phenotype model, which enables the characterization of the role of these genetic polymorphisms on TB pathogenesis. This study further illustrates the utility of such a model for disentangling complex traits. C. C. Whalen and S. K. Iyengar contributed equally as senior authors of this work.     

Unique helical conformation of the fourth cytoplasmic loop of the CB1 cannabinoid receptor in a negatively charged environment

The proximal portion of the C-terminus of the CB(1) cannabinoid receptor is a primary determinant for G-protein activation. A 17 residue proximal C-terminal peptide (rodent CB1 401-417), the intracellular loop 4 (IL4) peptide, mimicked the receptor's G-protein activation domain. Because of the importance of the cationic amino acids to G-protein activation, the three-dimensional structure of the IL4 peptide in a negatively charged sodium dodecyl sulfate (SDS) micellar environment has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR) spectroscopy and distance geometry calculations. Unambiguous proton NMR assignments were carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints were used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which were refined using restrained energy minimization and dynamics. In water, the IL4 peptide prefers an extended conformation, whereas in SDS micelles, 3(10)-helical conformation is induced. The predominance of 3(10)-helical domain structure in SDS represents a unique difference compared with structure in alternative environments, which can significantly impact global electrostatic surface potential on the cytoplasmic surface of the CB(1) receptor and might influence the signal to the G-proteins.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

The isolation of reticulocyte-free human red blood cells

We depleted reticulocytes from erythrocytes of both sickle cell disease (SCD) subjects and healthy controls by four methods: fluorescence-activated cell sorting (FACS), Miltenyi immunomagnetic depletion (MACS), a combination of these methods (FACS + MACS) and Percoll density separation. The efficiency of these methods was assessed by new methylene blue staining and manual enumeration of the reticulocytes. FACS sorted erythrocytes from reticulocytes based on size and granularity, as well as the absence of dsDNA staining. MACS depleted reticulocytes from erythrocytes based on the immunoaffinity to CD36 and CD71. Reticulocytes from healthy controls were depleted to 相似文献   

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Membrane‐induced structure of novel human tachykinin hemokinin‐1 (hHK1)

PPT‐C encoded hemokinin‐1(hHK‐1) of Homo sapiens (TGKASQFFGLM) is a structurally distinct neuropeptide among the tachykinin family that participate in the NK‐1 receptor downstream signaling processes. Subsequently, signal transduction leads to execution of various effector functions which includes aging, immunological, and central nervous system (CNS) regulatory actions. However the conformational pattern of ligand receptor binding is unclear. The three‐dimensional structure of the hemokinin‐1 in aqueous and micellar environment has been studied by one and two‐dimensional proton nuclear magnetic resonance (2D 1H‐NMR spectroscopy) and distance geometry calculations. Data shows that hemokinin‐1 was unstructured in aqueous environment; anionic detergent SDS induces α‐helix formation. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient‐COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (CYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Helical conformation is observed from residue K3‐M11. The conformational range of the peptide revealed by NMR studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared to that of Substance P potent NK1 agonist. Thus the report provides a structural insight to study hHK‐1‐NK1 interaction that is essential for hHK1 based signaling events. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 702–710, 2015.