Complement-induced impairment of innate immunity during sepsis

This study defines the molecular basis for defects in innate immunity involving neutrophils during cecal ligation/puncture (CLP)-induced sepsis in rats. Blood neutrophils from CLP rats demonstrated defective phagocytosis and defective assembly of NADPH oxidase, the latter being due to the inability of p47(phox) to translocate from the cytosol to the cell membrane of neutrophils after cell stimulation by phorbol ester (PMA). The appearance of these defects was prevented by in vivo blockade of C5a in CLP rats. In vitro exposure of neutrophils to C5a led to reduced surface expression of C5aR and defective assembly of NADPH oxidase, as defined by failure in phosphorylation of p47(phox) and its translocation to the cell membrane, together with failure in phosphorylation of p42/p44 mitogen-activated protein kinases. These data identify a molecular basis for defective innate immunity involving neutrophils during sepsis.     

Conformations of nucleotides bound to wild type and Y78F mutant yeast guanylate kinase: proton two-dimensional transferred NOESY measurements

Wild type and Y78F mutant yeast guanylate kinase (GKy) were studied to investigate the effects of a site-directed mutation on bound substrate conformations. Previously published work showed that Y78 is involved in GMP binding and that the Y78F mutant has 30-fold weaker GMP binding and 2 orders of magnitude less activity, than the wild type. Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) and guanosine conformations of guanosine 5'-monophosphate (GMP) bound to wild type and Y78F mutant yeast guanylate kinase in the complexes GKy x Mg(II)ATP, GKy x Mg(II)ADP, GKy x GMP, and GKy x Mg(II)ADP x [U-13C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations. For adenyl nucleotides in wild type complexes, all glycosidic torsion angles, chi, were 54 +/- 5 degrees. In Y78F mutant complexes, adenyl nucleotide glycosidic torsion angles were 55 +/- 5 degrees (GKy x MgATP) and 49 +/- 5 degrees (GKy x MgADP). Thus, the adenyl nucleotides bind similarly for both the wild type and Y78F mutant complexes. However, in the fully constrained, two-substrate complexes, GKy x Mg(II)ADP x [U-13C]GMP, the guanyl glycosidic torsion angle, chi, is 50 +/- 5 degrees with the wild type and 83 +/- 5 degrees with the Y78F mutant. This difference suggests that an unfavorable torsion may be a large part of the mechanism for significantly weaker GMP binding to reaction complexes of the Y78F mutant.     

Three-dimensional solution structures of the chromodomains of cpSRP43

Chloroplasts contain a unique signal recognition particle (cpSRP). Unlike the cytoplasmic forms, the cpSRP lacks RNA but contains a conserved 54-kDa GTPase and a novel 43-kDa subunit (cpSRP43). Recently, three functionally distinct chromodomains (CDs) have been identified in cpSRP43. In the present study, we report the three-dimensional solution structures of the three CDs (CD1, CD2, and CD3) using a variety of triple resonance NMR experiments. The structure of CD1 consists of a triple-stranded beta-sheet segment. The C-terminal helical segment typically found in the nuclear chromodomains is absent in CD1. The secondary structural elements in CD2 and CD3 include a triple-stranded antiparallel beta-sheet and a C-terminal helix. Interestingly, the orientation of the C-terminal helix is significantly different in the structures of CD2 and CD3. Critical comparison of the structures of the chromodomains of cpSRP43 with those found in nuclear chromodomain proteins revealed that the diverse protein-protein interactions mediated by the CDs appear to stem from the differences that exist in the surface charge potentials of each CD. Results of isothermal titration calorimetry experiments confirmed that only CD2 is involved in binding to cpSRP54. The negatively charged C-terminal helix in CD2 possibly plays a crucial role in the cpSRP54-cpSRP43 interaction.     

In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases

Two proteinase inhibitors (PIs), CapA1 and CapA2, were purified from Capsicum annum Linn. Var. Phule Jyoti leaves and assessed for their in vitro and in vivo activity against Helicoverpa armigera gut proteinases (HGPs). Both the inhibitors exhibited molecular weights of about 12 kDa with inhibitory activity against bovine trypsin and chymotrypsin indicating presence of probable two-inhibitor repeats of PIN II family. CapA1 and CapA2 inhibited 60-80% HGP (azocaseinolytic) activity of fourth instar larvae feeding on various host plants while 45-65% inhibition of HGP activity of various instars (II to VI) larvae reared on artificial diet. The partial purification of HGP isoforms, their characterization with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-like activity (68-91%) of HGPs was sensitive to C. annum PIs while 39-85% chymotrypsin-like activity of HGPs was insensitive to these inhibitors. The feeding of C. annum leaf extracts and two purified PIs in various doses to H. armigera larvae for two successive generations through artificial diet demonstrated their potential in inhibiting larval growth and development, delay in pupation period and dramatic reduction in fecundity and fertility. This is the first report-demonstrating efficacy of C. annum PIs against insect gut proteinases as well as larval growth and development of H. armigera.     

Plasmodium falciparum: genetic diversity of C-terminal region of MSP-1 in isolates from Indian sub-continent

Malaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that merozoite surface protein-1 (MSP-1) is an effective target eliciting a protective immune response. The MSP-1(42) region comprising two EGF-like domains is involved in generating protective immune response in humans and other experimental animals. Searching for point mutations in this region is essential in view of vaccine development. We have investigated the sequence variations in Plasmodium falciparum MSP-1 carboxy terminal region in field isolates from different regions in India. Our study reveals the presence of eight variant types of MSP-1(19) in the Indian sub-continent, which comprise of E-TSR-L, Q-TSR-L, E-TSG-L, Q-KNG-L, Q-KNG-F, E-KNG-L, E-KNG-F, and E-KYG-F. The last named allele is a novel variant being reported for the first time.     

Characterization of two midgut proteinases of Helicoverpa armigera and their interaction with proteinase inhibitors

Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants.     

Genotoxicity evaluation of polluted ground water in human peripheral blood lymphocytes using the comet assay

This paper presents the genotoxicity experiments with the ground water collected from an area under the influence of textile dyeing and bleaching industries in Tirupur, Tamilnadu, India. The alkaline single cell gel electrophoresis (SCGE) assay was performed in vitro with human peripheral blood lymphocytes. The cells were exposed to two doses of non-volatile organic agents extracted from ground water samples. Ground water samples were collected from 12 locations distributed in and around Tirupur and extracts were taken at different pHs (without pH adjustment and acidic pH 2.0). The persistence of the DNA damage after exposure to the organic extracts was also studied. All the samples were found to contain substances capable of inducing DNA damage in human lymphocytes. Extracts from acidified waters (pH=2.0) were found to induce more DNA damage than extracts from without pH adjustment (natural pH). The DNA damage was not fully repaired after incubation for 2h at 37 degrees C. The chemical characterization of the sub-fractions revealed the existence of aromatic amines in the extracts, which may be responsible for the DNA damaging activity of the water samples. The results of this investigation demonstrate the application of the comet assay in environmental monitoring studies.     

The expression of proteins involved in digestion and detoxification are regulated in Helicoverpa armigera to cope up with chlorpyrifos insecticide

Helicoverpa armigera is a key pest in many vital crops, which is mainly controlled by chemical strategies. To manage this pest is becoming challenging due to its ability and evolution of resistance against insecticides. Further, its subsequent spread on nonhost plant is remarkable in recent times. Hence, decoding resistance mechanism against phytochemicals and synthetic insecticides is a major challenge. The present work describes that the digestion, defense and immunity related enzymes are associated with chlorpyrifos resistance in H. armigera. Proteomic analysis of H. armigera gut tissue upon feeding on chlorpyrifos containing diet (CH) and artificial diet (AD) using nano‐liquid chromatography–mass spectrometry identified upregulated 23‐proteins in CH fed larvae. Database searches combined with gene ontology analysis revealed that the identified gut proteins engrossed in digestion, proteins crucial for immunity, adaptive responses to stress, and detoxification. Biochemical and quantitative real‐time polymerase chain reaction analysis of candidate proteins indicated that insects were struggling to get nutrients and energy in presence of CH, while at the same time endeavoring to metabolize chlorpyrifos. Moreover, we proposed a potential processing pathway of chlorpyrifos in H. armigera gut by examining the metabolites using gas chromatography–mass spectrometry. H. armigera exhibit a range of intriguing behavioral, morphological adaptations and resistance to insecticides by regulating expression of proteins involved in digestion and detoxification mechanisms to cope up with chlorpyrifos. In these contexts, as gut is a rich repository of biological information; profound analysis of gut tissues can give clues of detoxification and resistance mechanism in insects.