CD8-mediated protection against Ebola virus infection is perforin dependent

CD8 T cells have been shown to play an important role in the clearance and protection against fatal Ebola virus infection. In this study, we examined the mechanisms by which CD8 T cells mediate this protection. Our data demonstrate that all normal mice infected s.c. with a mouse-adapted Ebola virus survived the infection, as did 100% of mice deficient in Fas and 90% of those deficient in IFN-gamma. In contrast, perforin-deficient mice uniformly died after s.c. challenge. Perforin-deficient mice failed to clear viral infection even though they developed normal levels of neutralizing anti-Ebola Abs and 5- to 10-fold higher levels of IFN-gamma than control mice. Using MHC class I tetramers, we have also shown that perforin-deficient mice have 2- to 4-fold higher numbers of Ebola-specific CD8s than control mice. These findings suggest that the clearance of Ebola virus is perforin-dependent and provide an additional example showing that this basic immunologic mechanism is not limited to the clearance of noncytopathic viruses.     

X-ray analysis of Mycobacterium smegmatis Dps and a comparative study involving other Dps and Dps-like molecules

The structure of the DNA binding protein from starved cells from Mycobacterium smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M.smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches that they possess. The M.smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular 3-fold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M.smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces do not need to be between layers of protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.     

GalR represses galP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: a GalR negative control mutant

GalR represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of GalR and RNA polymerase to DNA, and real-time kinetics of base-pair distortion (isomerization) showed that the equilibrium dissociation constant of RNA polymerase-P1 closed complexes is largely unaffected in the presence of saturating GalR, indicating that mutual antagonism (steric hindrance) of the regulator and the RNA polymerase does not occur at this promoter. In fluorescence kinetics with 2-AP labeled P1 DNA, GalR inhibited the slower of the two-step base-pair distortion process. We isolated a negative control GalR mutant, S29R, which while bound to the operator DNA was incapable of repression of P1. Based on these results and previous demonstration that repression requires the C-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase, we propose that GalR establishes contact with alpha-CTD at the last resolved isomerization intermediate, forming a kinetic trap.     

MiGenes: a searchable interspecies database of mitochondrial proteins curated using gene ontology annotation

MOTIVATION: There has been an explosion of interest in the role of mitochondria in programmed cell death and other fundamental pathological processes underlying the development of human diseases. Nevertheless, the inventory of mitochondrial proteins encoded in the nuclear genome remains incomplete, providing an impediment to mitochondrial research at the interface with systems biology. We created the MiGenes database to further define the scope of the mitochondrial proteome in humans and model organisms including mice, rats, flies and worms as well as budding and fission yeasts. MiGenes is intended to stimulate mitochondrial research using model organisms. SUMMARY: MiGenes is a large-scale relational database that is automatically updated to keep pace with advances in mitochondrial proteomics and is curated to assure that the designation of proteins as mitochondrial reflects gene ontology (GO) annotations supported by high-quality evidence codes. A set of postulates is proposed to help define which proteins are authentic components of mitochondria. MiGenes incorporates >1160 new GO annotations to human, mouse and rat protein records, 370 of which represent the first GO annotation reflecting a mitochondrial localization. MiGenes employs a flexible search interface that permits batchwise accession number searches to support high-throughput proteomic studies. A web interface is provided to permit members of the mitochondrial research community to suggest modifications in protein annotations or mitochondrial status.     

Arabidopsis inflorescence architecture requires the activities of KNOX-BELL homeodomain heterodimers

In flowering plants, post-embryonic development is mediated by the activity of shoot and root apical meristems. Shoot architecture results from activity of the shoot apical meristem (SAM), which initiates primordia, including leaves, internodes and axillary meristems, repetitively from its flanks. Axillary meristems can develop into secondary shoots or flowers. In Arabidopsis, two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), expressed in the SAM, encode DNA-binding proteins that are essential for specifying floral primordia and establishing early internode patterning events during inflorescence development. Biochemical studies show that PNY associates with the knotted1-like homeobox (KNOX) proteins, SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). PNY-BP heterodimers are essential for establishing early internode patterning events, while PNY-STM heterodimers are critical for SAM function. In this report, we examined the role of PNY, PNF and STM during development. First, we show that PNF interacts with STM and BP indicating that PNY and PNF are redundant functioning proteins. Inflorescence development, but not vegetative development, is sensitive to the dosage levels of PNY, PNF and STM. Characterization of stm-10, a weak allele in the Columbia ecotype, indicates that STM is also involved in floral specification and internode development. Our examination of the genetic requirements for PNY, PNF and STM demonstrates that these KNOX–BELL heterodimers control floral specification, internode patterning and the maintenance of boundaries between initiating floral primordia and the inflorescence meristem.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Synergistic effect of conjugated linolenic acid isomers against induced oxidative stress,inflammation and erythrocyte membrane disintegrity in rat model

Background

α-Eleostearic acid and punicic acid, two typical conjugated linolenic acid (CLnA) isomers present in bitter gourd and snake gourd oil respectively, exhibit contrasting cis-trans configuration which made them biologically important.

Methods

Rats were divided into six groups. Group 1 was control and group 2 was treated control. Rats in the groups 3 and 4 were treated with mixture of α-eleostearic acid and punicic acid (1:1) (0.5% and 1.0% respectively) while rats in the groups 5 and 6 were treated with 0.5% of α-eleostearic acid and 0.5% of punicic acid respectively along with sodium arsenite by oral gavage once per day.

Results

Results showed that increase in nitric oxide synthase (NOS) activity, inflammatory markers expression, platelet aggregation, lipid peroxidation, protein oxidation, DNA damage and altered expression of liver X receptor-α (LXR-α) after arsenite treatment were restored with the supplementation of oils containing CLnA isomers. Altered activities of different antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and ferric reducing ability of plasma (FRAP) also restored after oil supplementation. Altered morphology and fluidity of erythrocyte membrane studied by atomic force and scanning electron microscopy, after stress induction were significantly improved due to amelioration in cholesterol/phospholipid ratio and fatty acid profile of membrane. Oils treatment also improved morphology of liver and fatty acid composition of hepatic lipid.

Conclusions

Overall two isomers showed synergistic antioxidant and anti-inflammatory effect against induced perturbations and membrane disintegrity.

General significance

Synergistic antioxidant and anti-inflammatory role of these CLnA isomers were established by this study.     

Biochemical and molecular analyses of copper-zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23(nmf308/nmf308) mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23(nmf308/nmf308) mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23(nmf308/nmf308) mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23(nmf308/nmf308) mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5-10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23(nmf308/nmf308) mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23(nmf308/nmf308) mouse is an excellent animal model for investigating the mechanisms involved in human AHL.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A(2) (RVVA-PLA(2)-I) from Daboia russelli venom

An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA(2)-I was non-lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA(2) enzyme. RVVA-PLA(2)-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA(2)-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA(2)-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA(2)s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA(2)-I suggested the correlation between catalytic and membrane damaging activities of this PLA(2) enzyme. Our study advocates that the presence of a large number of PLA(2)-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA(2) enzyme.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom

An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24 h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.