Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

Unexpected inhibition of S-adenosyl-L-homocysteine hydrolase by a guanosine nucleoside

A series of shape-modified flexible nucleosides ('fleximers', 1, 2, and 3) was modeled, synthesized and subsequently assayed against S-adenosyl-L-homocysteine hydrolase (SAHase). No inhibitory activity was observed for the adenosine fleximer, which served as a substrate, but moderate inhibitory activity was exhibited by the guanosine fleximers. This is the first known report of a guanosine nucleoside analogue possessing activity against SAHase.     

CO2 permeability of the rat erythrocyte membrane and its inhibition

We have studied the CO₂ permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs ¹⁸O-labelled CO₂. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO₃⁻ permeability. For normal rat erythrocytes, we find at 37 °C a CO₂ permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10⁻³cm/s. We studied whether the rat erythrocyte membrane possesses protein CO₂ channels similar to the human red cell membrane by applying the potential CO₂ channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO₂ permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO₂ channels that are responsible for at least 50% of its CO₂ permeability.     

Co-ordinated role of TLR3, RIG-I and MDA5 in the innate response to rhinovirus in bronchial epithelium

The relative roles of the endosomal TLR3/7/8 versus the intracellular RNA helicases RIG-I and MDA5 in viral infection is much debated. We investigated the roles of each pattern recognition receptor in rhinovirus infection using primary bronchial epithelial cells. TLR3 was constitutively expressed; however, RIG-I and MDA5 were inducible by 8-12 h following rhinovirus infection. Bronchial epithelial tissue from normal volunteers challenged with rhinovirus in vivo exhibited low levels of RIG-I and MDA5 that were increased at day 4 post infection. Inhibition of TLR3, RIG-I and MDA5 by siRNA reduced innate cytokine mRNA, and increased rhinovirus replication. Inhibition of TLR3 and TRIF using siRNA reduced rhinovirus induced RNA helicases. Furthermore, IFNAR1 deficient mice exhibited RIG-I and MDA5 induction early during RV1B infection in an interferon independent manner. Hence anti-viral defense within bronchial epithelium requires co-ordinated recognition of rhinovirus infection, initially via TLR3/TRIF and later via inducible RNA helicases.     

Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation

Recent studies suggest that superoxide dismutase (SOD1) may represent a major target of oxidative damage in neurodegenerative diseases. To test the possibility that oxidized species of wild-type (WT) SOD1 might be involved in pathogenic processes, we analyzed the properties of the WT human SOD1 protein after its oxidation in vivo or in vitro by hydrogen peroxide (H2O2) treatment. Using transfected Neuro2a cells expressing WT or amyotrophic lateral sclerosis-linked SOD1 species, we show that exposure to H2O2 modifies the properties of WT SOD1. Western blot analysis of immunoprecipitates from cell lysates revealed that, like mutant SOD1, oxidized WT SOD1 can be conjugated with poly-ubiquitin and can interact with Hsp70. Chromogranin B, a neurosecretory protein that interacts with mutant SOD1 but not with WT SOD1, was co-immunoprecipitated with oxidized WT SOD1 from lysates of Neuro2a cells treated with H2O2. Treatment of microglial cells (line BV2) with either oxidized WT SOD1 or mutant SOD1 recombinant proteins induced tumor necrosis factor-alpha and inducible nitric oxide synthase. Furthermore, exposure of cultured motor neurons to oxidized WT SOD1 caused dose-dependent cell death like mutant SOD1 proteins. These results suggest that WT SOD1 may acquire binding and toxic properties of mutant forms of SOD1 through oxidative damage.     

Human DNA polymerase kappa encircles DNA: implications for mismatch extension and lesion bypass

Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.     

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2010-31 January 2011

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross-tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii.     

The circadian <Emphasis Type="Italic">Clock</Emphasis> mutant mouse: impaired masking response to light

Synchronization of an internal clock (entrainment) and a direct response to light (masking) are complementary ways of restricting activity of an animal to day or night. The protein CLOCK has an important role in the oscillatory mechanism of mammalian pacemakers. Our data show that it is also involved in masking responses. Mice with the Clock/Clock mutation reduced their wheel running less than wildtypes when given 1-h light pulses of light (2–1,600 lx) in the night. With dimmer lights (<2 lx), there were no significant differences between mutant and wildtype mice. Impaired masking responses to light in Clock/Clock mice were confirmed in tests with ultradian light–dark cycles (3.5:3.5 h and 1:1 h). Tests with pulses of light longer than 1 h revealed that, although the mutants responded more slowly to light, they sustained the suppression of activity over the course of the 3-h tests better than wildtypes.