Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.     

Reusability of surfactant-coated Candida rugosa lipase immobilized in gelatin microemulsion-based organogels for ethyl isovalerate synthesis

In our previous study, a surfactant-coated Candida rugosa lipase immobilized in microemulsion-based organogels was exploited for the synthesis of ethyl isovalerate. In the present study, we are focusing on the effective reuse of lipase immobilized in microemulsion-based organogels (MBGs) in terms of retainment of the catalytic activity. As water is one of the co-products in esterification reactions, the removal of water becomes a priority to allow the reaction to work in the forward direction and to prevent back hydrolysis. Taking this fact into consideration, the lipase-containing microemulsion-based organogels were given pretreatment and/or several intermittent treatments with dry reverse micellar solution of AOT in organic solvent during repeated cycles of ester synthesis. The pretreated MBGs with dry reverse micellar solution exhibited lower water content and higher initial rates of esterification in comparison with untreated freshly prepared MBGs. The esterification efficiency of untreated MBGs started decreasing after 5 cycles of reuse and was almost completely lost by the end of the 8th cycle. In contrast, pretreated MBGs exhibited a gradual decrease in esterification efficiency after 5 cycles and retained about 80% of the initial activity at the end of the 8th cycle. The intermittent treatment of MBGs after every 3 cycles resulted in enhanced reusability of immobilized lipase for up to 9 cycles without significant loss in esterification activity, after which it resulted in a slow decrease in activity with about 27% lower activity at the end of the 12th cycle. Furthermore, the treatment conditions such as concentration of AOT in liquid dessicant and time of treatment were optimized with respect to our system. The granulated MBGs proved to be better in terms of initial esterification rates (1.2- fold) as compared with the pelleted MBGs.     

Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes

We have resolved and analyzed two proline-rich proteins isolated from the walls of soybean cells in culture. The proteins are similar in amino acid content, containing 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% tyrosine, and 10% glutamate. The proteins undergo a rearrangement or a limited cleavage in dilute NaOH, but are otherwise remarkably stable to a high concentration of alkali. We have cloned and sequenced a cDNA from soybean axes germinated for 31 hours (1A10-2) coding for a protein that closely corresponds in its amino acid content to that of the proline-rich proteins. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-Tyr-Lys-Pro-Pro-Val-Glu-Lys. Consequently, this class of proteins is referred to as repetitive proline-rich proteins, i.e., RPRP2 and RPRP3. We have also analyzed RNA gel blots with probes that discriminate between the new cDNA clone and a related cDNA previously reported [SbPRP1; Hong, Nagao, and Key (1987). J. Biol. Chem. 262, 8367-8376]. Messenger RNAs from young seedlings and from soybean suspension cultures correspond primarily to the new RPRP clone (1A10-2), whereas the predominant mRNA accumulating later in the roots corresponds to SbPRP1.     

Antioxidant activity of polar lichens from King George Island (Antarctica)

Antioxidant agents prevent reactive oxygen species, which can cause degenerative diseases. Natural antioxidants are preferred over many synthetic antioxidants, which can be toxic, for therapeutic applications. Five lichen species were collected from King George Island, Antarctica. Antioxidant activities as assessed by DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical and ABTS^•+ [2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate)] radical scavenging capacities were determined and compared with those of commercial standards BHA (butylated hydroxyanisole) and trolox [(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid]. The results indicated that two lichens exhibited comparatively high antioxidant activities with the remaining three exhibiting less activity. The antioxidant activity was concentration-dependent. When compared, the antioxidant activity of crude extracts from polar lichens to previously published data for tropical and temperate lichen species, we concluded that lichens of Antarctic origin may be the potent sources of strong antioxidant agents. Such species should be explored as novel sources of effective antioxidant metabolites.     

A rapid serological assay for prediction of Salmonella infection status in slaughter pigs using surface plasmon resonance

We present a rapid surface plasmon resonance-based serological assay for the detection of Salmonella Typhimurium infection in pigs using the Plasmonic((R)) SPR device. Lipopolysaccharide (LPS, 10 microg mL(-1)) from Salmonella Typhimurium was immobilised by self-assembly on a hydrophobic SPR chip. Using this LPS-coated chip, it was possible to bind and detect the anti-Salmonella Typhimurium antibodies in serum of pigs infected with the bacteria. The developed SPR assay is able to differentiate between sera obtained from pigs having low, medium, and high levels of Salmonella infection. A commercial ELISA kit was used to classify the sera for levels of Salmonella infection on the basis of optical density (OD%). A strong positive correlation was observed between the SPR-based assay and the ELISA (n=38, r=0.90, p<0.01). The sensitivity and specificity of the assay are 0.93 and 0.87, respectively. The SPR-based assay is label-free and does not require any sample preparation or dilution steps. The total analysis time is 45 min for each serum sample. The assay was found to be specific for Salmonella Typhimurium and shows no cross-reactivity to Salmonella Choleraesuis or Escherichia coli antibodies. As no sample preparation is required the developed assay has the potential to be used as a reliable tool for Salmonella monitoring programmes in pork production.     

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hg^r and 170 PPT^r IR72 plants from protoplasts through selection. 31 Hg^r and 70 PPT^r plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.     

IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

Recovery of Sugars from Ionic Liquid Biomass Liquor by Solvent Extraction

The dissolution of biomass into ionic liquids (ILs) has been shown to be a promising alternative biomass pretreatment technology, facilitating faster breakdown of cellulose through the disruption of lignin and the decrystallization of cellulose. Both biological and chemical catalysis have been employed to enhance the conversion of IL-treated biomass polysaccharides into monomeric sugars. However, biomass-dissolving ILs, sugar monomers, and smaller carbohydrate oligomers are all soluble in water. This reduces the overall sugar content in the recovered solid biomass and complicates the recovery and recycle of the IL. Near-complete recovery of the IL and the holocellulose is essential for an IL-based pretreatment technology to be economically feasible. To address this, a solvent extraction technique, based on the chemical affinity of boronates such as phenylboronic acid and naphthalene-2-boronic acid for sugars, was applied to the extraction of glucose, xylose, and cellobiose from aqueous mixtures of 1-ethyl-3-methylimidazolium acetate. It was shown that boronate complexes could extract up to 90% of mono- and disaccharides from aqueous IL solutions, 100% IL systems, and hydrolysates of corn stover containing IL. The use of boronate complexes shows significant potential as a way to recover sugars at several stages in ionic liquid biomass pretreatment processes, delivering a concentrated solution of fermentable sugars, minimizing toxic byproducts, and facilitating ionic liquid cleanup and recycle.     

The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer.

In Agrobacterium tumefaciens, a cis-active 24-base-pair sequence adjacent to the right border of the T-DNA, called overdrive, stimulates tumor formation by increasing the level of T-DNA processing. Recent results from our laboratory have suggested that the virC operon which enhances T-DNA processing probably does so because the VirC1 protein interacts with overdrive (N. Toro, A. Datta, M. Yanofsky, and E. W. Nester, Proc. Natl. Acad. Sci. USA 85:8558-8562, 1988). We report here the purification of the VirC1 protein from cells of Escherichia coli harboring a plasmid containing the coding sequences of the virC locus of the octopine Ti plasmid. By gel mobility shift and DNase I footprinting assays, we showed that this purified virC1 gene product binds to overdrive but not to the right border of T-DNA.     

Structural and functional characterization of type three secretion system ATPase PscN and its regulator PscL from Pseudomonas aeruginosa

Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with V_max of 3.9 ± 0.2 μmol/min/mg and K _m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN–PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN–PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN–PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN–PscL complex in T3SS mediated pathogenic pathway.