Examination of the premelting transition of DNA A-tracts using a fluorescent adenosine analogue

The fluorescent adenosine analogue 4-amino-8-(2-deoxy-beta-d-ribofuranosyl)-5'-O-dimethoxytrityl-6-methyl-7(8H)-pteridone (6MAP) has been used to perform residue specific analyses of DNA A-tracts during the premelting transition. DNA A-tracts, which exhibit sequence-induced curvature, adopt a B-DNA conformation as a function of increasing temperature. Fluorescence melting curves indicate that 6MAP is a more sensitive reporter of the premelting transition than UV absorption spectroscopy. Further, residue specific fluorescence analyses of A-tract and control duplexes reveal that some of the conformational changes associated with the premelting transition occur within A-tract regions. Analyses of the energetics of the premelting transition indicate that ApA steps make a larger enthalpic contribution to the premelting transition than ApT steps. To explore the effect of cations on the premelting transition, fluorescence melts were performed in the presence of NH(4)(+), Mg(2+), and low (0.05 M) and high (0.5 M) concentrations of Na(+). These studies show that the fluorescence intensity changes associated with the premelting transition are sensitive to cation type and concentration and are larger and more pronounced in the presence of 0.5 M Na(+), NH(4)(+), and Mg(2+). Incorporation of 6MAP into longer duplexes containing phased A-tracts shows that the local environment of adenosines in phased A-tracts is similar to that of individual A-tracts. Fluorescence quenching results indicate that ApA and ApT steps within A-tracts are less solvent exposed than their counterparts in control sequence isomers, possibly because of the narrowed minor groove of A-tract sequences.     

A new Mad2-interacting domain of Cdc20 is critical for the function of Mad2-Cdc20 complex in the spindle assembly checkpoint

Interaction between Mad2 and Cdc20 (cell division cycle 20) is a key event during spindle assembly checkpoint activation. In the past, an N-terminal peptide containing amino acid residues 111-150 of Cdc20 was shown to bind Mad2 much better than the full-length Cdc20 protein. Using co-localization, co-immunoprecipitation and peptide inhibition analysis with different deletion mutants of Cdc20, we identified another Mad2-binding domain on Cdc20 from amino acids 342-355 within the WD repeat region. An intervening region between these two domains interferes with its Mad2 binding when present individually with any of these two Mad2-binding sites. We suggest that these three domains together determine the overall strength of Mad2 binding with Cdc20. Functional analysis suggests that an optimum Mad2 binding efficiency of Cdc20 is required during checkpoint arrest and release. Further, we have identified a unique polyhistidine motif with metal binding property adjacent to this second binding domain that may be important for maintaining the overall conformation of Cdc20 for its binding to Mad2.     

CpG oligonucleotides enhance the tumor antigen-specific immune response of an anti-idiotype antibody-based vaccine strategy in CEA transgenic mice

A murine monoclonal anti-idiotype (Id) antibody, 3H1 has been developed and characterized previously. Anti-Id 3H1 mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate antigen for CEA. 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colon cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. In this study, we have evaluated the efficacy of 3H1 as a tumor vaccine when admixed with a select CpG ODN 1826 in transgenic mice that express human CEA. The vaccine potential of 3H1 was also assessed in the presence of another widely used adjuvant, QS-21. 3H1 coupled to keyhole limpet hemocyanin (KLH) and mixed with Freund’s adjuvant (FA) was used as a gold standard in this system. 3H1 vaccination with different adjuvants induced both humoral and cellular anti-3H1, as well as anti-CEA immunity in CEA transgenic mice. The immune sera could lyse CEA-transfected murine colon carcinoma cells, C15 effectively in an antibody-dependent cellular cytotoxicity assay. The anti-CEA antibody responses were somewhat comparable in each adjuvant-treated group of mice, whereas cellular immune responses were significantly greater when CpG was used as an adjuvant. Splenocytes obtained from 3H1–CpG-immunized mice showed an increased proliferative CD4⁺ Th1-type T-cell response when stimulated in vitro with 3H1 or CEA and secreted elevated levels of Th1 cytokines (IL-2, IFN-γ). This vaccine also induced MHC class I antigen-restricted CD8⁺ T-cell responses. In a solid tumor model, C15 tumor growth was significantly inhibited by 3H1 vaccinations. In 3H1–CpG-vaccinated mice, the duration of survival was, however, longer compared to the 3H1–QS21-vaccinated mice. These findings suggest that 3H1-CpG vaccinations can break peripheral tolerance to CEA and induce protective antitumor immunity in this murine model transgenic for human CEA.     

Indomethacin inactivates gastric peroxidase to induce reactive-oxygen-mediated gastric mucosal injury and curcumin protects it by preventing peroxidase inactivation and scavenging reactive oxygen

We have investigated the mechanism of indomethacin-induced gastric ulcer caused by reactive oxygen species (ROS) and the gastroprotective effect of curcumin thereon. Curcumin dose-dependently blocks indomethacin-induced gastric lesions, showing 82% protection at 25 mg/kg. Indomethacin-induced oxidative damage by ROS as shown by increased lipid peroxidation and thiol depletion is almost completely blocked by curcumin. Indomethacin causes nearly fivefold increase in hydroxyl radical (()OH) and significant inactivation of gastric mucosal peroxidase to elevate endogenous H(2)O(2) and H(2)O(2)-derived ()OH, which is prevented by curcumin. In vitro studies indicate that indomethacin inactivates peroxidase irreversibly only in presence of H(2)O(2) by acting as a suicidal substrate. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) protects the peroxidase, indicating involvement of indomethacin radical in the inactivation. Indomethacin radical was also detected in the peroxidase-indomethacin-H(2)O(2) system as DMPO adduct (a(N) = 15 G, a(beta)(H) = 16 G) by electron spin resonance spectroscopy. Curcumin protects the peroxidase in a concentration-dependent manner and consumes H(2)O(2) for its oxidation as a suitable substrate of the peroxidase, thereby blocking indomethacin oxidation. Curcumin can also scavenge ()OH in vitro. We suggest that curcumin protects gastric damage by efficient removal of H(2)O(2) and H(2)O(2) -derived ()OH by preventing peroxidase inactivation by indomethacin.     

The crystal structure of an octapeptide repeat of the Prion protein in complex with a Fab fragment of the POM2 antibody

Prion diseases are progressive, infectious neurodegenerative disorders caused primarily by the misfolding of the cellular prion protein (PrP^c) into an insoluble, protease‐resistant, aggregated isoform termed PrP^sc. In native conditions, PrP^c has a structured C‐terminal domain and a highly flexible N‐terminal domain. A part of this N‐terminal domain consists of 4–5 repeats of an unusual glycine‐rich, eight amino acids long peptide known as the octapeptide repeat (OR) domain. In this article, we successfully report the first crystal structure of an OR of PrP^c bound to the Fab fragment of the POM2 antibody. The structure was solved at a resolution of 2.3 Å by molecular replacement. Although several studies have previously predicted a β‐turn‐like structure of the unbound ORs, our structure shows an extended conformation of the OR when bound to a molecule of the POM2 Fab indicating that the bound Fab disrupts any putative native β turn conformation of the ORs. Encouraging results from several recent studies have shown that administering small molecule ligands or antibodies targeting the OR domain of PrP result in arresting the progress of peripheral prion infections both in ex vivo and in in vivo models. This makes the structural study of the interactions of POM2 Fab with the OR domain very important as it would help us to design smaller and tighter binding OR ligands.     

Oilseed rape seeds with ablated defence cells of the glucosinolate-myrosinase system. Production and characteristics of double haploid MINELESS plants of Brassica napus L

Oilseed rape and other crop plants of the family Brassicaceae contain a unique defence system known as the glucosinolate-myrosinase system or the 'mustard oil bomb'. The 'mustard oil bomb' which includes myrosinase and glucosinolates is triggered by abiotic and biotic stress, resulting in the formation of toxic products such as nitriles and isothiocyanates. Myrosinase is present in specialist cells known as 'myrosin cells' and can also be known as toxic mines. The myrosin cell idioblasts of Brassica napus were genetically reprogrammed to undergo controlled cell death (ablation) during seed development. These myrosin cell-free plants have been named MINELESS as they lack toxic mines. This has led to the production of oilseed rape with a significant reduction both in myrosinase levels and in the hydrolysis of glucosinolates. Even though the myrosinase activity in MINELESS was very low compared with the wild type, variation was observed. This variability was overcome by producing homozygous seeds. A microspore culture technique involving non-fertile haploid MINELESS plants was developed and these plants were treated with colchicine to produce double haploid MINELESS plants with full fertility. Double haploid MINELESS plants had significantly reduced myrosinase levels and glucosinolate hydrolysis products. Wild-type and MINELESS plants exhibited significant differences in growth parameters such as plant height, leaf traits, matter accumulation, and yield parameters. The growth and developmental pattern of MINELESS plants was relatively slow compared with the wild type. The characteristics of the pure double haploid MINELESS plant are described and its importance for future biochemical, agricultural, dietary, functional genomics, and plant defence studies is discussed.     

Neem Leaf Glycoprotein Prophylaxis Transduces Immune Dependent Stop Signal for Tumor Angiogenic Switch within Tumor Microenvironment

We have reported that prophylactic as well as therapeutic administration of neem leaf glycoprotein (NLGP) induces significant restriction of solid tumor growth in mice. Here, we investigate whether the effect of such pretreatment (25µg/mice; weekly, 4 times) benefits regulation of tumor angiogenesis, an obligate factor for tumor progression. We show that NLGP pretreatment results in vascular normalization in melanoma and carcinoma bearing mice along with downregulation of CD31, VEGF and VEGFR2. NLGP pretreatment facilitates profound infiltration of CD8⁺ T cells within tumor parenchyma, which subsequently regulates VEGF-VEGFR2 signaling in CD31⁺ vascular endothelial cells to prevent aberrant neovascularization. Pericyte stabilization, VEGF dependent inhibition of VEC proliferation and subsequent vascular normalization are also experienced. Studies in immune compromised mice confirmed that these vascular and intratumoral changes in angiogenic profile are dependent upon active adoptive immunity particularly those mediated by CD8⁺ T cells. Accumulated evidences suggest that NLGP regulated immunomodulation is active in tumor growth restriction and normalization of tumor angiogenesis as well, thereby, signifying its clinical translation.     

Social Cohesion,Social Participation,and HIV Related Risk among Female Sex Workers in Swaziland

Social capital is important to disadvantaged groups, such as sex workers, as a means of facilitating internal group-related mutual aid and support as well as access to broader social and material resources. Studies among sex workers have linked higher social capital with protective HIV-related behaviors; however, few studies have examined social capital among sex workers in sub-Saharan Africa. This cross-sectional study examined relationships between two key social capital constructs, social cohesion among sex workers and social participation of sex workers in the larger community, and HIV-related risk in Swaziland using respondent-driven sampling. Relationships between social cohesion, social participation, and HIV-related risk factors were assessed using logistic regression. HIV prevalence among the sample was 70.4% (223/317). Social cohesion was associated with consistent condom use in the past week (adjusted odds ratio [AOR]  = 2.25, 95% confidence interval [CI]: 1.30–3.90) and was associated with fewer reports of social discrimination, including denial of police protection. Social participation was associated with HIV testing (AOR = 2.39, 95% CI: 1.36–4.03) and using condoms with non-paying partners (AOR = 1.99, 95% CI: 1.13–3.51), and was inversely associated with reported verbal or physical harassment as a result of selling sex (AOR = 0.55, 95% CI: 0.33–0.91). Both social capital constructs were significantly associated with collective action, which involved participating in meetings to promote sex worker rights or attending HIV-related meetings/ talks with other sex workers. Social- and structural-level interventions focused on building social cohesion and social participation among sex workers could provide significant protection from HIV infection for female sex workers in Swaziland.     

Salt-Induced Remodeling of Spatially Restricted Clathrin-Independent Endocytic Pathways in Arabidopsis Root

Endocytosis is a ubiquitous cellular process that is characterized well in animal cells in culture but poorly across intact, functioning tissue. Here, we analyze endocytosis throughout the Arabidopsis thaliana root using three classes of probes: a lipophilic dye, tagged transmembrane proteins, and a lipid-anchored protein. We observe a stratified distribution of endocytic processes. A clathrin-dependent endocytic pathway that internalizes transmembrane proteins functions in all cell layers, while a sterol-dependent, clathrin-independent pathway that takes up lipid and lipid-anchored proteins but not transmembrane proteins is restricted to the epidermal layer. Saline stress induces a third pathway that is clathrin-independent, nondiscriminatory in its choice of cargo, and operates across all layers of the root. Concomitantly, small acidic compartments in inner cell layers expand to form larger vacuole-like structures. Plants lacking function of the Rab-GEF (guanine nucleotide exchange factor) VPS9a (vacuolar protein sorting 9A) neither induce the third endocytic pathway nor expand the vacuolar system in response to salt stress. The plants are also hypersensitive to salt. Thus, saline stress reconfigures clathrin-independent endocytosis and remodels endomembrane systems, forming large vacuoles in the inner cell layers, both processes correlated by the requirement of VPS9a activity.