Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently

We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.     

Regulatory T cells control autoimmunity in vivo by inducing apoptotic depletion of activated pathogenic lymphocytes

Clinical autoimmunity requires both activation of self-reactive T cells as well as a failure of peripheral tolerance mechanisms. We previously identified one such mechanism that involves regulatory T cells recognizing TCR V beta 8.2 chain-derived peptides in the context of MHC. How this regulation affects the fate of target V beta 8.2(+) T lymphocytes in vivo that mediate experimental autoimmune encephalomyelitis has remained unknown. The present study using immunoscope and CFSE-labeling analysis demonstrates that the expansion of regulatory CD4 and CD8 T cells in vivo results in apoptotic depletion of the dominant, myelin basic protein-reactive V beta 8.2(+) T cells, but not subdominant V beta 13(+) T cells. The elimination of only activated T cells by this negative feedback mechanism preserves the remainder of the naive V beta 8.2(+) T cell repertoire and at the same time results in protection from disease. These studies are the first in clearly elucidating the fate of myelin basic protein-specific encephalitogenic T cells in vivo following regulation.     

Temperature range of thermodynamic stability for the native state of reversible two-state proteins

The difference between the heat (T(G)) and the cold (T(G)') denaturation temperatures defines the temperature range (T(Range)) over which the native state of a reversible two-state protein is thermodynamically stable. We have performed a correlation analysis for thermodynamic parameters in a selected data set of structurally nonhomologous single-domain reversible two-state proteins. We find that the temperature range is negatively correlated with the protein size and with the heat capacity change (DeltaC(p)) but is positively correlated with the maximal protein stability [DeltaG(T(S))]. The correlation between the temperature range and maximal protein stability becomes highly significant upon normalization of the maximal protein stability with protein size. The melting temperature (T(G)) also shows a negative correlation with protein size. Consistently, T(G) and T(G)' show opposite correlations with DeltaC(p), indicating a dependence of the T(Range) on the curvature of the protein stability curve. Substitution of proteins in our data set with their homologues and arbitrary addition or removal of a protein in the data set do not affect the outcome of our analysis. Simulations of the thermodynamic data further indicate that T(Range) is more sensitive to variations in curvature than to the slope of the protein stability curve. The hydrophobic effect in single domains is the principal reason for these observations. Our results imply that larger proteins may be stable over narrower temperature ranges and that smaller proteins may have higher melting temperatures, suggesting why protein structures often differentiate into multiple substructures with different hydrophobic cores. Our results have interesting implications for protein thermostability.     

Quorum-sensing inhibitory compounds from extremophilic microorganisms isolated from a hypersaline cyanobacterial mat

In this study, extremely halophilic and moderately thermophilic microorganisms from a hypersaline microbial mat were screened for their ability to produce antibacterial, antidiatom, antialgal, and quorum-sensing (QS) inhibitory compounds. Five bacterial strains belonging to the genera Marinobacter and Halomonas and one archaeal strain belonging to the genus Haloterrigena were isolated from a microbial mat. The strains were able to grow at a maximum salinity of 22–25 % and a maximum temperature of 45–60 °C. Hexanes, dichloromethane, and butanol extracts from the strains inhibited the growth of at least one out of nine human pathogens. Only butanol extracts of supernatants of Halomonas sp. SK-1 inhibited growth of the microalga Dunaliella salina. Most extracts from isolates inhibited QS of the acyl homoserine lactone producer and reporter Chromobacterium violaceum CV017. Purification of QS inhibitory dichloromethane extracts of Marinobacter sp. SK-3 resulted in isolation of four related diketopiperazines (DKPs): cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Leu), cyclo(l-Pro-l-isoLeu), and cyclo(l-Pro-d-Phe). QS inhibitory properties of these DKPs were tested using C. violaceum CV017 and Escherichia coli-based QS reporters (pSB401 and pSB1075) deficient in AHL production. Cyclo(l-Pro-l-Phe) and cyclo(l-Pro-l-isoLeu) inhibited QS-dependent production of violacein by C. violaceum CV017. Cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Leu), and cyclo(l-Pro-l-isoLeu) reduced QS-dependent luminescence of the reporter E. coli pSB401 induced by 3-oxo-C6-HSL. Our study demonstrated the ability of halophilic and moderately thermophilic strains from a hypersaline microbial mat to produce biotechnologically relevant compounds that could be used as antifouling agents.     

Survivability of parthenogenetic embryos following in vivo transfer in naturally synchronized Capra hircus

The present study was conducted to see the in vivo developmental potency of caprine parthenogenetic embryos generated in a modified way. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and then activated by 7% ethanol followed by 2 mM 6-dimethyl amino purine (6-DMAP) and embryo development was recorded. Early stage parthenogenetic embryos (two to four cells) were surgically transferred in recipients (10). The pregnancy diagnosis was done by nonreturn to oestrus, ultrasonography (USG), and progesterone estimation. The levels of progesterone were above normal values (1 ng/ml) of pregnancy and fall below the level of pregnancy just before retuned to oestrus. Progesterone profile revealed that out of ten recipients (G1–G10), four goats (G1, G2, G3, and G5) returned to oestrus after 43?±?7.29 (Mean?±?SE) d of embryo transfer and six goats (G4, G6, G7, G8, G9, and G10) did not return to cycle even after 70 d of embryo transfer. In three recipients (G4, G5, and G6), the USG on day 40 revealed that there was fluid filled uterine body with solid fetus-like structure. These might be dead fetus and had started resorption. The progesterone profile also corroborated the assumption of pregnancy in these animals. Authors believe that this may be the first report on in vivo diploid parthenogenetic embryo development in caprine species.     

Anticonvulsant Effect of Asparagus racemosus Willd. in a Mouse Model of Catamenial Epilepsy

Neurochemical Research - Asparagus racemosus Willd. (Family Liliaceae), also known as female reproductive tonic, is traditionally used across the Sub-Himalayan region in Uttarakhand, India for...     

Role of Bacterial Communities in the Natural Suppression of Rhizoctonia solani Bare Patch Disease of Wheat (Triticum aestivum L.)

Rhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga, Pedobacter, Oxalobacteriaceae (Duganella and Massilia), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia-inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.     

Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

Evaluation of Salmonella Porins as a Broad Spectrum Vaccine Candidate

Porins were prepared from smooth strain of Salmonella typhi 0–901 and chemotype of rough mutant of S. typhimurium Ra-30. Mice were immunized with both the porin preparations in different groups and challenged with S. typhimurium LT2–71 and S. enteritidis SH-1269. Porin immunized mice showed significant protection (P <0.01) against challenge with homologous as well as heterologous strains. Hence, the use of porins may be attempted in future to protect against salmonellosis.