Enhancement of thermal stability and inhibition of protein aggregation by osmolytic effect of hydroxyproline

A combination of spectroscopic, calorimetric, and microscopic studies to understand the effect of hydroxyproline on the thermal stability, conformation, biological activity, and aggregation of proteins has been investigated. Significantly increased protein stability and suppression of aggregation is achieved in the presence of hydroxyproline. For example, exceptional increase in the thermal stability of lysozyme up to 26.4 degrees C and myoglobin up to 31.8 degrees C is obtained in the presence of hydroxyproline. The increased thermal stability of the proteins is observed to be accompanied with significant rise of the catalytic activity. Hydroxyproline is observed to prevent lysozyme fibril formation in vitro. Fluorescence and circular dichroism studies indicate induction of tertiary structures of the studied proteins in the presence of hydroxyproline. Preferential hydration of the native state is found to be crucial for the mechanism of protein stabilization by hydroxyproline. We compared the effect of hydroxyproline to that of proline and observed similar increase in the activity and suppression of protein aggregation. The results demonstrate the use of hydroxyproline as a protein stabilizer and in the prevention of protein aggregation and fibril formation.     

Equimolar Mixture of 2,2,2-Trifluoroethanol and 4-Chloro-1-butanol is a Stronger Inducer of Molten Globule State: Isothermal Titration Calorimetric and Spectroscopic Studies

A mixture of 4-chloro-1-butanol and 2,2,2-Trifluoroethanol (TFE) has been used to generate Molten globule (MG) state of structurally homologous but functionally different proteins bovine α-lactalbumin and hen egg-white lysozyme. The thermal denaturation was done using UV–Visible spectroscopy. From UV–Visible profile, thermal transition was not observed beyond a particular concentration. There was an indication of molten globule state in case of α-lactalbumin from circular dichroism experiments. By intrinsic tryptophan fluorescence, acrylamide and potassium iodide quenching, 8-anilino-naphthalene sulfonic acid (ANS) binding and energy transfer studies the presence of molten globule state was confirmed. Quantitative characterization of MG state and determining the binding thermodynamics of ANS to the MG state was done using Isothermal Titration Calorimetry (ITC). Results show that α-lactalbumin exists in MG state at a particular concentration but lysozyme does not show features of MG state.     

Cerebroprotective effects of hydrogen sulfide in homocysteine-induced neurovascular permeability: Involvement of oxidative stress,arginase, and matrix metalloproteinase-9

An elevated level of homocysteine (Hcy) leads to hyperhomocysteinemia (HHcy), which results in vascular dysfunction and pathological conditions identical to stroke symptoms. Hcy increases oxidative stress and leads to increase in blood–brain barrier permeability and leakage. Hydrogen sulfide (H₂S) production during the metabolism of Hcy has a cerebroprotective effect, although its effectiveness in Hcy-induced neurodegeneration and neurovascular permeability is less explored. Therefore, the current study was designed to perceive the neuroprotective effect of exogenous H ₂S against HHcy, a cause of neurodegeneration. To test this hypothesis, we used four groups of mice: control, Hcy, control + sodium hydrosulfide hydrate (NaHS), and Hcy + NaHS, and an HHcy mice model in Swiss albino mice by giving a dose of 1.8 g of dl -Hcy/L in drinking for 8–10 weeks. Mice that have 30 µmol/L Hcy were taken for the study, and a H ₂S supplementation of 20 μmol/L was given for 8 weeks to all groups of mice. HHcy results in the rise of the levels of superoxide and nitrite, although a concomitant decrease in the level of superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, and arginase in oxidative stress and a concomitant decrease in the endogenous level of H ₂S. Although H ₂S supplementation ameliorated, the effect of HHcy and the levels of H ₂S returned to the average level in HHcy animals supplemented with H ₂S. Interestingly, H ₂S supplementation ameliorated neurovascular remodeling and neurodegeneration. Thus, our study suggested that H ₂S could be a beneficial therapeutic candidate for the treatment of Hcy-associated neurodegeneration, such as stroke and neurovascular disorders.     

Solid-state NMR relaxation studies of Australian spider silks

Solid-state NMR techniques were used to study two different types of spider silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. A comparison of (13)C-T(1) and (1)H-T(1rho) solid-state NMR relaxation data of the Ala Calpha, Ala Cbeta, Gly Calpha, and carbonyl resonances revealed subtle differences between dragline and cocoon silk. (13)C-T(1rho) and (1)H-T(1) relaxation experiments showed significant differences between silks of the two species with possible structural variations. Comparison of our data to previous (13)C-T(1) relaxation studies of silk from Nephila clavipes (A. Simmons et al., Macromolecules, 1994, Vol. 27, pp. 5235-5237) also supports the finding that differences in molecular mobility of dragline silk exist between species. Interspecies differences in silk structure may be due to different functional properties. Relaxation studies performed on wet (supercontracted) and dry silks showed that the degree of hydration affects relaxation properties, and hence changes in molecular mobility are correlated with functional properties of silk.     

Suppressor cell activity of intestinal mucosal leucocytes from buffalo (Bubalus bubalis arni)

Suppressor activity of buffalo intestinal intraepithelial leucocytes and lamina propria leucocytes was induced by Concanavalin A, and was assayed against the mitogenic response of autologous and allogenic leucocytes to mitogens. Appreciable suppression was observed with 25 micrograms ConA/ml on the proliferative activity of the responder cells cocultured at a ratio of < 2:1 (suppressor:responder cell). Mitomycin C treatment of intestinal leucocytes did not totally vanish the viability and functionality of leucocytes.     

Cutting edge: clustered AU-rich elements are the target of IL-10-mediated mRNA destabilization in mouse macrophages

In the present study we show that IL-10-mediated inhibition of inflammatory gene expression can be mediated by an AU-rich element (ARE) cluster present in the 3' untranslated region (3'UTR) of sensitive genes. A series of chloramphenicol acetyl transferase (CAT) reporter gene constructs were prepared in which different fragments from the IL-10-sensitive KC mRNA 3'UTR were placed downstream of the coding region of the reporter gene CAT. CAT mRNA containing the KC 3'UTR was markedly destabilized as compared with the control CAT mRNA, and the decay rate was further increased in cells stimulated with IL-10. The KC 3'UTR contains an ARE cluster and three isolated ARE motifs. The ARE cluster spanning nucleotides 378-399 appeared to be both necessary and sufficient to mediate sensitivity to IL-10 because a 116-nucleotide fragment that contains the cluster conferred sensitivity, while mutation of the sequence between positions 378 and 399 eliminated sensitivity. The destabilizing effect of IL-10 was relatively selective, as the stability of chimeric CAT mRNAs was not modulated in cells treated with IFN-gamma or IL-4.     

Interaction of 2,2,2-trifluoroethanol with proteins: calorimetric, densimetric and surface tension approach

The thermal denaturation of hen egg-white lysozyme was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various pH values using micro differential scanning calorimetry. Quantitative thermodynamic parameters accompanying the thermal transitions were evaluated. It is observed that thermal unfolding of lysozyme in the presence of TFE upto a concentration of 4.0 mol dm(-3) follows a two-state denaturation mechanism as indicated by the equality of van't Hoff and calorimetric enthalpies. The finer details of interaction were studied by measuring the partial molar volume of some constituent amino acids and glycine peptides from water to aqueous TFE at 298.15 K. The physico-chemical properties of aqueous TFE: apparent molar heat capacities, apparent molar volumes and surface tension were measured to understand the intrinsic properties of the cosolvent as well. From the correlation among the thermal unfolding data on lysozyme in aqueous TFE, calculated preferential interaction parameters, physico chemical properties of aqueous TFE and partial molar volumes of transfer, it is concluded that both solvent mediated effect and direct interaction constitute the mechanism of TFE-protein interactions.     

Conserved simple sequence repeats for the Limnanthaceae (Brassicales)

The Limnanthaceae (Order Brassicales) is a family of 18 taxa of Limnanthes (meadowfoam) native to California, Oregon, and British Columbia. Cultivated meadowfoam (L. alba Benth.), a recently domesticated plant, has been the focus of research and development as an industrial oilseed for three decades. The goal of the present research was to develop several hundred simple sequence repeat (SSR) markers for genetic mapping, molecular breeding, and genomics research in wild and cultivated meadowfoam taxa. We developed 389 SSR markers for cultivated meadowfoam by isolating and sequencing 1,596 clones from L. alba genomic DNA libraries enriched for AG _n or AC _n repeats, identifying one or more unique SSRs in 696 clone sequences, and designing and testing primers for 624 unique SSRs. The SSR markers were screened for cross- taxa utility and polymorphisms among ten of 17 taxa in the Limnanthaceae; 373 of these markers were polymorphic and 106 amplified loci from every taxon. Cross-taxa amplification percentages ranged from 37.3% in L. douglasii ssp. rosea (145/389) to 85.6% in L. montana (333/389). The SSR markers amplified 4,160 unique bands from 14 genotypes sampled from ten taxa (10.7 unique bands per SSR marker), of which 972 were genotype-specific. Mean and maximum haplotype heterozygosities were 0.71 and 0.90, respectively, among six L. alba genotypes and 0.63 and 0.93, respectively, among 14 genotypes (ten taxa). The SSR markers supply a critical mass of high-throughput DNA markers for biological and agricultural research across the Limnanthaceae and open the way to the development of a genetic linkage map for meadowfoam (x = 5).Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by O. Savolainen     

Cellware--a multi-algorithmic software for computational systems biology

The intracellular environment of a cell hosts a wide variety of enzymatic reactions, diffusion events, molecular binding, polymerization and metabolic channeling. To transform these biological events into a computational framework, distinct modeling strategies are required. While currently no tool is capable of capturing all these events, progress is being made to create an integrated environment for the modeling community. To address this niche requirement, Cellware has been developed to offer a multi-algorithmic environment for modeling and simulating both deterministic and stochastic events in the cell. AVAILABILITY: The software is available for free and can be downloaded from http://www.bii.a-star.edu.sg/sbg/cellware