Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds

The introduction of disulfide bonds has been used as a strategy to enhance the stability of Bacillus circulans xylanase. The transition temperature of the S100C/N148C (DS1), V98C/A152C (DS2), and A1GC/G187,C188 (cXl) in comparison to the wild type was increased by 5.0, 4.1 and 3.8 degrees C, respectively. Interestingly, a combination of two disulfide bonds of DS1 and cXl (cDS1, circular disulfide 1) led to a 12 degrees C increase in the transition temperature. Importantly, an increase in the melting point and DeltaDeltaG values of the cDS1 mutant was cooperative. These results suggest that the mechanism of stabilization by disulfide bonds under irreversible denaturation condition is achieved through: (1) a change in the rate-limiting step on the denaturation pathway; (2) destabilizing the unfolded state without affecting the relative rate constants on the denaturation pathway (like cXl mutant); and (3) or combination of the two (cDS1 mutant).     

Accurate intraocular pressure prediction from applanation response data using genetic algorithm and neural networks

The fact that Goldmann applanation tonometry does not accurately account for individual corneal elastic stiffness often leads to inaccuracy in the measurement of intraocular pressure (IOP). IOP should account not only for the effect of central corneal thickness (CCT) but should also account for other corneal biomechanical factors. A computational method for accurate and reliable determination of IOP is investigated with a modified applanation tonometer in this paper. The proposed method uses a combined genetic algorithm/neural network procedure to match the clinically measured applanation force-displacement history with that obtained from a nonlinear finite element simulation of applanation. An additional advantage of the proposed method is that it also provides the ability to determine CCT and material properties of the cornea from the same applanation response data. The performance of the proposed method has been demonstrated through a parametric study and via comparison with a well known clinical case. The proposed method is also shown to be computationally efficient, which is an important practical consideration for clinical application.     

A theoretical elucidation of bilirubin interaction with HSA's lysines: first electrostatic binding site in IIA subdomain

The electrostatic interaction of amino acid lysines 190, 195 and 199 of human serum albumin (HSA) with bilirubin have been investigated using molecular dynamic simulations, QM and QM/MM minimization methods. In this study two methodological approaches have been employed. In the first approach X-ray structure and the structure obtained from the molecular dynamic simulation of subdomain IIA of HSA in vacuum have been utilized. Interactions have been evaluated with the segment 186-200 of the cited subdomain. Calculations on the X-ray structure of above segment indicate an effective interaction of the lysine 195 with bilirubin, although that of the lysine 190 is also found considerable in this structure. Performing simulation in vacuum, it has been revealed that except for the lysine 195, the other two lysine residues (190 and 199) could not be considered as centers of interaction. Such finding, which is in accord with experimental data, lends support to the procedure employed in this study. NBO analyses suggest that tasks to achieve a structure indicating bilirubin interaction with the lysine 195 from the 186-200 segment extracted from X-ray structure, results in a structure that lacks any electrostatic interaction. In fact, it has been found that the stability of the latter species can be attributed to the H-bonding interaction of the glutamate 188 with both bilirubin and the lysine 195. Further NBO analysis on the structure of the same species, while achieved after molecular dynamic simulation on subdomain IIA in vacuum has revealed that a favorable electrostatic interaction between the lysine 195 and bilirubin has occurred. Besides, H-bonding interaction of the glutamate 188 with bilirubin has been evident in the same species. For the second approach, presence of water molecules and ions has been considered to simulate condensed medium. Applying docking, conformational sampling, and QM/MM minimization steps in sequence, a structure has been achieved which presents a specific interaction between epsilon-NH3(+) group of the lysine 195 residue and the lactam oxygen atom of bilirubin. NBO analyses suggest that above electrostatic interaction is combined with hydrogen bonding interaction between same two groups. Moreover, a hydrogen bond between oxygen atom of bilirubin's acetate group and alpha-NH group of lysine 195 has been observed. Molecular orbital calculations have been presented which support the NBO analyses.     

Hollow fiber liquid phase microextraction followed by high performance liquid chromatography for determination of ultra-trace levels of Se(IV) after derivatization in urine,plasma and natural water samples

In the present work, a simple and high sensitive method based on hollow fiber liquid phase microextraction (HF-LPME) was developed followed by high performance liquid chromatography (HPLC) for determination of ultra-trace amounts of Se(IV) after derivatization in biological and natural water samples. Se(IV) was complexed with o-phenylenediamine to form piazselenol. The formed piazselenol was extracted into 20 μL of 1-octanol located in the lumen of a hollow fiber and the solution was injected into HPLC-UV for analysis. Using the Taguchi method, an orthogonal array design (OAD), OA₁₆ (4⁵) was employed to optimize the HF-LPME of piazselenol. The effect of five experimental factors (each factor at four levels) including the volume of the organic phase, extraction time, pH of the solution, stirring rate and ionic strength on the extraction efficiency of piazselenol was studied and optimized. The maximum extraction efficiency of piazselenol was obtained at 20 μL of 1-octanol as the extracting solvent, 30 min extraction time, pH 2, stirring rate of 500 rpm and 30% (w/v) NaCl. Under the optimum conditions, preconcentration factors up to 130 were achieved and the relative standard deviation (%RSD) of the method was <3.7% for different concentrations of Se(IV). The calibration curves were obtained in the ranges of 0.2–100 and 0.05–10 μg L^?1 for the 11 and 50 mL of the sample volumes with reasonable linearity, respectively (r² > 0.995). The limits of detection (LOD) were 0.1 and 0.02 μg L^?1 for the 11 and 50 mL sample volumes, respectively (S/N = 3). Finally, the applicability of the proposed method was evaluated by the extraction and determination of Se(IV) in the plasma, urine and water samples.     

Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.     

Significance of inducible nitric oxide synthase in acute myocarditis caused by Trypanosoma cruzi (Tulahuen strain)

Chagas' disease, caused by Trypanosoma cruzi, is associated with myocarditis and expression of myocardial cytokines and inducible nitric oxide synthase (NOS2). To assess the functional significance of NOS2 in murine Chagas' disease, we infected NOS2 knockout (NOS2(-/-)) and C57BL/6x129sv (wild type) mice with the Tulahuen strain of T. cruzi. Serial transthoracic echocardiography was performed to assess the progression of left and right ventricular dysfunction in infected mice. Uninfected wild type and NOS2(-/-) mice served as controls. At day 10 post-infection (p.i.), infected wild type mice had larger left ventricular end-diastolic diameter (2.52+/-0.14-vs-2.11+/-0.06 mm, P<0.02) and right ventricle (0.6+/-0.2-vs-0 visual grade, P<0.02) as compared with uninfected wild type mice. At day 19 p.i., compared with uninfected controls, infected wild type mice had larger left ventricular end-diastolic diameter (3.30+/-0.29-vs-2.11+/-0.07 mm), left ventricular end-systolic diameter (1.86+/-0.29-vs-0.88+/-0.05 mm), right ventricle (1.6+/-0.2-vs-0 visual grade), lower heart rate (413+/-27-vs-557+/-25 beats per min), left ventricular relative wall thickness (0.44+/-0.05-vs-0.64+/-0.03) and fractional shortening (45+/-4-vs-57+/-2%) [P<0.05 for all]. In contrast, no differences in left ventricular end-diastolic diameter or fractional shortening were noted among infected and uninfected NOS2(-/-) mice at day 19 p.i. Compared with uninfected controls, infected NOS2(-/-) mice had significantly lower heart rate (272+/-23-vs-512+/-31 beats per min, P<0.01) and larger right ventricle (0.6+/-0.2-vs-0, P<0.05 visual grade). The magnitude of right ventricular dilation in NOS2(-/-) mice was less than that observed in infected wild type mice. At necropsy, the heart weight was greater (129+/-16-vs-109+/-7 mg, P=0.02) and myocardial inflammation more severe in infected wild type compared with infected NOS2(-/-) mice. Myocardial interleukin (IL)-1beta, IL-6, tumour necrosis factor-alpha, and interferon-gamma were induced in all infected mice. These data indicate that nitric oxide derived from NOS2 plays an important role in the development and progression of ventricular dilation and systolic dysfunction in acute murine chagasic myocarditis caused by infection with the Tulahuen strain.     

Maternal Milk Concentration of Zinc,Iron, Selenium,and Iodine and Its Relationship to Dietary Intakes

The dietary intake of zinc (Zn), iron (Fe), selenium (Se), and iodine (I) of 31 lactating Mexican–American women attending the Hidalgo County WIC program in Rio Grande Valley (RGV), Texas was estimated from 24-h dietary recall interviews. Milk samples were obtained from lactating mothers who had infants 3 months of age and younger. Milk samples were collected in two visits to assess change in breast milk composition after 1–3 months postpartum: group A—after 30–45 days and group B—75–90 days. Dietary intakes indicated that the study participants had significantly inadequate percent energy intakes than the DRI (Dietary Recommended Intakes) percent recommended kilocalorie values but protein intakes were substantially higher than the percent recommended values. The estimated percent Zn, Fe, Se, and I intakes were also significantly lower than the DRI percent recommended values. The lactating mothers consumed significantly less Zn, Se, and I when compared to the Recommended Dietary Allowances (RDA) even though Fe intake was higher than the RDA value. Breast milk concentration of Zn, Fe, and Se were in agreement within the range of representative values for Constituents of Human Milk but I has significantly less concentration than the representative value. There was no statistically significant correlation observed between dietary intake and milk concentration of Zn, Fe, Se, and I. This study compares the estimated dietary intake of zinc, iron, selenium, and iodine to the concentration of these trace elements in the maternal milk of lactating women of Mexican–American heritage who attend the Rio Grande Valley WIC clinic.     

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.     

Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts

Recently, development ofa caveolin-1-deficient (Cav-1 null) mouse model has allowed thedetailed analysis of caveolin-1's function in the context of awhole animal. Interestingly, we now report that the hearts ofCav-1 null mice are markedly abnormal, despite the fact that caveolin-1is not expressed in cardiac myocytes. However, caveolin-1 is abundantlyexpressed in the nonmyocytic cells of the heart, i.e., cardiacfibroblasts and endothelia. Quantitative imaging studies of Cav-1 nullhearts demonstrate a significantly enlarged right ventricular cavityand a thickened left ventricular wall with decreased systolic function.Histological analysis reveals myocyte hypertrophy withinterstitial/perivascular fibrosis. Because caveolin-1 is thought toact as a negative regulator of the p42/44 MAP kinase cascade, weperformed Western blot analysis with phospho-specific antibodies thatonly recognize activated ERK1/2. As predicted, the p42/44 MAP kinasecascade is hyperactivated in Cav-1 null heart tissue (i.e.,interstitial fibrotic lesions) and isolated cardiac fibroblasts. Inaddition, endothelial and inducible nitric oxide synthase levels aredramatically upregulated. Thus loss of caveolin-1 expression drivesp42/44 MAP kinase activation and cardiac hypertrophy.