Heterogeneity of equilibrium molten globule state of cytochrome c induced by weak salt denaturants under physiological condition.
ABSTRACT: While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO4) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (??400) and CD ([?]409), is a three-step process, N ? X ? D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ? X3 ? X2 ? X1 ? N.
Project description:Red alga dulse possesses a unique xylan, which is composed of a linear ?-(1?3)/?-(1?4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (?-(1?3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with ?-(1?4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed ?-(1?3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.
Project description:Calcium antagonists play an important role in clinical practice. However, most of them have serious side effects. We have synthesized a series of novel calcium antagonists, quaternary ammonium salt derivatives of haloperidol with N-p-methoxybenzyl (X1), N-m-methoxybenzyl (X2) and N-o-methoxybenzyl (X3) groups. The objective of this study was to investigate the bioactivity of these novel calcium antagonists, especially the vasodilation activity and cardiac side-effects. The possible working mechanisms of these haloperidol derivatives were also explored.Novel calcium antagonists were synthesized by amination. Compounds were screened for their activity of vasodilation on isolated thoracic aortic ring of rats. Their cardiac side effects were explored. The patch-clamp, confocal laser microscopy and the computer-fitting molecular docking experiments were employed to investigate the possible working mechanisms of these calcium antagonists.The novel calcium antagonists, X1, X2 and X3 showed stronger vasodilation effect and less cardiac side effect than that of classical calcium antagonists. They blocked L-type calcium channels with an potent effect order of X1 > X2 > X3. Consistently, X1, X2 and X3 interacted with different regions of Ca2+-CaM-CaV1.2 with an affinity order of X1 > X2 > X3.The new halopedidol derivatives X1, X2 and X3 are novel calcium antagonists with stronger vasodilation effect and less cardiac side effect. They could have wide clinical application.
Project description:Activins regulate numerous processes including inflammation and are synthesized as precursors consisting of a long N-terminal pro-region and a mature protein. Genomic human databases currently list three activin A (Act A) variants termed X1, X2 and X3. The X3 variant is the shortest, lacks N-terminal segments present in X1 and X2, and has been the focus of most past literature. Here, we asked whether these variants are expressed by human cells and tissues and what structural features are contained within their pro-regions. Human monocytic-like cells THP1 and U937 expressed X1 and X2 variants after exposure to phorbol ester or granulocyte-macrophage colony-stimulating factor, while X2 transcripts were present in placenta. Expression vectors encoding full length X2 or X3 variants resulted in production and secretion of biologically active Act A from cultured cells. Previous studies reported a putative HS-binding domain (HBD) in the X3 pro-region. Here, we identified a novel HBD with consensus HS-binding motifs near the N-terminal end of X1 and X2 pro-regions. Peptides encompassing this new domain interacted with substrate-bound HS with nanomolar affinity, while peptides from putative X3 HBD did not. In good agreement, full length X2 pro-region interacted with heparin-agarose, while the X3 pro-region did not. In sum, our study reveals that Act A variants are expressed by inflammatory cells and placenta and yield biological activity. The high affinity HBD in X1 and X2 pro-region and its absence in X3 could greatly influence overall Act A distribution, availability and activity in physiological and pathological circumstances.
Project description:Entelegyne spiders rarely show fusions yielding neo-Y chromosomes, which M. J. D. White attributed to a constraint in spiders, namely their proximal chiasma localization acting to upset meiotic segregation in males with fusions. Of the 75 taxa of Habronattus and outgroups studied, 47 have X1 X2 0 sex chromosomes in males, 10 have X1 X2 Y, 15 have X1 X2 X3 Y, 2 have X0, and one has both X1 X2 0 and X1 X2 X3 Y. Chromosome numbers and behavior suggest neo-Ys formed by an autosome-X fusion to make X1 X2 Y, with a second fusion to an autosome to make X1 X2 X3 Y. Phylogeny shows at least 8-15 gains (or possibly some losses) of neo-Y (i.e., X-autosome fusions), a remarkable number for such a small clade. In contrast to the many X-autosome fusions, at most one autosome-autosome fusion is indicated. Origins of neo-Y are correlated significantly with distal localization of chiasmata, supporting White's hypothesis that evolution of neo-Y systems is facilitated by looser pairing (distal chiasmata) at meiosis. However, an alternative (or contributing) explanation for the correlation is that X-autosome fusions were selected to permit isolation of male-favored alleles to the neo-Y chromosome, aided by distal chiasmata limiting recombination. This intralocus sexual conflict hypothesis could explain both the many X-autosome fusions, and the stunning complexity of male Habronattus courtship displays.
Project description:Metaphase karyotype investigation on two allopatric strains of Anopheles nitidus Harrison, Scanlon, and Reid (Diptera: Culicidae) was conducted in Thailand during 2011-2012. Five karyotypic forms, i.e., Form A (X1, Y1), Form B (X1, Y2), Form C (X2, Y3), Form D (X1, X3, Y4), and Form E (X1, X2, X3, Y5) were obtained from a total of 21 isofemale lines. Forms A, B, and C were confined to Phang Nga Province, southern Thailand, whereas Forms D and E were restricted to Ubon Ratchathani Province, northeastern Thailand. Cross-mating experiments among the five isofemale lines, which were representative of five karyotypic forms of An. nitidus, revealed genetic compatibility by providing viable progenies and synaptic salivary gland polytene chromosomes through F2 generations. The results suggest that the forms are conspecific, and An. nitidus comprises five cytological races. The very low intraspecific sequence variations (average genetic distances?=?0.002-0.008) of the nucleotide sequences in ribosomal DNA (internal transcribed spacer 2) and mitochondrial DNA (cytochrome c oxidase subunits I and II) among the five karyotypic forms were very good supportive evidence.
Project description:Owing to their high surface area, stability, and functional groups on the surface, iron oxide hydroxide nanoparticles have attracted attention as enzymatic support. In this work, a chemometric approach was performed, aiming at the optimization of the horseradish peroxidase (HRP) immobilization process on ?-FeOOH nanoparticles (NPs). The enzyme/NPs ratio (X1), pH (X2), temperature (X3), and time (X4) were the independent variables analyzed, and immobilized enzyme activity was the response variable (Y). The effects of the factors were studied using a factorial design at two levels (-1 and 1). The biocatalyst obtained was evaluated for the ferulic acid (FA) removal, a pollutant model. The materials were characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The SEM images indicated changes in material morphology. The independent variables X1 (-0.57), X2 (0.71), and X4 (0.42) presented the significance effects estimate. The variable combinations resulted in two significance effects estimates, X1*X2 (-0.57) and X2*X4 (0.39). The immobilized HRP by optimized conditions (X1 = 1/63 (enzyme/NPs ratio, X2 = pH 8, X4 = 60 °C, and 30 min) showed high efficiency for FA oxidation (82%).
Project description:We report a method for high throughput mapping of transcriptional enhancers, genomic regulatory regions that activate lineage-specific transcription. Since tissue-specific enhancers are bound by the transcriptional co-activator p300, we developed murine knock-in alleles that permit Cre-dependent, lineage-specific p300 in vivo biotinylation and high affinity pulldown on immobilized streptavidin. Subsequent next generation sequencing of p300-bound genomic DNA identified lineage-specific enhancers. By driving this system with lineage-specific Cre transgenes, we mapped enhancers active in embryonic endothelial cells/blood or skeletal muscle. Analysis of these enhancer sequences identified new transcription factor heterodimer motifs that likely regulate transcription in these lineages. Furthermore, we identified candidate enhancers that regulate adult heart- or lung- specific endothelial cell specialization. Overall design: ES_p300_bioChIP x2, E12.5 forebrain_p300bioChIP x2 and H3K27Ac ChIP x2, E12.5 heart_p300bioChIP x2 and H3K27Ac ChIP x2, E11.5_embryo_p300_bioChIP x3, E11.5_Tie2Cre_EC_p300-bioChIP x3, E13.5_Myf5Cre_muscle prognitors_p300-bioChIP x3, adult_heart_p300-bioChIP x2, adult heart_lung_p300-bioChIP x3, adult_heart_EC_p300-bioChIP x3, adult_lung_EC_p300-bioChIP x3, E12.5 Cardiomyocyte_ATAC-seq x2, E10.5_Tie2Cre-TRAP x1
Project description:Angelica keiskei Koidzumi (A. keiskei), as a Japanese edible herbal plant, enjoys a variety of biological activities due to the presence of numerous active compounds, especially flavonoids. This study aims for the optimization of ultrasound-assisted extraction (UAE) for flavonoids in A. keiskei and their antioxidant activity by using the response surface methodology (RSM). Single-factor experiments and a four-factor three-level Box-Behnken design (BBD) were performed to explore the effects of the following parameters on flavonoid extraction and antioxidant activity evaluation: ultrasonic temperature (X1), ultrasonic time (X2), ethanol concentration (X3) and liquid-solid ratio (X4). The optimum conditions of the combination of total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (DPPH-RSC) and ferric-reducing antioxidant power (FRAP) were as follows: X1 = 80 °C, X2 = 4 min, X3 = 78%, X4 = 35 mL/g, respectively. The experimental results provide a theoretical basis for the extensive utilization of A. keiskei and flavonoids extraction from A. keiskei as a potential source of antioxidants.
Project description:Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit ?-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.
Project description:<b>Objective:</b>We aimed to investigate bias in applying machine learning to predict real-world individual treatment effects.<br><br><b>Materials and methods:</b>Using a virtual patient cohort, we simulated real-world healthcare data and applied random forest and gradient boosting classifiers to develop prediction models. Treatment effect was estimated as the difference between the predicted outcomes of a treatment and a control. We evaluated the impact of predictors (ie, treatment predictors [X1], confounders [X2], treatment effects modifiers [X3], and other outcome risk factors [X4]) with known effects on treatment and outcome using real-world data, and outcome imbalance on predicting individual outcome. Using counterfactuals, we evaluated percentage of patients with biased predicted individual treatment effects.<br><br><b>Results:</b>The X4 had relatively more impact on model performance than X2 and X3 did. No effects were observed from X1. Moderate-to-severe outcome imbalance had a significantly negative impact on model performance, particularly among subgroups in which an outcome occurred. Bias in predicting individual treatment effects was significant and persisted even when the models had a 100% accuracy in predicting health outcome.<br><br><b>Discussion:</b>Inadequate inclusion of the X2, X3, and X4 and moderate-to-severe outcome imbalance may affect model performance in predicting individual outcome and subsequently bias in predicting individual treatment effects. Machine learning models with all features and high performance for predicting individual outcome still yielded biased individual treatment effects.<br><br><b>Conclusions:</b>Direct application of machine learning might not adequately address bias in predicting individual treatment effects. Further method development is needed to advance machine learning to support individualized treatment selection.