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关键词： Tissue  

摘要： Thyroglobulin is a glycoiodoprotein that is produced by thyroid follicular cells;it is stored in follicles in structures known as colloids. The main function of this protein is to stock the hormones triiodothyronine (T3) and thyroxine (T4) until the body requires them. This study aims to demonstrate that infrared spectral imaging with appropriate multivariate analysis can reveal biochemical changes in this glycoprotein. The results achieved herein point out biochemical differences in the colloid samples obtained from normal and goiter patients including glycosylation and changes in the secondary conformational structure. We have presented the first spectral histopathology-based method to detect biochemical differences in thyroid colloids, such as TG iodination, glycosylation, and changes in the secondary structure in normal and goiter patients. The observed changes in the colloids were mainly due to the alterations in amide I and amide II (secondary conformation of proteins) and there is a correlation with different glycosylation between normal and goiter tissues.

关键词： Aspergillus terreus   allergen   triose-phosphate isomerase   proteomics   chromatography   circular dichroism   epitope mapping   airway inflammation   immunoglobulin E (IgE)   immunoglobulin G (IgG)   circular dichroism (CD)  

摘要： Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.

关键词： Lipids   Peptides and proteins   Bacteria   Crystal structure   Biosynthesis  

摘要： Lipopolysaccharide (LPS) is a crucial component in the outer membrane of Gram-negative bacteria that contributes to both pathogenicity as well as immunity against pathogenic bacteria. Typical LPS contains GlcN disaccharide as the core of lipid A. However, some bacteria such as Acidithiobacillus ferrooxidans and Leptospira interrogans contain GlcN3N in lipid A instead. This modification has been shown to dampen the host immune response and increase resistance to antimicrobial peptides. Therefore, investigation of the enzymes responsible for the biosynthesis of GlcN3N has promising applications in the development of vaccines, antibiotics, or usage of the enzymes in chemoenzymatic synthesis of modified LPS. Here, we describe biochemical and structural investigation of GnnA from A. ferrooxidans (Af GnnA) that is responsible for oxidation of UDP-GlcNAc, which subsequently undergoes transamination to produce UDP-GlcNAc3N as a precursor for LPS biosynthesis. Af GnnA is specific for NAD(+) and UDP-GlcNAc. The crystal structures of Af GnnA in combination with molecular dynamics simulation and mutational analysis suggest the substrate recognition mode and the catalytic mechanism. K91 or H164 is a potential catalytic base in the oxidation reaction. The results will not only provide insights into the biosynthesis of unusual LPS but will also lay the foundation for development of more immunogenic vaccines, novel antibiotics, or utilization of GnnA in the synthesis of UDP-sugars or modified LPS.

关键词： lipid A   lipopolysaccharide (LPS)   Toll-like receptor 4 (TLR4)   inflammation   tolerance   macrophage   natural killer cells (NK cells)   colitis   acyloxyacyl hydrolase   AOAH   CD14  

摘要： Animals can sense the presence of microbes in their tissues and mobilize their own defenses by recognizing and responding to conserved microbial structures (often called microbe-associated molecular patterns (MAMPs)). Successful host defenses may kill the invaders, yet the host animal may fail to restore homeostasis if the stimulatory microbial structures are not silenced. Although mice have many mechanisms for limiting their responses to lipopolysaccharide (LPS), a major Gram-negative bacterial MAMP, a highly conserved host lipase is required to extinguish LPS sensing in tissues and restore homeostasis. We review recent progress in understanding how this enzyme, acyloxyacyl hydrolase (AOAH), transforms LPS from stimulus to inhibitor, reduces tissue injury and death from infection, prevents prolonged post-infection immunosuppression, and keeps stimulatory LPS from entering the bloodstream. We also discuss how AOAH may increase sensitivity to pulmonary allergens. Better appreciation of how host enzymes modify LPS and other MAMPs may help prevent tissue injury and hasten recovery from infection.

关键词： Isocitrate lyase   Bacillus cereus   Metal utilization   Crystal structure  

摘要： The glyoxylate cycle is an important anabolic pathway and acts under a C-2 compound (such as acetic acid) rich condition in bacteria. The isocitrate lyase (ICL) enzyme catalyzes the first step in the glyoxylate cycle, which is the cleavage of isocitrate to glyoxylate and succinate. This enzyme is a metalo-enzyme that contains an Mg2+ or a Mn(2+)ion at the active site for enzyme catalysis. We expressed and purified ICL from Bacillus cereus (BcICL) and investigated its biochemical properties and metal usage through its enzyme activity and stability with various divalent metal ion. Based on the results, BcICL mainly utilized the Mg2+ ion for enzyme catalysis as well as the Mn2+, Ni2+ and Co2+ ions. To elucidate its molecular mechanisms, we determined the crystal structure of BcICL at 1.79 angstrom. Through this structure, we analyzed a tetrameric interaction of the protein. We also determined the BcICL structure in complex with both the metal and its products, glyoxylate and succinate at 2.50 angstrom resolution and revealed each ligand binding modes. (C) 2020 Elsevier Inc. All rights reserved.

关键词： Hu-PPase   Pyrophosphatase   Crystal structure   Cell proliferation  

摘要： Inorganic pyrophosphatase (PPase) plays an essential role in energy conservation and provides energy for many biosynthetic pathways. Here, we present two three-dimensional structures of PPase from Homo sapiens (Hu-PPase) at 2.38 angstrom and 3.40 angstrom in different crystallization conditions. One of the Hu-PPase structures complex of two magnesium metal ions was determined to be a monomer (Hu-PPase-mono) here, while the other one to be a dimer-dimer (Hu-PPase-dd). In each asymmetric unit of Hu-PPase-mono, there are four alpha-helices and ten beta-strands and folds as a barrel structure, and the active site contains two magnesium ions. Like PPases from many species, we found that Hu-PPase was able to undergo self-assembly. To our surprise, disruption of the self-assembly of Hu-PPase did not influence its enzymatic activity or the ability to promote cell growth. Our work uncovered that different structure forms of Hu-PPase and found that the pyrophosphatase activity of Hu-PPase is independent of its self-assembly. (C) 2020 Elsevier Inc. All rights reserved.

关键词： methoxylated quercetin   glucuronidation   glucuronosyltransferases   phase II metabolites   semi-synthesis   intrinsic reaction coordinate   molecular modelling  

摘要： Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'-O-methyl-quercetin-3-O-beta-D-glucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4'-O-methyl-quercetin-7-O-beta-D-glucuronide (4), 3-O-methyl-quercetin-3'-O-beta-D-glucuronide (5), and 4'-O-methyl-quercetin-3'-O-beta-D-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results;3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. A mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.

关键词： Interaction   Lipid accumulation   Feeding trial   Two-way ANOVA  

摘要： An 8-week feeding trial was conducted to comprehensively investigate the effects of dietary phosphorus level and stocking density on tiger puffer. A 3 x 3 factorial design was used. The three dietary phosphorus levels were 0.68% (LP), 0.98% (MP), and 1.31% (HP) of dry matter, of which the available phosphorus level was 0.44%, 0.76%, and 1.06%, respectively. The three stocking density grades were 1.53 (LD), 2.30 (MD), and 3.06 (HD) kg m(-3). The feeding trial was conducted in an indoor flow-through seawater system. Three replicate tanks were used for each group. The results showed that HP resulted in the highest growth rate. Higher dietary phosphorus levels reduced the whole-body lipid content, serum cholesterol contents, and hepatic mRNA expression of lipid metabolism-related genes such as ACAC beta, DGAT1, and PPAR alpha 2. High stocking density levels reduced the growth rate and feed intake, but rarely affected other physiological parameters. Significant interactions were observed between effects of dietary phosphorus level and stocking density on phosphorus and calcium metabolism, but not between effects of both variables on the growth performance. Synergetic enhancement of phosphorus excretion by high phosphorus level and high stocking density was observed. These results shed new lights into the phosphorus physiology of marine fish and the interactions between phosphorus nutrition and rearing condition.

关键词： Pesticides   Phytoplankton   Flow cytometry   PAM fluorometry   Lipid classes  

摘要： The occurrence of pesticides and their mixtures in the environment can alter the ecological relationships between aquatic food chains. Since fipronil and 2,4-dichlorophenoxyacetic acid (2,4-D) are commonly found together in Brazilian water bodies, the present study aimed to investigate through an integrative approach the toxicity mechanisms of environmentally relevant concentrations of pesticides Regent (R) 800 WG (active ingredient - a.i. fipronil), DMA (R) 806 BR (a.i. 2,4-D) isolated and in mixtures on the green alga Raphidocelis subcapitata using multiple parameters: physiological (growth rate and chlorophyll a fluorescence), morphological (cell complexity and size), biochemical (composition of lipid classes) and related to the photosynthetic activity (variable fluorescence, the maximum quantum yield of the photosystem II - PSII - and the efficiency of the oxygen evolving complex - OEC - of PSII). The results indicated that fipronil significantly inhibited algal population growth, increased the chlorophyll a content (observed by fluorescence), cell size and lipid class content of triacylglycerol (TAG), free fatty acid (FFA) and acetone mobile polar lipid (AMPL) and, on the other hand, decreased variable fluorescence of algae. The tested concentrations of 2,4-D increased the chlorophyll a fluorescence, the cell size and the lipid classes TAG and FFA. The pesticide mixtures have had more effects on algae than isolated compounds, causing alterations in all parameters analyzed, including photosynthetic activity (maximum quantum yield and efficiency of the oxygen evolving complex of the PSII), in which no alterations were observed for the toxicity of the single pesticides. The results suggest that these analyses are important to evaluate pesticide toxicity mechanisms in ecological risk assessments of tropical regions. Thus, here we demonstrate the importance of using multiple parameters in ecotoxicological studies to obtain a better understanding of the toxicity of t

关键词： Leishmania donovani   Aspartyl tRNA synthetase   Enzyme kinetics   Immunomodulation   Structural characterization   Pyrimidine-derived inhibitors  

摘要： Aminoacyl tRNA synthetases (aaRSs) are integral components of protein biosynthesis along with several non-canonical cellular processes. Inhibition studies of aaRSs presented these enzymes as promising drug targets in many pathogens, however aspartyl tRNA synthetase has not been studied in trypanosomatids despite its essentiality. Hence, full-length ORF of Leishmania donovani aspartyl tRNA synthetase (LdaspRS) was cloned and purified to homogeneity followed by molecular mass determination. The aminoacylation assay established that the purified protein performs its function optimally at physiological pH and temperature. The kinetic parameters of LdaspRS revealed the affinity of L-aspartate towards the enzyme to be very much lower than the cofactor. Our study also highlights the moonlighting function of LdaspRS to stimulate the pro-inflammatory cytokines and nitric oxide generation by host macrophage. Furthermore, CD and intrinsic tryptophan fluorescence measurements showed the changes in structural conformation at varying pH, denaturants and ligands. The modelled LdaspRS structure presented all the specific characteristics of class II aaRSs, while in silico study suggested binding of pyrimidine-derived inhibitors in its cofactor binding site with high affinity followed by validation using MD simulation. Altogether, this study could provide a platform for exploring LdaspRS to develop potential therapeutics against leishmaniasis. (C) 2020 Elsevier B.V. All rights reserved.