ResearchPad - tryptophan https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Ontogenetic changes in energetic reserves, digestive enzymes, amino acid and energy content of <i>Lithodes santolla</i> (Anomura: Lithodidae): Baseline for culture]]> https://www.researchpad.co/article/elastic_article_14500 The southern king crab (SKC) Lithodes santolla is an important commercial species in southern South America. Fishing pressure has caused the deterioration of its stocks. Currently, culture techniques are being developed for producing SKC juveniles to enhance the natural population and to recover the fishing stock. Therefore, it is necessary to know about physiology, energetic and nutritional requirements for SKC maintenance in hatchery. Thus, this study aims to evaluate the biochemical and physiological changes in the midgut gland, muscle and hemolymph of juveniles, pre-adults and adults of wild SKC. The energetic reserves, digestive enzymes activity, amino acid profile and energy were quantified in twelve juveniles, ten pre-adult, and ten adult crabs. Juveniles showed high glycogen and low lipids in the midgut gland, and low proteins and low lactate in muscle. In the hemolymph, juveniles had high lipids. Pre-adults had high glycogen and lipids in the midgut gland, and both high protein and lactate in muscle. In the hemolymph, pre-adults had high lipids. Adults had low glycogen and high lipids in midgut gland, and both high proteins and high lactate in muscle. In hemolymph, adults had high glucose and lactate. Juveniles and pre-adults had high proteinase activity, whereas adults had high lipase activity. Major essential amino acids of SKC were arginine, methionine, and tryptophan, and the non-essential amino acids were glycine, aspartic acid and glutamic acid. On another hand, SKC had similar energy in the midgut gland and muscle, regardless of the ontogenetic stage. Moreover, we demonstrated that the biochemical energy calculation underestimates the actual measured values by a calorimeter. Thus, our results help to understand the physiological changes, energetic and nutritional requirements of L. santolla, and this study is a baseline for research on diet formulation for maintaining this species under culture conditions.

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<![CDATA[A precedented nuclear genetic code with all three termination codons reassigned as sense codons in the syndinean Amoebophrya sp. ex Karlodinium veneficum]]> https://www.researchpad.co/article/5c818e8fd5eed0c484cc2557

Amoebophrya is part of an enigmatic, diverse, and ubiquitous marine alveolate lineage known almost entirely from anonymous environmental sequencing. Two cultured Amoebophrya strains grown on core dinoflagellate hosts were used for transcriptome sequencing. BLASTx using different genetic codes suggests that Amoebophyra sp. ex Karlodinium veneficum uses the three typical stop codons (UAA, UAG, and UGA) to encode amino acids. When UAA and UAG are translated as glutamine about half of the alignments have better BLASTx scores, and when UGA is translated as tryptophan one fifth have better scores. However, the sole stop codon appears to be UGA based on conserved genes, suggesting contingent translation of UGA. Neither host sequences, nor sequences from the second strain, Amoebophrya sp. ex Akashiwo sanguinea had similar results in BLASTx searches. A genome survey of Amoebophyra sp. ex K. veneficum showed no evidence for transcript editing aside from mitochondrial transcripts. The dynein heavy chain (DHC) gene family was surveyed and of 14 transcripts only two did not use UAA, UAG, or UGA in a coding context. Overall the transcriptome displayed strong bias for A or U in third codon positions, while the tRNA genome survey showed bias against codons ending in U, particularly for amino acids with two codons ending in either C or U. Together these clues suggest contingent translation mechanisms in Amoebophyra sp. ex K. veneficum and a phylogenetically distinct instance of genetic code modification.

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<![CDATA[S100A4 inhibits cell proliferation by interfering with the S100A1-RAGE V domain]]> https://www.researchpad.co/article/5c75ac75d5eed0c484d087e3

The Ca2+-dependent human S100A4 (Mts1) protein is part of the S100 family. Here, we studied the interactions of S100A4 with S100A1 using nuclear magnetic resonance (NMR) spectroscopy. We used the chemical shift perturbed residues from HSQC to model S100A4 and S100A1 complex with HADDOCK software. We observed that S100A1 and the RAGE V domain have an analogous binding area in S100A4. We discovered that S100A4 acts as an antagonist among the RAGE V domain and S100A1, which inhibits tumorigenesis and cell proliferation. We used a WST-1 assay to examine the bioactivity of S100A1 and S100A4. This study could possibly be beneficial for evaluating new proteins for the treatment of diseases.

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<![CDATA[Cholesterol re-organisation and lipid de-packing by arginine-rich cell penetrating peptides: Role in membrane translocation]]> https://www.researchpad.co/article/5c521850d5eed0c484797b72

Cell penetrating peptides (CPPs) are able to transport hydrophilic molecules inside cells. To reach the cytosol, the peptide associated with a cargo must cross the plasma or the endosomal membrane. Different molecular mechanisms for peptide internalisation into cells have been proposed and it is becoming clear that the cellular internalisation mechanisms are different depending on the peptide sequence and structure and the target membrane. Herein, the penetration of three peptides into large unilamellar vesicles were studied: the homeodomain derived 16-residues penetratin, nona-arginine (R9), and a small peptide containing 6 arginine and 3 tryptophan residues (RW9). The membrane models were composed of phospholipids from natural sources containing different molecular species. We observed that among the three peptides, only the amphipathic peptide RW9 was able to cross the membrane vesicles in the liquid disordered state. The changes in the distribution of the previously characterized cholesterol-pyrene probe show that cholesterol-pyrene molecules dissociate from clusters upon membrane interaction with the three peptides and that the cholesterol environment becomes more disordered in the presence of RW9. Finally, we studied the effect of the peptides on lipid ordering on giant plasma membrane vesicles. The amphipathic peptides RW9 and its longer homologue RW16 induced lipid de-packing in plasma membrane vesicles. Overall, the data suggest that a disordered membrane favours the translocation of RW9, that the membrane cholesterol is redistributed during peptide interaction, and that the peptide amphipathic character is important to increase membrane fluidity and peptide membrane translocation.

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<![CDATA[Experimental approach to study the effect of mutations on the protein folding pathway]]> https://www.researchpad.co/article/5c46655dd5eed0c484518ccc

Is it possible to compare the physicochemical properties of a wild-type protein and its mutant form under the same conditions? Provided the mutation has destabilized the protein, it may be more correct to compare the mutant protein under native conditions to the wild-type protein destabilized with a small amount of the denaturant. In general, is it appropriate to compare the properties of proteins destabilized by different treatments: mutations, pH, temperature, and denaturants like urea? These issues have compelled us to search for methods and ways of presentation of experimental results that would allow a comparison of mutant forms of proteins under different conditions and lead to conclusions on the effect of mutations on the protein folding/unfolding pathway. We have studied equilibrium unfolding of wild-type bovine carbonic anhydrase II (BCA II) and its six mutant forms using different urea concentrations. BCA II has been already studied in detail and is a good model object for validating new techniques. In this case, time-resolved fluorescence spectroscopy was chosen as the basic research method. The main features of this experimental method allowed us to compare different stages of unfolding of studied proteins and prove experimentally that a single substitution of the amino acid in three mutant forms of BCA II affected the native state of the protein but did not change its unfolding pathway. On the contrary, the inserted disulfide bridge in three other mutant forms of BCA II affected the protein unfolding pathway. An important result of this research is that we have validated the new approach allowing investigation of the effect of mutations on the folding of globular proteins, because in this way it is possible to compare proteins in the same structural states rather than under identical conditions.

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<![CDATA[Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress]]> https://www.researchpad.co/article/5c1028d2d5eed0c4842482d4

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

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<![CDATA[Biomarkers of Environmental Enteropathy, Inflammation, Stunting, and Impaired Growth in Children in Northeast Brazil]]> https://www.researchpad.co/article/5989da4eab0ee8fa60b8d481

Critical to the design and assessment of interventions for enteropathy and its developmental consequences in children living in impoverished conditions are non-invasive biomarkers that can detect intestinal damage and predict its effects on growth and development. We therefore assessed fecal, urinary and systemic biomarkers of enteropathy and growth predictors in 375 6–26 month-old children with varying degrees of malnutrition (stunting or wasting) in Northeast Brazil. 301 of these children returned for followup anthropometry after 2-6m. Biomarkers that correlated with stunting included plasma IgA anti-LPS and anti-FliC, zonulin (if >12m old), and intestinal FABP (I-FABP, suggesting prior barrier disruption); and with citrulline, tryptophan and with lower serum amyloid A (SAA) (suggesting impaired defenses). In contrast, subsequent growth was predicted in those with higher fecal MPO or A1AT and also by higher L/M, plasma LPS, I-FABP and SAA (showing intestinal barrier disruption and inflammation). Better growth was predicted in girls with higher plasma citrulline and in boys with higher plasma tryptophan. Interactions were also seen with fecal MPO and neopterin in predicting subsequent growth impairment.

Biomarkers clustered into markers of 1) functional intestinal barrier disruption and translocation, 2) structural intestinal barrier disruption and inflammation and 3) systemic inflammation. Principle components pathway analyses also showed that L/M with %L, I-FABP and MPO associate with impaired growth, while also (like MPO) associating with a systemic inflammation cluster of kynurenine, LBP, sCD14, SAA and K/T. Systemic evidence of LPS translocation associated with stunting, while markers of barrier disruption or repair (A1AT and Reg1 with low zonulin) associated with fecal MPO and neopterin.

We conclude that key noninvasive biomarkers of intestinal barrier disruption, LPS translocation and of intestinal and systemic inflammation can help elucidate how we recognize, understand, and assess effective interventions for enteropathy and its growth and developmental consequences in children in impoverished settings.

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<![CDATA[Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes]]> https://www.researchpad.co/article/5989d9dcab0ee8fa60b67ec1

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal’s lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene’s promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species.

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<![CDATA[IDO1 and IDO2 Non-Synonymous Gene Variants: Correlation with Crohn's Disease Risk and Clinical Phenotype]]> https://www.researchpad.co/article/5989db21ab0ee8fa60bcf69c

Background

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Genetic polymorphisms can confer CD risk and influence disease phenotype. Indoleamine 2,3 dioxygenase-1 (IDO1) is one of the most over-expressed genes in CD and mediates potent anti-inflammatory effects via tryptophan metabolism along the kynurenine pathway. We aimed to determine whether non-synonymous polymorphisms in IDO1 or IDO2 (a gene paralog) are important either as CD risk alleles or as modifiers of CD phenotype.

Methods

Utilizing a prospectively collected database, clinically phenotyped CD patients (n = 734) and non-IBD controls (n = 354) were genotyped for established IDO1 and IDO2 non-synonymous single nucleotide polymorphisms (SNPs) and novel genetic variants elucidated in the literature. Allelic frequencies between CD and non-IBD controls were compared. Genotype-phenotype analysis was conducted. IDO1 enzyme activity was assessed by calculating the serum kynurenine to tryptophan ratio (K/T).

Results

IDO1 SNPs were rare (1.7% non-IBD vs 1.1% CD; p = NS) and not linked to Crohn's disease diagnosis in this population. IDO1 SNPs did however associate with a severe clinical course, presence of perianal disease, extraintestinal manifestations and a reduced serum K/T ratio during active disease suggesting lower IDO1 function. IDO2 minor allele variants were common and one of them, rs45003083, associated with reduced risk of Crohn's disease (p = 0.025). No IDO2 SNPs associated with a particular Crohn's disease clinical phenotype.

Conclusions

This work highlights the functional importance of IDO enzymes in human Crohn's disease and establishes relative rates of IDO genetic variants in a US population.

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<![CDATA[Histidine 352 (His352) and Tryptophan 355 (Trp355) Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol]]> https://www.researchpad.co/article/5989da76ab0ee8fa60b96cce

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser357 and Trp355 as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys335, Gln337 and Trp355 were predicted to bind the 7-OH, 2-OCH3 and 17-OCH3 of SECO. Site-directed mutagenesis of Cys335, Gln337, His352, Trp355 and Ser357, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp355 was substituted to Ala355 and Gly355 or when changing His352 to Asp352, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp355 and His352 are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.

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<![CDATA[Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells]]> https://www.researchpad.co/article/5989db08ab0ee8fa60bc9075

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

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<![CDATA[Rational Structure-Based Rescaffolding Approach to De Novo Design of Interleukin 10 (IL-10) Receptor-1 Mimetics]]> https://www.researchpad.co/article/5989dabaab0ee8fa60bae74c

Tackling protein interfaces with small molecules capable of modulating protein-protein interactions remains a challenge in structure-based ligand design. Particularly arduous are cases in which the epitopes involved in molecular recognition have a non-structured and discontinuous nature. Here, the basic strategy of translating continuous binding epitopes into mimetic scaffolds cannot be applied, and other innovative approaches are therefore required. We present a structure-based rational approach involving the use of a regular expression syntax inspired in the well established PROSITE to define minimal descriptors of geometric and functional constraints signifying relevant functionalities for recognition in protein interfaces of non-continuous and unstructured nature. These descriptors feed a search engine that explores the currently available three-dimensional chemical space of the Protein Data Bank (PDB) in order to identify in a straightforward manner regular architectures containing the desired functionalities, which could be used as templates to guide the rational design of small natural-like scaffolds mimicking the targeted recognition site. The application of this rescaffolding strategy to the discovery of natural scaffolds incorporating a selection of functionalities of interleukin-10 receptor-1 (IL-10R1), which are relevant for its interaction with interleukin-10 (IL-10) has resulted in the de novo design of a new class of potent IL-10 peptidomimetic ligands.

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<![CDATA[Effect of N-Ethylmaleimide as a Blocker of Disulfide Crosslinks Formation on the Alkali-Cold Gelation of Whey Proteins]]> https://www.researchpad.co/article/5989d9d8ab0ee8fa60b669a0

N-ethylmaleimide (NEM) was used to verify that no new disulfide crosslinks were formed during the fascinating rheology of the alkali cold-gelation of whey proteins, which show Sol-Gel-Sol transitions with time at pH > 11.5. These dynamic transitions involve the formation and subsequent destruction of non-covalent interactions between soluble whey aggregates. Therefore, incubation of aggregates with NEM was expected not to affect much the rheology. Experiments show that very little additions of NEM, such as 0.5 mol per mol of protein, delayed and significantly strengthened the metastable gels formed. Interactions between whey protein aggregates were surprisingly enhanced during incubation with NEM as inferred from oscillatory rheometry at different protein concentrations, dynamic swelling, Trp fluorescence and SDS-PAGE measurements.

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<![CDATA[Selective Photocatalytic Disinfection by Coupling StrepMiniSog to the Antibody Catalyzed Water Oxidation Pathway]]> https://www.researchpad.co/article/5989da00ab0ee8fa60b73b64

For several decades reactive oxygen species have been applied to water quality engineering and efficient disinfection strategies; however, these methods are limited by disinfection byproduct and catalyst-derived toxicity concerns which could be improved by selectively targeting contaminants of interest. Here we present a targeted photocatalytic system based on the fusion protein StrepMiniSOG that uses light within the visible spectrum to produce reactive oxygen species at a greater efficiency than current photosensitizers, allowing for shorter irradiation times from a fully biodegradable photocatalyst. The StrepMiniSOG photodisinfection system is unable to cross cell membranes and like other consumed proteins, can be degraded by endogenous digestive enzymes in the human gut, thereby reducing the consumption risks typically associated with other disinfection agents. We demonstrate specific, multi-log removal of Listeria monocytogenes from a mixed population of bacteria, establishing the StrepMiniSOG disinfection system as a valuable tool for targeted pathogen removal, while maintaining existing microbial biodiversity.

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<![CDATA[Inter-Protein Sequence Co-Evolution Predicts Known Physical Interactions in Bacterial Ribosomes and the Trp Operon]]> https://www.researchpad.co/article/5989db37ab0ee8fa60bd3718

Interaction between proteins is a fundamental mechanism that underlies virtually all biological processes. Many important interactions are conserved across a large variety of species. The need to maintain interaction leads to a high degree of co-evolution between residues in the interface between partner proteins. The inference of protein-protein interaction networks from the rapidly growing sequence databases is one of the most formidable tasks in systems biology today. We propose here a novel approach based on the Direct-Coupling Analysis of the co-evolution between inter-protein residue pairs. We use ribosomal and trp operon proteins as test cases: For the small resp. large ribosomal subunit our approach predicts protein-interaction partners at a true-positive rate of 70% resp. 90% within the first 10 predictions, with areas of 0.69 resp. 0.81 under the ROC curves for all predictions. In the trp operon, it assigns the two largest interaction scores to the only two interactions experimentally known. On the level of residue interactions we show that for both the small and the large ribosomal subunit our approach predicts interacting residues in the system with a true positive rate of 60% and 85% in the first 20 predictions. We use artificial data to show that the performance of our approach depends crucially on the size of the joint multiple sequence alignments and analyze how many sequences would be necessary for a perfect prediction if the sequences were sampled from the same model that we use for prediction. Given the performance of our approach on the test data we speculate that it can be used to detect new interactions, especially in the light of the rapid growth of available sequence data.

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<![CDATA[The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of Chlamydia trachomatis]]> https://www.researchpad.co/article/5989d9e0ab0ee8fa60b6953b

Chlamydia trachomatis is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected in vitro using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for C. trachomatis co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit C. trachomatis replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects C. trachomatis from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known Homo sapiens proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.

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<![CDATA[α-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of α-Mangostin to HSA or TRF]]> https://www.researchpad.co/article/5989da35ab0ee8fa60b86232

In order to obtain the biological active compound, α-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of α-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of α-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (1H NMR and 13C NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained α-mangostin was 95.6% as determined by HPLC analysis. The binding of native α-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that α-mangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to α-mangostin is 6.4832×105 L/mol and 1.4652×105 L/mol at 298 K and 7.8619×105 L/mol and 1.1582×105 L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between α-mangostin and HSA or TRF were also obtained by virtue of the Förster theory of non-radiation energy transfer. The effect of α-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between α-mangostin and HSA is hydrophobic interactions, while the main interaction between α-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.

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<![CDATA[Tryptophan confers resistance to SDS-associated cell membrane stress in Saccharomyces cerevisiae]]> https://www.researchpad.co/article/5c900d3ed5eed0c48407e416

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Previously, we have shown that deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate; thus, we implemented a suppressor gene screening revealing that the overexpression of TAT2 tryptophan permease rescues cell growth in sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that trp1-1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil when cells are grown on rich media. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as Calcofluor White, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discovered that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, we conclude that internal concentration of tryptophan and tyrosine makes cells more resistant to detergent such as sodium dodecyl sulfate.

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<![CDATA[Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdccc0

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

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<![CDATA[Gene modification of the acetate biosynthesis pathway in Escherichia coli and implementation of the cell recycling technology to increase L-tryptophan production]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be1116

The implementation of a novel cell recycling technology based on a special disk centrifuge during microbial fermentation process can continuously separate the product and harmful intermediates, while maintaining the cell viability owing to the installed cooling system. Acetate accumulation is an often encountered problem in L-tryptophan fermentation by Escherichia coli. To extend our previous studies, the current study deleted the key genes underlying acetate biosynthesis to improve l-tryptophan production. The deletion of the phosphotransacetylase (pta)–acetate kinase (ackA) pathway in a gltB (encoding glutamate synthase) mutant of E. coli TRTHB, led to the highest production of l-tryptophan (47.18 g/L) and glucose conversion rate (17.83%), with a marked reduction in acetate accumulation (1.22 g/L). This strain, TRTHBPA, was then used to investigate the effects of the cell recycling process on L-tryptophan fermentation. Four different strategies were developed concerning two issues, the volume ratio of the concentrated cell solution and clear solution and the cell recycling period. With strategy I (concentrated cell solution: clear solution, 1: 1; cell recycling within 24–30 h), L-tryptophan production and the glucose conversion rate increased to 55.12 g/L and 19.75%, respectively, 17.55% and 10.77% higher than those without the cell recycling. In addition, the biomass increased by 13.52% and the fermentation period was shortened from 40 h to 32 h. These results indicated that the cell recycling technology significantly improved L-tryptophan production by E. coli.

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