An efficient pyrrolysyl-tRNA synthetase for economical production of MeHis-containing enzymes.

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

Combined assessment of lysine and N-acetyl cadaverine levels assist as a potential biomarker of the smoker periodontitis.

Periodontitis is an inflammatory condition of supporting structures of teeth leading to attachment and bone loss. Cigarette smoking is the single most important and modifiable risk factor with 5 to 20-fold susceptibility for periodontal diseases. Reverse smoking is a peculiar habit of smoking where the lit end is kept inside the mouth, which is predominant in the northern coastal districts of Andhra Pradesh. Polyamines are biologically active amines involved in tissue regeneration and modulation of inflammation. The study aimed to evaluate polyamines and check their utility as a marker in detection of periodontitis among different groups. Total polyamine levels showed significant increase in reverse smokers with periodontitis when compared to the other groups. Qualitative analysis by thin layer chromatography showed three polyamine bands with varying intensity among the different groups. Mass spectrometric and NMR analyses of the three bands identified them as N1, N8-diacetyl spermidine, N-acetyl cadaverine and lysine. Most significantly elevated levels of lysine was observed in the smoker and reverse smoker periodontitis groups when compared to healthy and non-smoker periodontitis groups. The significantly elevated levels of N-acetyl cadaverine could be responsible for the more destruction of periodontium in the reverse smoker group. Antioxidant potential decreased significantly in different smoker periodontitis groups. The present study suggests that the quantitative analysis of salivary polyamines, lysine and N-acetyl cadaverine can aid as an easy noninvasive diagnostic method for assessing the periodontal status, especially in smokers.

Identification of Aggregation Mechanism of Acetylated PHF6* and PHF6 Tau Peptides Based on Molecular Dynamics Simulations and Markov State Modeling.

The microtubule-associated protein tau (MAPT) has a critical role in the development and preservation of the nervous system. However, tau's dysfunction and accumulation in the human brain can lead to several neurodegenerative diseases, such as Alzheimer's disease, Down's syndrome, and frontotemporal dementia. The microtubule binding (MTB) domain plays a significant, important role in determining the tau's pathophysiology, as the core of paired helical filaments PHF6* (275VQIINK280) and PHF6 (306VQIVYK311) of R2 and R3 repeat units, respectively, are formed in this region, which promotes tau aggregation. Post-translational modifications, and in particular lysine acetylation at K280 of PHF6* and K311 of PHF6, have been previously established to promote tau misfolding and aggregation. However, the exact aggregation mechanism is not known. In this study, we established an atomic-level nucleation-extension mechanism of the separated aggregation of acetylated PHF6* and PHF6 hexapeptides, respectively, of tau. We show that the acetylation of the lysine residues promotes the formation of ß-sheet enriched high-ordered oligomers. The Markov state model analysis of ac-PHF6* and ac-PHF6 aggregation revealed the formation of an antiparallel dimer nucleus which could be extended from both sides in a parallel manner to form mixed-oriented and high-ordered oligomers. Our study describes the detailed mechanism for acetylation-driven tau aggregation, which provides valuable insights into the effect of post-translation modification in altering the pathophysiology of tau hexapeptides.

Assessment of Mitochondrial Dysfunctions After Sirtuin Inhibition.

Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), with the latter being grouped into four phylogenetic classes. The class III of the HDAC family, the sirtuins (SIRTs), contributes to gene expression, genomic stability, cell metabolism, and tumorigenesis. Thus, several specific SIRT inhibitors (SIRTi) have been developed to target cancer cell proliferation. Here we provide an overview of methods to study SIRT-dependent cell metabolism and mitochondrial functionality. The chapter describes metabolic flux analysis using Seahorse analyzers, methods for normalization of Seahorse data, flow cytometry and fluorescence microscopy to determine the mitochondrial membrane potential, mitochondrial content per cell and mitochondrial network structures, and Western blot analysis to measure mitochondrial proteins.

Quantitative Assessment of Arsenite-Induced Perturbation of Ubiquitinated Proteome.

Arsenic contamination in food and groundwater constitutes a public health concern for more than 200 million people worldwide. Individuals chronically exposed to arsenic through drinking and ingestion exhibit a higher risk of developing cancers and cardiovascular diseases. Nevertheless, the underlying mechanisms of arsenic toxicity are not fully understood. Arsenite is known to bind to and deactivate RING finger E3 ubiquitin ligases; thus, we reason that a systematic interrogation about how arsenite exposure modulates global protein ubiquitination may reveal novel molecular targets for arsenic toxicity. By employing liquid chromatography-tandem mass spectrometry, in combination with stable isotope labeling by amino acids in cell culture (SILAC) and immunoprecipitation of di-glycine-conjugated lysine-containing tryptic peptides, we assessed the alterations in protein ubiquitination in GM00637 human skin fibroblast cells upon arsenite exposure at the entire proteome level. We observed that arsenite exposure led to altered ubiquitination of many proteins, where the alterations in a large majority of ubiquitination events are negatively correlated with changes in expression of the corresponding proteins, suggesting their modulation by the ubiquitin-proteasomal pathway. Moreover, we observed that arsenite exposure confers diminished ubiquitination of a rate-limiting enzyme in cholesterol biosynthesis, HMGCR, at Lys248. We also revealed that TRC8 is the major E3 ubiquitin ligase for HMGCR ubiquitination in HEK293T cells, and the arsenite-induced diminution of HMGCR ubiquitination is abrogated upon genetic depletion of TRC8. In summary, we systematically characterized arsenite-induced perturbations in a ubiquitinated proteome in human cells and found that the arsenite-elicited attenuation of HMGCR ubiquitination in HEK293T cells involves TRC8.

Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay.

Sodium taurocholate cotransporting polypeptide (NTCP), which is highly expressed in the sinusoidal membrane of hepatocytes, maintains bile acid homeostasis and participates in the hepatic disposition of a variety of endogenous substances as well as xenobiotics. Manifested by the involvement of organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) in the hepatic uptake of statin drugs, sinusoidal membrane transporters play an important role in the pharmacokinetics and pharmacodynamics of these agents. It has been speculated that NTCP may function as an alternative pathway for statin hepatic uptake, complementary to OATP1B1 and OATP1B3. In the current study, we produced stable NTCP-expressing human embryonic kidney 293 (HEK293) cells and developed a fluorescence-based assay using flow cytometry for measuring NTCP transport with chenodeoxycholyl-(Nε-7-nitrobenz-2-oxa-1,3-diazole)-lysine (CDCA-NBD) as the substrate. NTCP-mediated CDCA-NBD transport was time-dependent and exhibited typical Michaelis-Menten kinetics, with a Km of 6.12 µM. Compounds known to interact with NTCP, including chenodeoxycholic acid and taurocholic acid, displayed concentration-dependent inhibition of NTCP-mediated CDCA-NBD transport. We report here a systematic evaluation of the interaction between statins and the NTCP transporter. Utilizing this system, several statins were either found to inhibit NTCP-dependent transport or act as substrates. We find a good correlation between the reported lipophilicity of statins and their ability to inhibit NTCP. The objective was to develop a higher-throughput system to evaluate potential inhibitors such as the statins. The in vitro assays using CDCA-NBD as fluorescent substrate are convenient, rapid, and have utility in screening drug candidates for potential drug-NTCP interactions.

Large-Scale Assessment of Bioinformatics Tools for Lysine Succinylation Sites.

Lysine succinylation is a form of posttranslational modification of the proteins that play an essential functional role in every aspect of cell metabolism in both prokaryotes and eukaryotes. Aside from experimental identification of succinylation sites, there has been an intense effort geared towards the development of sequence-based prediction through machine learning, due to its promising and essential properties of being highly accurate, robust and cost-effective. In spite of these advantages, there are several problems that are in need of attention in the design and development of succinylation site predictors. Notwithstanding of many studies on the employment of machine learning approaches, few articles have examined this bioinformatics field in a systematic manner. Thus, we review the advancements regarding the current state-of-the-art prediction models, datasets, and online resources and illustrate the challenges and limitations to present a useful guideline for developing powerful succinylation site prediction tools.

Large-scale comparative assessment of computational predictors for lysine post-translational modification sites.

Lysine post-translational modifications (PTMs) play a crucial role in regulating diverse functions and biological processes of proteins. However, because of the large volumes of sequencing data generated from genome-sequencing projects, systematic identification of different types of lysine PTM substrates and PTM sites in the entire proteome remains a major challenge. In recent years, a number of computational methods for lysine PTM identification have been developed. These methods show high diversity in their core algorithms, features extracted and feature selection techniques and evaluation strategies. There is therefore an urgent need to revisit these methods and summarize their methodologies, to improve and further develop computational techniques to identify and characterize lysine PTMs from the large amounts of sequence data. With this goal in mind, we first provide a comprehensive survey on a large collection of 49 state-of-the-art approaches for lysine PTM prediction. We cover a variety of important aspects that are crucial for the development of successful predictors, including operating algorithms, sequence and structural features, feature selection, model performance evaluation and software utility. We further provide our thoughts on potential strategies to improve the model performance. Second, in order to examine the feasibility of using deep learning for lysine PTM prediction, we propose a novel computational framework, termed MUscADEL (Multiple Scalable Accurate Deep Learner for lysine PTMs), using deep, bidirectional, long short-term memory recurrent neural networks for accurate and systematic mapping of eight major types of lysine PTMs in the human and mouse proteomes. Extensive benchmarking tests show that MUscADEL outperforms current methods for lysine PTM characterization, demonstrating the potential and power of deep learning techniques in protein PTM prediction. The web server of MUscADEL, together with all the data sets assembled in this study, is freely available at http://muscadel.erc.monash.edu/. We anticipate this comprehensive review and the application of deep learning will provide practical guide and useful insights into PTM prediction and inspire future bioinformatics studies in the related fields.

Evaluation of starter dietary digestible lysine level on broilers raised under a sex-separated or straight-run housing regime, part 2: Economics of sex separation and digestible lysine level for maximum returns.

The objective of this experiment was to determine the maximum net returns digestible lysine (dLys) levels (MNRL) when maintaining the ideal amino acid ratio for starter diets of broilers raised sex separate or comingled (straight-run). A total of 3,240 Ross 708 chicks was separated by sex and placed in 90 pens by 2 rearing types: sex separate (36 males or 36 females) or straight-run (18 males + 18 females). Each rearing type was fed 6 starter diets (25 d) formulated to have dLys levels between 1.05 and 1.80%. A common grower diet with 1.02% of dLys was fed from 25 to 32 days. Body weight gain (BWG) and feed intake were assessed at 25 and 32 d for performance evaluation. Additionally, at 26 and 33 d, 4 birds per pen were sampled for carcass yield evaluation. Data were modeled using response surface methodology in order to estimate feed intake and whole carcass weight at 1,600 g live BW. Returns over feed cost were estimated for a 1.8-million-broiler complex of each rearing system under 9 feed/meat price scenarios. Results indicated that females needed more feed to reach market weight, followed by straight-run birds, and then males. At medium meat and feed prices, female birds had MNRL at 1.07% dLys, whereas straight-run and males had MNRL at 1.05%. As feed and meat prices increased, females had MNRL increased up to 1.15% dLys. Sex separation resulted in increased revenue under certain feed and meat prices, and before sex separation cost was deducted. When the sexing cost was subtracted from the returns, sex separation was not shown to be economically viable when targeting birds for light market BW.

In vitro assessment of skin sensitization, photosensitization and phototoxicity potential of commercial glyphosate-containing formulations.

This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging.

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic cross-links, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692cm-1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p<0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high-performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture.

Quantitative assessment of chemical artefacts produced by propionylation of histones prior to mass spectrometry analysis.

Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot-gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain "Arg-C like" digestion products that are more suitable for LC-MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. is a quantitative assessment of methyl-esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059-2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl-esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl-esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation.

Quantitative assessment of organ distribution of dietary protein-bound 13 C-labeled Nɛ -carboxymethyllysine after a chronic oral exposure in mice.

SCOPE: Nɛ -Carboxymethyl-lysine (CML) is a prominent advanced glycation end-product which is not only found in vivo but also in food. It is known that a percentage of the dietary CML (dCML) is absorbed into the circulation and only partly excreted in the urine. Several studies have tried to measure how much dCML remains in tissues. However obstacles to interpreting the data have been found. METHODS AND RESULTS: A new protocol which discriminates dCML from native CML (nCML) has been developed. Three CML isotopes with different mass-to-charge ratios were used: nCML Nε -carboxymethyl-L-lysine, dCML Nε -[13 C]carboxy[13 C]methyl-L-lysine and internal standard Nε -carboxymethyl-L-[4,4,5,5-2 H4 ]lysine. Wild-type (n = 7) and RAGE-/- (n = 8) mice were fed for 30 days with either a control, or a BSA-bound dCML-enriched diet. Organs were analyzed for nCML and dCML using liquid chromatography-tandem mass spectrometry. Mice exposed to dCML showed an accumulation in all tissues tested except fat. The rate of deposition was high (81-320 µgdCML /g dry matter) in kidneys, intestine, and lungs and low (<5 µg/g) in heart, muscle, and liver. This accumulation was not RAGE dependent. CONCLUSION: The kidney is not the only organ affected by the accumulation of dCML. Its high accumulation in other tissues and organs may also, however, have important physiological consequences.

Chromatin Unfolding by Epigenetic Modifications Explained by Dramatic Impairment of Internucleosome Interactions: A Multiscale Computational Study.

Histone tails and their epigenetic modifications play crucial roles in gene expression regulation by altering the architecture of chromatin. However, the structural mechanisms by which histone tails influence the interconversion between active and inactive chromatin remain unknown. Given the technical challenges in obtaining detailed experimental characterizations of the structure of chromatin, multiscale computations offer a promising alternative to model the effect of histone tails on chromatin folding. Here we combine multimicrosecond atomistic molecular dynamics simulations of dinucleosomes and histone tails in explicit solvent and ions, performed with three different state-of-the-art force fields and validated by experimental NMR measurements, with coarse-grained Monte Carlo simulations of 24-nucleosome arrays to describe the conformational landscape of histone tails, their roles in chromatin compaction, and the impact of lysine acetylation, a widespread epigenetic change, on both. We find that while the wild-type tails are highly flexible and disordered, the dramatic increase of secondary-structure order by lysine acetylation unfolds chromatin by decreasing tail availability for crucial fiber-compacting internucleosome interactions. This molecular level description of the effect of histone tails and their charge modifications on chromatin folding explains the sequence sensitivity and underscores the delicate connection between local and global structural and functional effects. Our approach also opens new avenues for multiscale processes of biomolecular complexes.

Determination of protein and amino acid requirements of lactating sows using a population-based factorial approach.

Determination of appropriate nutritional requirements is essential to optimize the productivity and longevity of lactating sows. The current recommendations for requirements do not consider the large variation between animals. Therefore, the aim of this study was to determine the amino acid recommendations for lactating sows using a stochastic modeling approach that integrates population variation and uncertainty of key parameters into establishing nutritional recommendations for lactating sows. The requirement for individual sows was calculated using a factorial approach by adding the requirement for maintenance and milk. The energy balance of the sows was either negative or zero depending on feed intake being a limiting factor. Some parameters in the model were sow-specific and others were population-specific, depending on state of knowledge. Each simulation was for 1000 sows repeated 100 times using Monte Carlo simulation techniques. BW, back fat thickness of the sow, litter size (LS), average litter gain (LG), dietary energy density and feed intake were inputs to the model. The model was tested using results from the literature, and the values were all within ±1 s.d. of the estimated requirements. Simulations were made for a group of low- (LS=10 (s.d.=1), LG=2 kg/day (s.d.=0.6)), medium- (LS=12 (s.d.=1), LG=2.5 kg/day (s.d.=0.6)) and high-producing (LS=14 (s.d.=1), LG=3.5 kg/day (s.d.=0.6)) sows, where the average requirement was the result. In another simulation, the requirements were estimated for each week of lactation. The results were given as the median and s.d. The average daily standardized ileal digestible (SID) protein and lysine requirements for low-, medium- and high-producing sows were 623 (CV=2.5%) and 45.1 (CV=4.8%); 765 (CV=4.9%) and 54.7 (CV=7.0%); and 996 (CV=8.5%) and 70.8 g/day (CV=9.6%), respectively. The SID protein and lysine requirements were lowest at week 1, intermediate at week 2 and 4 and the highest at week 3 of lactation. The model is a valuable tool to develop new feeding strategies by taking into account the variable requirement between groups of sows and changes during lactation. The inclusion of between-sow variation gives information on safety margins when developing new dietary recommendations of amino acids and protein for lactating sows.

Neighboring Genes Show Correlated Evolution in Gene Expression.

When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking.

Changes in the amount of lysine in protective proteins and immune cells after a systemic response to dead Escherichia coli: implications for the nutritional costs of immunity.

The nutritional demands of the immune system may result in tradeoffs with competing processes such as growth and reproduction. The magnitude of the nutritional costs of immunity is largely unknown. Thus, we examine the lysine content of the systemic components of the immune system in adult male chickens (Gallus gallus domesticus) in a healthy condition (maintenance) and following a robust Escherichia coli-specific immune response. Lysine was used as a metric, because it is found both in leukocytes and in protective proteins. The dynamics of subsets of leukocytes were monitored in primary and secondary immune tissues (thymus, bone marrow, and spleen) that would be expected to be involved in the response following iv injection of E. coli. The systemic immune system at maintenance has the same lysine content as 332 average-sized feathers, 16% of an egg, or 5.4% of a pectoralis muscle from an adult chicken. During the acute-phase response to E. coli, the additional lysine needed would equal 355 feathers, 17% of an egg, or 5.5% of a pectoralis muscle. The acute-phase proteins accounted for the greatest proportion of lysine in the immune system at maintenance and the proportion increased substantially during an acute-phase response. Hypertrophy of the liver required more lysine than all of the leukocytes and protective proteins that were produced during the acute-phase response. Size of the liver and levels of protein during the acute phase returned to normal during the time when the adaptive response began to utilize significant quantities of lysine. The catabolism would release a surfeit of lysine to provision the anabolic processes of the adaptive response, thus making proliferation of lymphocytes and production of immunoglobulins very cheap.

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei.

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme-substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈6.1kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240-325cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2-2.3kcal/mol to set the enzyme into a catalytically competent form.

Effects of the comminution rate and microbial contamination of particles in the rumen on in situ estimates of protein and amino acid digestion of expeller palm kernel and rapeseed meal.

BACKGROUND: Microbial corrected effective in situ estimates of ruminal undegraded fraction (RU) and intestinal effective digestibility (IED) of dry matter (DM), crude protein (CP) and amino acids (AA) of expeller palm kernel (EPK) and rapeseed meal (RSM) were measured on three rumen- and duodenum-cannulated wethers using ¹5N labelling techniques and considering ruminal rates of comminution (k(c)) and outflow (k(p)) of particles. RESULTS: The lack of k(c) and microbial correction overestimated the RU of DM by 4.91% (EPK) and 9.88% (RSM). The lack of this correction also overestimated in both feeds the RU of CP, individual and total (TAA) AA as well as the IED of DM, CP, TAA and most AA. RU estimates were higher for CP than for TAA, but the opposite was observed for IED. The intestinal digested fraction was higher for CP than for TAA: 17.4% (EPK) and 13.8% (RSM). Digestion led to large changes in the essential AA profile in both feeds. CONCLUSION: The lack of k(c) and microbial correction as well as CP-based results leads to considerable overestimations in the protein use of both feeds. Digestion aggravates the lysine deficiency of EPK but has global positive effects in the absorbed profile of RSM.

The balance of striatal feedback transmission is disrupted in a model of parkinsonism.

Inhibitory connections among striatal projection neurons (SPNs) called "feedback inhibition," have been proposed to endow the striatal microcircuit with computational capabilities, such as motor sequence selection, filtering, and the emergence of alternating network states. These properties are disrupted in models of Parkinsonism. However, the impact of feedback inhibition in the striatal network has remained under debate. Here, we test this inhibition at the microcircuit level. We used optical and electrophysiological recordings in mice and rats to demonstrate the action of striatal feedback transmission in normal and pathological conditions. Dynamic calcium imaging with single-cell resolution revealed the synchronous activation of a pool of identified SPNs by antidromic stimulation. Using bacterial artificial chromosome-transgenic mice, we demonstrate that the activated neuron pool equally possessed cells from the direct and indirect basal ganglia pathways. This pool inhibits itself because of its own GABA release when stimuli are frequent enough, demonstrating functional and significant inhibition. Blockade of GABAA receptors doubled the number of responsive neurons to the same stimulus, revealing a second postsynaptic neuron pool whose firing was being arrested by the first pool. Stronger connections arise from indirect SPNs. Dopamine deprivation impaired striatal feedback transmission disrupting the ability of a neuronal pool to arrest the firing of another neuronal pool. We demonstrate that feedback inhibition among SPNs is strong enough to control the firing of cell ensembles in the striatal microcircuit. However, to be effective, feedback inhibition should arise from synchronized pools of SPNs whose targets are other SPNs pools.