Mitochondrial Alpha-Keto Acid Dehydrogenase Complexes: Recent Developments on Structure and Function in Health and Disease.

The present work delves into the enigmatic world of mitochondrial alpha-keto acid dehydrogenase complexes discussing their metabolic significance, enzymatic operation, moonlighting activities, and pathological relevance with links to underlying structural features. This ubiquitous family of related but diverse multienzyme complexes is involved in carbohydrate metabolism (pyruvate dehydrogenase complex), the citric acid cycle (α-ketoglutarate dehydrogenase complex), and amino acid catabolism (branched-chain α-keto acid dehydrogenase complex, α-ketoadipate dehydrogenase complex); the complexes all function at strategic points and also participate in regulation in these metabolic pathways. These systems are among the largest multienzyme complexes with at times more than 100 protein chains and weights ranging up to ~10 million Daltons. Our chapter offers a wealth of up-to-date information on these multienzyme complexes for a comprehensive understanding of their significance in health and disease.

Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase.

Clinically relevant disease-causing variants of the human dihydrolipoamide dehydrogenase (hLADH, hE3), a common component of the mitochondrial α-keto acid dehydrogenase complexes, were characterized using a multipronged approach to unravel the molecular pathomechanisms that underlie hLADH deficiency. The G101del and M326V substitutions both reduced the protein stability and triggered the disassembly of the functional/obligate hLADH homodimer and significant FAD losses, which altogether eventually manifested in a virtually undetectable catalytic activity in both cases. The I12T-hLADH variant proved also to be quite unstable, but managed to retain the dimeric enzyme form; the LADH activity, both in the forward and reverse catalytic directions and the affinity for the prosthetic group FAD were both significantly compromised. None of the above three variants lent themselves to an in-depth structural analysis via X-ray crystallography due to inherent protein instability. Crystal structures at 2.89 and 2.44 Å resolutions were determined for the I318T- and I358T-hLADH variants, respectively; structure analysis revealed minor conformational perturbations, which correlated well with the residual LADH activities, in both cases. For the dimer interface variants G426E-, I445M-, and R447G-hLADH, enzyme activities and FAD loss were determined and compared against the previously published structural data.

Population genetic features of calving interval of the Limousin beef cattle breed in Hungary.

Variance, covariance components, heritability, breeding values (BV) and genetic trends in calving interval (CI) of the Limousin population in Hungary were evaluated. A total of 3,008 CI data of 779 cows from three herds in 1996-2016 were processed. For influencing effects GLM method, for population genetic parameters and BV estimation BLUP animal model, for trend analyses linear regression was applied. The average CI obtained was 378.8 ± 3.1 days. The variance distribution components of the phenotype were as follow: age of cow at calving 34.30%, season of calving 26.09%, year of calving 23.00%, sire 7.45%, herd 3.23%, sex of calf 0.33% and type of calving 0.30%. The heritability of CI proved to be low (h2 d = 0.04 ± 0.02 and 0.03 ± 0.02; h2 m = 0.01 ± 0.02). The repeatability was low (R = 0.03 ± 0.02). Based on the phenotypic trend calculation, the CI of cows decreased by an average of 0.60 days per year (R 2 = 0.19; P < 0.05). In case of genetic trend calculation, the average BV of sires in CI increased 0.07 and 0.17 days per year (R 2 = 0.23 and 0.27; P < 0.05).

Underlying molecular alterations in human dihydrolipoamide dehydrogenase deficiency revealed by structural analyses of disease-causing enzyme variants.

Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially inactive hE3 variants. Here we report the crystal structure of wild-type hE3 at an unprecedented high resolution of 1.75 Å and the structures of six disease-causing hE3 variants at resolutions ranging from 1.44 to 2.34 Å. P453L proved to be the most deleterious substitution in structure as aberrations extensively compromised the active site. The most prevalent G194C-hE3 variant primarily exhibited structural alterations close to the substitution site, whereas the nearby cofactor-binding residues were left unperturbed. The G426E substitution mainly interfered with the local charge distribution introducing dynamics to the substitution site in the dimer interface; G194C and G426E both led to minor structural changes. The R460G, R447G and I445M substitutions all perturbed a solvent accessible channel, the so-called H+/H2O channel, leading to the active site. Molecular pathomechanisms of enhanced reactive oxygen species (ROS) generation and impaired binding to multienzyme complexes were also addressed according to the structural data for the relevant mutations. In summary, we present here for the first time a comprehensive study that links three-dimensional structures of disease-causing hE3 variants to residual hLADH activities, altered capacities for ROS generation, compromised affinities for multienzyme complexes and eventually clinical symptoms. Our results may serve as useful starting points for future therapeutic intervention approaches.

Functional Comparison of Blood-Derived Human Neural Progenitor Cells.

Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although the establishment, stabilization, and directed differentiation of iPSC lines are rather expensive and time-demanding. However, interrupted reprogramming by omitting the stabilization of iPSCs may allow for the generation of a plastic stage of the cells and thus provide a shortcut to derive NPSCs directly from tissue samples. Here, we demonstrate a method to generate shortcut NPCs (sNPCs) from blood mononuclear cells and present a detailed comparison of these sNPCs with NPCs obtained from the same blood samples through stable iPSC clones and a subsequent neural differentiation (classical NPCs-cNPCs). Peripheral blood cells were obtained from a schizophrenia patient and his two healthy parents (a case-parent trio), while a further umbilical cord blood sample was obtained from the cord of a healthy new-born. The expression of stage-specific markers in sNPCs and cNPCs were compared both at the protein and RNA levels. We also performed functional tests to investigate Wnt and glutamate signaling and the oxidative stress, as these pathways have been suggested to play important roles in the pathophysiology of schizophrenia. We found similar responses in the two types of NPCs, suggesting that the shortcut procedure provides sNPCs, allowing an efficient screening of disease-related phenotypes.

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.

[Diagnostics of inborn errors of metabolism: laboratory approaches]. / Ritka örökletes anyagcsere-betegségek diagnosztikája: laboratóriumi vizsgálati megközelítések.

Inherited errors of metabolism are rare genetic disorders characterized by diverse clinical and biochemical phenotypes. The complexity of signs and symptoms often presents a challenge for both clinicians and laboratory specialists. In many cases, prevention of permanent neurological symptoms or death in patients presenting these disorders is dependent on early diagnosis and introduction of appropriate therapy. For professionals it is indispensable to be familiar with the major clinical signs of inborn errors of metabolism and with the necessary and available laboratory studies to achieve an early diagnosis. The review tries to give a way of approach, diagnostic algorithm of laboratory measurements for the correct diagnosis in inherited errors of metabolism. The combination of biochemical and clinical signs, results of special metabolic investigations represent a portentous challenge in general practice. For the correct diagnosis of an inherited error of metabolism, the teamwork between clinicians and laboratory specialists is indispensable. Orv Hetil. 2017; 158(48): 1903-1907.

Quantitative Analytical Method for the Determination of Biotinidase Activity in Dried Blood Spot Samples.

Biotinidase activity assay is included in most newborn screening protocols, and the positive results are confirmed by quantitative enzyme activity measurements. In our study, we describe a new quantitative analytical method for the determination of biotinidase activity using the blood sample deposited onto filter paper as the assay medium, by predepositing N-biotinyl-p-aminobenzoic acid onto the standard sample collection paper. The analysis of the assay mixture requires a simple extraction step from a dried blood spot followed by the quantification of product by LC-MS. The method provides a simple and reliable enzyme assay method that enables the rapid diagnosis of biotinidase deficiency (BD). Out of the measured 36 samples, 13 were healthy with lower enzyme activities, 16 were patients with partial BD, and 7 were patients with profound BD with residual activity below 10%. Expression of enzyme activity in percentage of mean activity of negative controls allows comparison of the different techniques. The obtained results are in good agreement with activity data determined from both dried blood spots and serum samples, giving an informative diagnostic value.

EFFECT OF STORAGE TEMPERATURE ON THE STABILITY OF TOTAL PARENTERAL NUTRITION ADMIXTURES PREPARED FOR INFANTS.

Physical, chemical and microbiological stability of total parenteral nutrient (TPN) admixtures was studied as a function of storage time and temperature. Particle size analysis and zeta potential measurements were carried out to evaluate the possible changes in the kinetic stability of the emulsions as a function of storage time and temperature. The concentration changes of the applied additives, those of the ascorbic acid and L-alanyl-L-glutamine, were also determined under different storage conditions. Our results indicate that there were no significant differences in the particle size and zeta potential values of admixtures stored at the three examined temperatures. The best results were obtained in the case of admixtures stored at 30°C temperature. Rapid decomposition of vitamin C was found while the glutamine showed adequate stability as a function of storage time and temperature. According to the results of the physicochemical examinations 10-day storage period of this type of TPN admixtures can be accepted at room temperature. Their storage does not require refrigeration (2-8°C) thus they can be administered without special preheating ensuring better physiological tolerance. Ascorbic acid can be added to the system preceding the administration to the patient because of its rapid decomposition.

[The severity of gestational diabetes mellitus affects microvascular dysfunction measured three years after pregnancy that may be related to increased oxidative stress]. / A gestatiós diabetes mellitus súlyossága befolyásolja a három évvel a szülést követoen mért microvascularis diszfunkció mértékét, amely kapcsolatban állhat az emelkedett szisztémás oxidatív stresszel.

INTRODUCTION: Oxidative-nitrative stress and poly(ADP-ribose) polymerase activation observed in gestational diabetes may play role in the increased cardiovascular risk in later life. AIM: The present study aimed to examine the influence of the severity of previous gestational diabetes (insulin need) on vascular function three years after delivery. Furthermore, the authors investigated the relation of vascular function with oxidative-nitrative stress and poly(ADP-ribose) polymerase activation. METHOD: Macrovascular function was measured by applanation tonometry; microvascular reactivity was assessed by provocation tests during Laser-Doppler flowmetry in 40 women who had gestational diabetes 3 years before the study. Oxidative-nitrative stress and poly(ADP-ribose) polymerase activity in blood components were determined by colorimetry and immunohistochemistry. RESULTS: Three years after insulin treated gestational diabetes impaired microvascular function and increased oxidative stress was observed compared to mild cases. CONCLUSIONS: The severity of previous gestational diabetes affects microvascular dysfunction that is accompanied by elevated oxidative stress. Nitrative stress and poly(ADP-ribose) polymerase activity correlates with certain vascular factors not related to the severity of the disease.

Chiral analysis of amino acid neurotransmitters and neuromodulators in mouse brain by CE-LIF.

Chiral CE method has been developed for quantitative determination of d-amino acid modulators of NMDA glutamate receptor; d-serine and d-aspartate along with l-glutamate and l-aspartate in biological samples. These ligands are suggested to be involved in regulation of NMDA receptor related brain functions, such as neurogenesis, neuronal plasticity, and memory formation. For sensitive determination of the amino acids LIF detection was chosen, and a fluorogenic reagent, 7-fluoro-4-nitro-2,1,3-benzoxadiazole was used for derivatization. An amino-modified ß-CD, 6-monodeoxy-6-mono(3-hydroxy)propylamino-ß-CD (HPA-ß-CD) was applied as chiral selector. Determinations were accomplished in a polyacrylamide coated capillary and reverse polarity was used for the analysis of the negatively charged analytes. The method was optimized and validated; 6 mM HPA-ß-CD in 50 mM HEPES buffer, pH 7 was appropriate to achieve baseline separation of the analytes. The limit of quantification with acceptable accuracy is 0.05 µM for both d-amino acids. The method was used for the determination of d-aspartate and d-serine content in various brain regions of adult mice.

The effect of alcohol on speech production.

The present study investigates the effect of acute alcohol consumption on speech in Hungarian subjects. The measures used to reveal these effects were tongue-twisters, which were grouped according to their linguistic features. The number and type of speech errors while uttering the tongue-twisters were compared between intoxicated and sober conditions. The results showed that subjects made more speech errors in alcohol influenced than in sober states in all types of the tongue-twisters except for those using foreign words. Changes in the articulation rate, number of pauses and fundamental frequency were investigated as well. In the intoxicated state, no changes were observed in fundamental frequency and articulation rate, while the number of pauses increased.

Do early meanings of negation map onto a fully-fledged negation concept in infancy?

Young children acquire an amazing knowledge base, rapidly learning from, and even going beyond the observable evidence. They arrive at forming abstract concepts and generalizations and recruit logical operations. The question whether young infants can already rely on abstract logical operations, such as disjunction or negation, or whether these operations emerge gradually over development has recently become a central topic of interest. Here we target this question by focusing on infants' early understanding of negation. According to one view, negation comprehension is initially restricted to a narrow range of meanings (such as rejection or non-existence) and only much later infants develop a broader understanding that maps onto a fully-fledged negation concept. Alternatively, however, infants may rely on a fully-fledged negation concept from early on, but some forms of negation may pose more mapping and processing difficulties than others. Here we tested infants' understanding of two syntactically and semantically different forms of negation, existential negation and propositional denial in a language (Hungarian) that has a separate negative particle for each, and thus the two negation forms can be directly compared. We engaged 15- and 18-month-old infants in a search task where they had to find a toy in one out of two locations based on verbal utterances referring to the object at one of the locations involving existential negation (Nincsen - not.be.3SG) or propositional denial (Nem itt van - not here be.3SG). In Experiments 1-3 we found a parallel development for these two kinds of negation. 18-month-olds successfully comprehended both, while 15-month-olds were at chance for both. In Experiment 4 we excluded the possibility that 15-month-olds' chance performance is explained by task-related difficulties, as they succeeded in a similar, but nonverbal task. Thus, 15-month-olds likely still have not solved the mapping for the two negation forms. The parallel performance of the two age groups with the two negation types (failing or succeeding on both) is consistent with the hypothesis that different forms of negation rely on similar conceptual underpinnings already in early development.

Shear rate induced viscosity change of human blood samples and blood mimicking fluids.

Purpose: The aim of this study was to measure blood and blood mimicking fluids viscosity at different shear rates (on the interval of 0.1-5000 1/s and 0.1-10000 1/s) while taking into consideration the measuring device's capability and blood's characteristics. We also provided the measurement results of the most accurate measuring program. Methods: We measured blood samples from five donors, and four different blood mimicking fluid compositions. The measurements were done on an Anton Paar Physica MCR301 rotational rheometer with two measuring programs varying in the shear rate intervals, the number of measuring points and the measuring point durations. Results: The results confirmed the significant shear thinning and thixotropic effects of blood. Blood mimicking fluids also had these characteristics. The measured blood viscosity values are in agreement with those of the literature. Conclusions: It can be concluded that the step test program was able to give more stable results as the measured torque was over the nominal limit of 0.05 ìNm over 0.1 1/s and over the selected torque limit of 0.5 ìNm over 31.6 1/s. Blood mimicking fluid measurement results were different from that of the literature due to different measuring conditions. The sample consisting of water, glycerol and starch mimicked well blood's behaviour and viscosity values at 37 degrees Celsius.

Impact of Coffee Intake on Measures of Wellbeing in Mice.

Coffee intake is increasingly recognized as a life-style factor associated with the preservation of health, but there is still a debate on the relative effects of caffeinated and decaffeinated coffee. We now tested how the regular drinking of caffeinated and decaffeinated coffee for 3 weeks impacted on the behavior of male and female adult mice. Males drinking caffeinated coffee displayed statistically significant lower weight gain, increased sensorimotor coordination, greater motivation in the splash test, more struggling in the forced swimming test, faster onset of nest building, more marble burying and greater sociability. Females drinking caffeinated coffee displayed statistically significant increased hierarchy fighting, greater self-care and motivation in the splash test and faster onset of nest building. A post-hoc two-way ANOVA revealed sex-differences in the effects of caffeinated coffee (p values for interaction between the effect of caffeinated coffee and sex) on the hierarchy in the tube test (p = 0.044; dominance), in the time socializing (p = 0.044) and in the latency to grooming (p = 0.048; selfcare), but not in the marble burying test (p = 0.089). Intake of decaffeinated coffee was devoid of effects in males and females. Since caffeine targets adenosine receptors, we verified that caffeinated but not decaffeinated coffee intake increased the density of adenosine A1 receptors (A1R) and increased A1R-mediated tonic inhibition of synaptic transmission in the dorsolateral striatum and ventral but not dorsal hippocampus, the effects being more evident in the ventral hippocampus of females and striatum of males. In contrast, caffeinated and decaffeinated coffee both ameliorated the antioxidant status in the frontal cortex. It is concluded that caffeinated coffee increases A1R-mediated inhibition in mood-related areas bolstering wellbeing of both males and females, with increased sociability in males and hierarchy struggling and self-care in females.

Lipoamide dehydrogenase (LADH) deficiency: medical perspectives of the structural and functional characterization of LADH and its pathogenic variants.

(Dihydro)lipoamide dehydrogenase (LADH) deficiency is an autosomal recessive genetic metabolic disorder. It generally presents with an onset in the neonatal age and premature death. The clinical picture usually involves metabolic decompensation and lactic acidosis that lead to neurological, cardiological, and/or hepatological outcomes. Severity of the disease is due to the fact that LADH is a common E3 subunit to the pyruvate, alpha-ketoglutarate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes and is also part of the glycine cleavage system; hence, a loss in LADH activity adversely affects several central metabolic pathways simultaneously. The severe clinical manifestations, however, often do not parallel the LADH activity loss, which implies the existence of auxiliary pathological pathways; stimulated reactive oxygen species (ROS) production as well as dissociation from the relevant multienzyme complexes proved to be auxiliary exacerbating pathomechanisms for selected disease-causing LADH mutations. This review provides an overview on the therapeutic challenges of inherited metabolic diseases, structural and functional characteristics of the mitochondrial alpha-keto acid dehydrogenase complexes, molecular pathogenesis and structural basis of LADH deficiency, and relevant potential future medical perspectives.

Effects of Chronic Caffeine Consumption on Synaptic Function, Metabolism and Adenosine Modulation in Different Brain Areas.

Adenosine receptors mainly control synaptic function, and excessive activation of adenosine receptors may worsen the onset of many neurological disorders. Accordingly, the regular intake of moderate doses of caffeine antagonizes adenosine receptors and affords robust neuroprotection. Although caffeine intake alters brain functional connectivity and multi-omics analyses indicate that caffeine intake modifies synaptic and metabolic processes, it is unclear how caffeine intake affects behavior, synaptic plasticity and its modulation by adenosine. We now report that male mice drinking caffeinated water (0.3 g/L) for 2 weeks were behaviorally indistinguishable (locomotion, mood, memory) from control mice (drinking water) and displayed superimposable synaptic plasticity (long-term potentiation) in different brain areas (hippocampus, prefrontal cortex, amygdala). Moreover, there was a general preservation of the efficiency of adenosine A1 and A2A receptors to control synaptic transmission and plasticity, although there was a tendency for lower levels of endogenous adenosine ensuring A1 receptor-mediated inhibition. In spite of similar behavioral and neurophysiological function, caffeine intake increased the energy charge and redox state of cortical synaptosomes. This increased metabolic competence likely involved a putative increase in the glycolytic rate in synapses and a prospective greater astrocyte-synapse lactate shuttling. It was concluded that caffeine intake does not trigger evident alterations of behavior or of synaptic plasticity but increases the metabolic competence of synapses, which might be related with the previously described better ability of animals consuming caffeine to cope with deleterious stimuli triggering brain dysfunction.

Metabonomics of newborn screening dried blood spot samples: a novel approach in the screening and diagnostics of inborn errors of metabolism.

A novel, single stage high resolution mass spectrometry-based method is presented for the population level screening of inborn errors of metabolism. The approach proposed here extends traditional electrospray tandem mass spectrometry screening by introducing nanospray ionization and high resolution mass spectrometry, allowing the selective detection of more than 400 individual metabolic constituents of blood including acylcarnitines, amino acids, organic acids, fatty acids, carbohydrates, bile acids, and complex lipids. Dried blood spots were extracted using a methanolic solution of isotope labeled internal standards, and filtered extracts were electrosprayed using a fully automated chip-based nanospray ion source in both positive and negative ion mode. Ions were analyzed using an Orbitrap Fourier transformation mass spectrometer at nominal mass resolution of 100,000. Individual metabolic constituents were quantified using standard isotope dilution methods. Concentration threshold (cutoff) level-based analysis allows the identification of newborns with metabolic diseases, similarly to the traditional electrospray tandem mass spectrometry (ESI-MS/MS) method; however, the detection of additional known biomarkers (e.g., organic acids) results in improved sensitivity and selectivity. The broad range of detected analytes allowed the untargeted multivariate statistical analysis of spectra and identification of additional diseased states, therapeutic artifacts, and damaged samples, besides the metabolic disease panel.

Current Approaches in Molecular Enzymology.

Enzymes are the main executioners of living organisms [...].

Generation of Human Neural Progenitors from Blood Samples by Interrupted Reprogramming.

Human neuronal cell cultures are essential tools for biological and preclinical studies of our nervous system. Since we have very limited access to primary human neural samples, derivation of proliferative neural progenitor cells (NPCs) from cells harvested by minimally invasive sampling is a key issue. Here we describe a "shortcut" method to establish proliferative NPC cultures directly from peripheral blood mononuclear cells (PBMCs) via interrupted reprogramming. In addition, we provide procedures to characterize the NPC stage.