The Catalytic Mechanism of Electron-Bifurcating Electron Transfer Flavoproteins (ETFs) Involves an Intermediary Complex with NAD\u3csup\u3e+\u3c/sup\u3e

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum. The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family

Neurodevelopmental Disruption of Cortico-Striatal Function Caused by Degeneration of Habenula Neurons

The habenula plays an important role on cognitive and affective functions by regulating monoamines transmission such as the dopamine and serotonin, such that its dysfunction is thought to underlie a number of psychiatric conditions. Given that the monoamine systems are highly vulnerable to neurodevelopmental insults, damages in the habenula during early neurodevelopment may cause devastating effects on the wide-spread brain areas targeted by monoamine innervations.Using a battery of behavioral, anatomical, and biochemical assays, we examined the impacts of neonatal damage in the habenula on neurodevelopmental sequelae of the prefrontal cortex (PFC) and nucleus accumbens (NAcc) and associated behavioral deficits in rodents. Neonatal lesion of the medial and lateral habenula by ibotenic acid produced an assortment of behavioral manifestations consisting of hyper-locomotion, impulsivity, and attention deficit, with hyper-locomotion and impulsivity being observed only in the juvenile period, whereas attention deficit was sustained up until adulthood. Moreover, these behavioral alterations were also improved by amphetamine. Our study further revealed that impulsivity and attention deficit were associated with disruption of PFC volume and dopamine (DA) receptor expression, respectively. In contrast, hyper-locomotion was associated with decreased DA transporter expression in the NAcc. We also found that neonatal administration of nicotine into the habenula of neonatal brains produced selective lesion of the medial habenula. Behavioral deficits with neonatal nicotine administration were similar to those caused by ibotenic acid lesion of both medial and lateral habenula during the juvenile period, whereas they were different in adulthood.Because of similarity between behavioral and brain alterations caused by neonatal insults in the habenula and the symptoms and suggested neuropathology in attention deficit/hyperactivity disorder (ADHD), these results suggest that neurodevelopmental deficits in the habenula and the consequent cortico-striatal dysfunctions may be involved in the pathogenesis and pathophysiology of ADHD

Low catalytic activity is insufficient to induce disease pathology in triosephosphate isomerase (TPI) deficiency

Triosephosphate isomerase (TPI) deficiency is a fatal genetic disorder characterized by hemolytic anemia and neurological dysfunction. Although the enzyme defect in TPI was discovered in the 1960s, the exact etiology of the disease is still debated. Some aspects indicate the disease could be caused by insufficient enzyme activity, whereas other observations indicate it could be a protein misfolding disease with tissue-specific differences in TPI activity. We generated a mouse model in which exchange of a conserved catalytic amino acid residue (isoleucine to valine, Ile170Val) reduces TPI specific activity without affecting the stability of the protein dimer. TPIIle170Val/Ile170Val mice exhibit an approximately 85% reduction in TPI activity consistently across all examined tissues, which is a stronger average, but more consistent, activity decline than observed in patients or symptomatic mouse models that carry structural defect mutant alleles. While monitoring protein expression levels revealed no evidence for protein instability, metabolite quantification indicated that glycolysis is affected by the active site mutation. TPIIle170Val/Ile170Val mice develop normally and show none of the disease symptoms associated with TPI deficiency. Therefore, without the stability defect that affects TPI activity in a tissue-specific manner, a strong decline in TPI catalytic activity is not sufficient to explain the pathological onset of TPI deficiency

Fluid challenges in intensive care: the FENICE study A global inception cohort study

Fluid challenges (FCs) are one of the most commonly used therapies in critically ill patients and represent the cornerstone of hemodynamic management in intensive care units. There are clear benefits and harms from fluid therapy. Limited data on the indication, type, amount and rate of an FC in critically ill patients exist in the literature. The primary aim was to evaluate how physicians conduct FCs in terms of type, volume, and rate of given fluid; the secondary aim was to evaluate variables used to trigger an FC and to compare the proportion of patients receiving further fluid administration based on the response to the FC.This was an observational study conducted in ICUs around the world. Each participating unit entered a maximum of 20 patients with one FC.2213 patients were enrolled and analyzed in the study. The median [interquartile range] amount of fluid given during an FC was 500 ml (500-1000). The median time was 24 min (40-60 min), and the median rate of FC was 1000 [500-1333] ml/h. The main indication for FC was hypotension in 1211 (59 %, CI 57-61 %). In 43 % (CI 41-45 %) of the cases no hemodynamic variable was used. Static markers of preload were used in 785 of 2213 cases (36 %, CI 34-37 %). Dynamic indices of preload responsiveness were used in 483 of 2213 cases (22 %, CI 20-24 %). No safety variable for the FC was used in 72 % (CI 70-74 %) of the cases. There was no statistically significant difference in the proportion of patients who received further fluids after the FC between those with a positive, with an uncertain or with a negatively judged response.The current practice and evaluation of FC in critically ill patients are highly variable. Prediction of fluid responsiveness is not used routinely, safety limits are rarely used, and information from previous failed FCs is not always taken into account

Characteristics of back pain in Polish youth depending on place of residence

Introduction and objective. Spinal pain (SP) has become a very common problem in contemporary societies and occurs in adults, youths, and even children. The aim of the study was to determine whether the prevalence and characteristics of SP, as well as the determinants leading to SP in Polish youths, vary depending on the place of residence. The determinants considered were out-of-school physical activity (PA) and physical work (PW), time spent at a computer and TV, and the level of psychosocial development expressed as the level of anxiety-trait. The restricting effect of the experienced SP on everyday functioning of youths was also analysed. Materials and method. A cross-sectional, population-based study comprising 502 village residents (VR) and 1,593 city residents (CR) aged 13–19, residing in 3 random Polish provinces. The tools used were a custom-designed survey and the State-Trait Anxiety Inventory (STAI) filled by students during classes. Results. The frequency and localisation of SP, the time and circumstances in which SP episodes occur, as well as the influence of the pain on everyday functioning, are the same in youths living in villages and youths living in cities. Slight differences in the correlation between the prevalence of SP and determinants of SP development related to lifestyle depending on the place of residence were observed. However, in both VR and CR the occurrence of SP is significantly correlated negatively with the level of psychosocial development. Conclusions. At present, the conditions of living in a rural and in an urban environment in Poland pose no specific threat determining the occurrence of SP in youths, and increasing the negative effect of the pain on the functioning of youths

Wplyw wybranych substancji chemicznych na akumulacje amoniaku i proliny w czasie starzenia cietych lisci funkii [Hosta 'Undulata Erromena']

W doświadczeniach określono wpływ pożywki standardowo stosowanej do przedłużania trwałości kwiatów ciętych cytrynian 8-hydroksychinoliny + 2% sacharoza (8HQC + 2% S) oraz benzyloadeniny (BA) i kwasu giberelinowego (GA₃) na zawartość amoniaku i proliny w czasie starzenia ciętych liści funkii ‘Undulata Erromena’. Wykazano, iż w czasie starzenia ciętych liści tego gatunku, przetrzymywanych w wodzie destylowanej, następuje prawie 2,5-krotny wzrost zawartości amoniaku, który pociąga za sobą prawie proporcjonalny, bo 2,4-krotny wzrost poziomu proliny. Poziom obu komponentów w liściach funkii uzależniony był jednak od rodzaju zastosowanej substancji chemicznej. Wstawienie liści bezpośrednio po zbiorze do pożywki standardowej spowodowało 4-krotny wzrost poziomu amoniaku i prawie 3-krotny proliny w stosunku do stanu wyjściowego. Zastosowanie BA w formie 24-godzinnego kondycjonowania powstrzymało wzrost zawartości obu komponentów w ciętych liściach funkii, utrzymując ich zawartość na poziomie zbliżonym do wyjściowego. Zawartość amoniaku i proliny była istotnie wyższa przy zastosowaniu GA₃ niż BA.The contents of ammonium and proline in ageing cut leaves of Hosta ‘Undulata Erromena’ affected by the standard preservative used to prolong cut flower longevity (composed of 8HQC 200 mg·dm⁻³ + sucrose 2%) and benzyladenine (BA) or gibberellic acid (GA₃) was examined. In leaves kept held in water the 2.5 fold increase in ammonium content was observed as compared to the initial level and almost a similar rise in proline content occurred in the senescing leaves. In leaves kept held in the preservative solution increase in ammonium and proline contents was 4-fold and 3-fold, respectively. BA application as a 24 hour puls treatment stopped both ammonium and proline accumulation, maintaining their contents at the initial level. BA prevented ammonium and proline accumulation more effectively than GA₃ did