Free AMP Research Articles

Longitudinal diffusion barriers imposed by myofilaments and mitochondria in murine cardiac myocytes.

Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases from 30 to 2,000, roughly as expected for pores with porin channel dimensions. Bodipy-FL ATP diffuses >40-fold slower than in free water at 25°C. From several fluorophores analyzed, bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. Unbound fluorophores diffuse at 0.5-8 × 10-7 cm2/s (5-80 μm2/s). Analysis of Na/K pump and veratridine-modified Na channel currents suggests that Na diffusion is nearly unrestricted at 35°C (time constant for equilibration with the pipette tip, ∼20 s). Using multiple strategies, we estimate that at 35°C, ATP diffuses four to eight times slower than in free water. To address whether restrictions are caused more by protein or membrane networks, we verified first that a protein gel, 10 g% gelatin, restricts diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is less restricted than in intact myocytes. Notably, myofilaments extracted similarly from skeletal (diaphragm) myocytes are less restrictive. Solute diffusion in myocytes with sarcolemma permeabilized by β-escin (80 µM) is similar to diffusion in intact myocytes. Restrictions are strain-dependent, being twofold greater in BL6 myocytes than in CD1/J6/129svJ myocytes. Furthermore, longitudinal diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, VDAC1, than in WT CD1/J6/129svJ myocytes. Thus, mitochondria networks restrict long-range diffusion while presumably optimizing nucleotide transfer between myofilaments and mitochondria. We project that diffusion restrictions imposed by both myofilaments and the outer mitochondrial membrane are important determinants of total free cytoplasmic AMP and ADP (∼10 μM). However, the capacity of diffusion to deliver ATP to myofilaments remains ∼100-fold greater than ATP consumption.

Hidden Pool of Cardiac Adenine Nucleotides That Controls Adenosine Production.

Myocardial ischemic adenosine production decreases in subsequent events that may blunt its protective functions. To test the relation between total or mitochondrial cardiac adenine nucleotide pool (TAN) on the energy status with adenosine production, Langendorff perfused rat hearts were subjected to three protocols: 1 min ischemia at 40 min, 10 min ischemia at 50 min, and 1 min ischemia at 85 min in Group I; additional infusion of adenosine (30 µM) for 15 min after 10 min ischemia in Group I-Ado, and 1 min ischemia at 40 and 85 min in the controls (Group No I). A 31P NMR and an HPLC were used for the analysis of nucleotide and catabolite concentrations in the heart and coronary effluent. Cardiac adenosine production in Group I measured after 1 min ischemia at 85 min decreased to less than 15% of that at 40 min in Group I, accompanied by a decrease in cardiac ATP and TAN to 65% of the initial results. Adenosine production at 85 min was restored to 45% of that at 40 min in Group I-Ado, accompanied by a rebound of ATP and TAN by 10% vs. Group I. Mitochondrial TAN and free AMP concentrations paralleled that of total cardiac TAN. Changes in energy equilibrium or mitochondrial function were minor. This study highlights that only a fraction of the cardiac adenine nucleotide pool is available for adenosine production, but further studies are necessary to clarify its nature.

AMP functionalized curdlan nanoparticles as a siRNA carrier: Synthesis, characterization and targeted delivery via adenosine A2B receptor

Receptor-mediated endocytosis has been used for tissue targeted delivery of short interfering RNA (siRNA) drugs. Herein, we investigated adenosine receptor (AR) as a candidate for receptor-mediated siRNA internalization. We synthesized adenosine functionalized cationic curdlan derivatives (denote CuAMP polymers). One of these polymers, CuAMP4, efficiently delivered siRNA to breast cancer cells expressing high level of A2B receptor. The internalization of siRNA loaded CuAMP4 by cancer cells was inhibited by free AMP as well as endocytosis inhibitors. Moreover, knockdown of A2BR by siRNA, or pre-treatment of the cells with anti-A2BR antibody, strongly inhibited the cellular uptake of CuAMP4. Our findings confirmed that A2BR can be utilized for cell type specific siRNA delivery, and CuAMP4 NP may be a promising delivery system for cancer cell targeted delivery of therapeutic siRNAs.

Lipid-Polymer Hybrid Nanoparticles Enhance the Potency of Ampicillin against Enterococcus faecalis in a Protozoa Infection Model.

Enterococcus faecalis (E. faecalis) biofilms are implicated in endocarditis, urinary tract infections, and biliary tract infections. Coupled with E. faecalis internalization into host cells, this opportunistic pathogen poses great challenges to conventional antibiotic therapy. The inability of ampicillin (Amp) to eradicate bacteria hidden in biofilms and intracellular niches greatly reduces its efficacy against complicated E. faecalis infections. To enhance the potency of Amp against different forms of E. faecalis infections, Amp was loaded into Lipid-Polymer hybrid Nanoparticles (LPNs), a highly efficient nano delivery platform consisting of a unique combination of DOTAP lipid shell and PLGA polymeric core. The antibacterial activity of these nanoparticles (Amp-LPNs) was investigated in a protozoa infection model, achieving a much higher multiplicity of infection (MOI) compared with studies using animal phagocytes. A significant reduction of total E. faecalis was observed in all groups receiving 250 μg/mL Amp-LPNs compared with groups receiving the same concentration of free Amp during three different interventions, simulating acute and chronic infections and prophylaxis. In early intervention, no viable E. faecalis was observed after 3 h LPNs treatment whereas free Amp did not clear E. faecalis after 24 h treatment. Amp-LPNs also greatly enhanced the antibacterial activity of Amp at late intervention and boosted the survival rate of protozoa approaching 400%, where no viable protozoa were identified in the free Amp groups at the 40 h postinfection treatment time point. Prophylactic effectiveness with Amp-LPNs at a concentration of 250 μg/mL was exhibited in both bacteria elimination and protozoa survival toward subsequent infections. Using protozoa as a surrogate model for animal phagocytes to study high MOI infections, this study suggests that LPN-formulated antibiotics hold the potential to significantly improve the therapeutic outcome in highly complicated bacterial infections.

Coadministration of liposomal methylglyoxal increases the activity of amphotericin B against Candida albicans in leukopoenic mice

In this study, we investigated the therapeutic efficacy of a combination of liposomal amphotericin B (Lip-Amp B) and Methylglyoxal (Lip-MG) against Candida albicans in the leukopoenic mice. The antifungal efficacy of Lip-Amp B or Lip-MG or a combination of Lip-Amp B and Lip-MG was evaluated by the analysis of the survival rate and the fungal load in the treated mice. The immune-stimulatory effect of Lip-MG on macrophages was evaluated by analysing the secretion of proinflammatory cytokines. C. albicans infected mice treated at the doses of 1 and 2 mg/kg of Lip-Amp B showed 20% and 50% survival rates, respectively. Whereas the mice treated with free Amp B at the same doses died within 40 days of treatment. Interestingly, C. albicans infected mice treated with a combination of Lip-Amp B and Lip-MG had 70% survival rate on day 40 postinfection. Moreover, treatment of macrophages with Lip-MG increased their fungicidal activity and the secretion of proinflammatory cytokines, including TNF-α and IL-1β. These findings suggested that co-treatment with Lip-Amp B and Lip-MG had a synergistic effect and could be effective against C. albicans in immunocompromised subjects.

Skeletal muscle AMPK is not activated during 2h of moderate intensity exercise at ∼65% in endurance trained men.

Key points AMP‐activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise.However, we previously showed that, although AMPK activity increases by 8–10‐fold during ∼120 min of exercise at ∼65% V˙O2peak in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study).In a cross‐sectional study, we show that there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% V˙O2peak in endurance‐trained individuals.These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% V˙O2peak in endurance trained men.It is important that more energy is directed towards examining other potential regulators of exercise metabolism. AMP‐activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise. Indeed, AMPK is activated during exercise and activation of AMPK by 5‐aminoimidazole‐4‐carboxyamide‐ribonucleoside (AICAR) increases skeletal muscle glucose uptake and fat oxidation. However, we have previously shown that, although AMPK activity increases by 8–10‐fold during ∼120 min of exercise at ∼65% V˙O2peak in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study). In a cross‐sectional study, we examined whether there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% V˙O2peak in endurance‐trained individuals. Eleven untrained (UT; V˙O2peak = 37.9 ± 5.6 ml.kg−1 min−1) and seven endurance trained (ET; V˙O2peak = 61.8 ± 2.2 ml.kg−1 min−1) males completed 120 min of cycling exercise at 66 ± 4% V˙O2peak (UT: 100 ± 21 W; ET: 190 ± 15 W). Muscle biopsies were obtained at rest and following 30 and 120 min of exercise. Muscle glycogen was significantly (P < 0.05) higher before exercise in ET and decreased similarly during exercise in the ET and UT individuals. Exercise significantly increased calculated skeletal muscle free AMP content and more so in the UT individuals. Exercise significantly (P < 0.05) increased skeletal muscle AMPK α2 activity (4‐fold), AMPK αThr172 phosphorylation (2‐fold) and ACCβ Ser222 phosphorylation (2‐fold) in the UT individuals but not in the ET individuals. These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% V˙O2peak in endurance trained men.

Amphotericin B Loaded Polymeric Nanoparticles for Treatment of Leishmania Infections.

Fungal infections in immune-compromised patients are an important cause of mortality and morbidity. Amphotericin B (Amp B) is considered a powerful fungicidal drug but its clinical usage has certain limitations when administered intravenously due to its toxicity and poor solubility. In consideration of such challenges, in cutaneous leishmaniasis, the topical application of Amp B can be a safer option in many aspects. Thus, herein, biopolymer of polycaprolactone (PCL) nanoparticles (NPs) were developed with the loading of Amp B by nanoprecipitation for the treatment of topical leishmanial infections. Various parameters, such as concentration of PCL and surfactant Poloxamer 407, were varied in order to optimize the formation of nanoparticles for the loading of Amp B. The optimized formulation exhibited a mean hydrodynamic particle size of 183 nm with a spherical morphology and an encapsulation efficiency of 85%. The applications of various kinetic models reveal that drug release from nanoformulation follows Korsmeyer–Peppas kinetics and has a high diffusion exponent at a physiological pH of 7.4 as well a skin relevant pH = 5.5. The activity of the prepared nanoparticles was also demonstrated in Leishmania infected macrophages. The measured IC50 of the prepared nanoparticle formulation was observed to be significantly lower when compared to control free Amp B and AmBisome® for both L. tropica KWH23 and L. donovani amastigotes in order to demonstrate maximum parasite inhibition. The prepared topical nanoformulations are capable of providing novel options for the treatment of leishmaniasis, which can be possible after in vivo assays as well as the establishment of safety profiles.

Glucose Metabolism in Cardiac Hypertrophy and Heart Failure.

Glucose Metabolism in Cardiac Hypertrophy and Heart Failure.

Abstract 3862: Empowering research on ubiquitin and ubiquitin-like protein modification cascade using recombinant enzyme systems

Abstract Protein modifications by the covalent attachment of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) such as SUMO, ISG15 or NEDD8 participate in many critical cellular processes. These include cell-cycle regulation, DNA repair, endocytosis, mitophagy, oncogenesis, post-membrane-protein trafficking, antiviral pathways and most notably, proteasomal degradation of target proteins. Ubiquitination and modification by UBLs share a similar catalytic cascade which requires the sequential action of three classes of enzymes: E1 activating enzymes, E2 conjugating enzymes and E3 ligases. While Ub/UBLs are activated by E1 through an ATP-dependent thioester bond formation and transferred to E2 from E1, the exact mechanism of how they are subsequently attached to a protein substrate depends on the type of E3's containing characteristic catalytic domain - RING, HECT or RBR. It has been generally accepted that E3's mediate substrate specificity and E2's are the main determinants for selection of the lysine to construct the modifier chains, which thereby directly control the cellular fate of the substrate. Recent research has linked dysregulation of the Ub/UBLs modification system to numerous diseases including cancer, immunological disorders and neurodegeneration. The high substrate specificity provided by combinations of over 30 E2's and over 600 E3's in the human genome makes these enzymes emerging drug targets. In response to a growing market demand, SignalChem has developed an extensive array of products encompassing enzymes, Ub/UBL modifiers and substrates in the ubiquitination, SUMOylation, ISGylation and NEDDylation processes. With Promega's AMP-GloTM reagents and an optimized assay protocol, we have identified and validated a variety of functionally competent combinations of these enzyme components. With the established protocol, each enzyme in the catalytic cascade has been assessed for their specific activity towards generation of free AMP. In addition, inhibition profiles of all three enzymes have been obtained using the assay system, further demonstrating their potential to be used in high-throughput screening to identify potential lead compounds for drug discovery and development programs. Citation Format: Eric Yao, Shenshen Lai, Jun Yan. Empowering research on ubiquitin and ubiquitin-like protein modification cascade using recombinant enzyme systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3862.

Comparing the therapeutic efficacy of different amphotericin B-carrying delivery systems against visceral leishmaniasis

Amphotericin B (Amp) has been well-successfully used to treat against Leishmania infection, although high toxicity has been found in patients. In the present study, Amp was administered in Leishmania infantum-infected BALB/c mice by three distinct delivery systems aiming to compare their efficacy against challenge infection, as well as their side effects in a murine visceral leishmaniasis (VL) model. This product was administered in a Poloxamer P407 (Pluronic® F127)-based polymeric micelle system (Amp/M), in the Ambisome® formulation (Lip-Amp) or in a free format (free Amp). Glucantime® (Gluc) was used as a comparative drug. Aiming to evaluate different endpoints of the treatments, the efficacy of the compounds was investigated one and 15-days after the therapeutic regimens, determining the parasite load by a limiting dilution assay and a quantitative PCR (qPCR) technique, as well as evaluating the immune response generated in the infected and treated animals. In the results, Amp/M or Lip-Amp-treated mice presented the best outcomes, since significant parasite load reductions were found in the evaluated organs, as well as a parasite-specific Th1 immune response was observed in the animals. In addition, no hepatic or renal damage was found in these mice. On the other hand, free Amp or Gluc induced toxicity in the animals, which was associated with a low Th1 immune response. Comparatively, Amp/M was the most effective drug in our experimental model, and results showed that the Amp-carrying system could be considered as a future alternative in studies against VL.

Real-time detection of carboplatin using a microfluidic system.

A microfluidic sensor system based on a carbon nanotube-epoxy composite electrode was fabricated to allow detection of the presence of the anti-cancer drug carboplatin in healthy tissue in real time during chemotherapy. Detection of carboplatin was carried out by observing the effects of the drug on the differential pulse voltammetry of free purine bases using a novel carbon nanotube-epoxy composite electrode. In free solution these electrodes performed better than glassy carbon electrodes for oxidation of the free purine bases AMP and GMP, and than DNA-modified carbon nanotube-epoxy composite sensors for detection of carboplatin. On-line carboplatin detection was performed using a computer-controlled microfluidic platform. The methodology for on-line carboplatin detection was optimised in terms of the analysis time and to allow repeated carboplatin measurement using the same electrode. Microdialysis sampling and our microfluidic platform were combined to give a proof-of-concept system for real-time carboplatin detection with a limit of detection of 0.014 μM carboplatin in the sampled media. This paper is dedicated to Craig Lunte's pioneering work in analysis and microdialysis.

Silver Nanoparticle‐Embedded Polymersome Nanocarriers for the Treatment of Antibiotic‐Resistant Infections

The rapid rise and prevalence of bacterial genetic resistance to antibiotics is having a drastic impact on human health today. This study explored the development and optimization of a polymersome nanocarrier formed from a biodegradable diblock copolymer to overcome such antibiotic resistance. Here, the polymersomes (Ps) were designed with silver nanoparticles embedded in the particle's hydrophobic membrane bilayer, and an ampicillin solution encapsulated in the particle's aqueous core in order to provide a dual-mechanism, concentrated, and less cytotoxic localized treatment. These silver nanoparticle-embedded polymersomes (AgPs) were subsequently tested for bactericidal function against Escherichia coli transformed with a gene for ampicillin resistance (bla). Results showed for the first time that the AgPs killed the antibiotic-resistant bacteria, whereas free antibiotic, encapsulated antibiotic without the addition of the silver nanoparticles, and AgPs without the addition of ampicillin did not (Fig 1). Figure 1. The growth kinetics of ampicillin-resistant E. coli was measured over 24 hours in the presence of differing concentrations of (A) AgPs loaded with 160µg/mL amp and (B) control tests of: AgPs synthesized from PBS, Ps synthesized from 1mg/mL amp, 1mg/mL free amp, and no treatment (pure PBS). (C) Transmission electron micrographs reveal that the AgPs orient “silver nanoparticle first” in order to interact with the bacterial outer membrane (white arrows). In this manner, this study introduces a novel nanomaterial that can effectively treat problematic, antibiotic-resistant infections in an improved capacity which should be further examined for a wide range of medical applications.

Bifunctional ADP-dependent phosphofructokinase/glucokinase activity in the order Methanococcales--biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis.

In some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a divalent metal cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg(2+) concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein–ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADP-dependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases.

Metformin Increases Mitochondrial Energy Formation in L6 Muscle Cell Cultures

A popular hypothesis for the action of metformin, the widely used anti-diabetes drug, is the inhibition of mitochondrial respiration, specifically at complex I. This is consistent with metformin stimulation of glucose uptake by muscle and inhibition of gluconeogenesis by liver. Yet, mitochondrial inhibition is inconsistent with metformin stimulation of fatty acid oxidation in both tissues. In this study, we measured mitochondrial energy production in intact cells adapting an in vivo technique of phosphocreatine (PCr) formation following energy interruption ("PCr recovery") to cell cultures. Metformin increased PCr recovery from either dinitrophenol (DNP) or azide in L6 cells. We found that metformin alone had no effect on cell viability as measured by total ATP concentration, trypan blue exclusion, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. However, treatments with low concentrations of DNP or azide reversibly decreased ATP concentration. Metformin increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction during recovery from either agent. Viability measured by trypan blue exclusion indicated that cells were intact under these conditions. We also found that metformin increased free AMP and, to a smaller extent, free ADP concentrations in cells, an action that was duplicated by a structurally unrelated AMP deaminase inhibitor. We conclude that, in intact cells, metformin can lead to a stimulation of energy formation, rather than an inhibition.

Characterization of AK2 Gene Function in Zebrafish Hematopoiesis

Abstract 2185 Objective:The Adenylate Kinase (AK) gene family consists of 7 different members (AK1-7) that contribute to energy metabolism of the cells by converting ATP (or GTP) and free AMP to ADP (or GDP) and free ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 is uniquely located in the mitochondrial intermembrane space and has been implicated in Caspase 10-mediated apoptosis, although the published data remain controversial. More recently, it was demonstrated that mutations of the AK2 gene cause reticular dysgenesis, an autosomal recessive form of severe combined immunodeficiency (SCID). Reticular dysgenesis is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation resulting in overwhelming infections and high lethality in affected patients. Moreover, patients commonly present with bilateral sensorineural deafness. The mechanisms underlying the biological consequences of AK2-defieincy remain unclear and the generation and characterization study of model systems is expected to provide useful insigths. Ak2 gene-targeted mice have not been reported in the literature, but lines carrying homozygous inactivating retroviral insertions have been shown to be embryonically lethal (our unpublished observations).Because of the known advantages of zebrafish as model system for developmental studies and the similarities of hematopoiesis in zebrafish and higher vertebrates, we set out to investigate the function of the zebrafish ak2 gene in development, with particular emphasis on hematopoiesis. Results:Similar to humans, we found that two different alternatively spliced isoforms of the ak2 gene (Isoform A and Isoform B) are expressed in zebrafish. By Real-Time PCR and In situ Hybridization (ISH) we analyzed the expression of both ak2 isoforms during embryo development. Preliminary data indicate that Isoform A is more abundantly represented than Isoform B during embryo development. ISH analysis showed that the two isoforms have different spatial expression patterns. These data suggest different functionalities for ak2 isoforms during embryo development.To explore such hypothesis, we injected two different morpholinos (MOs) targeting the ak2 isoforms. Downregulation of both ak2 isoforms phenocopied the human disease and resulted in a strong reduction of developing lymphocytes. Moreover we observed a hypochromic phenotype that also suggested impairment of the erythroid lineage. ISH experiments are underway to better define the affected hematopoietic lineages. Interestingly, ak2 MOs-injected embryos showed also developmental defects beyond the hematopoietic system, such as abnormal jaw development. Future studies will focus on the characterization of the specific function of the alternatively spliced ak2 isoforms. Conclusions:We show that the transcription features of the AK2 gene are conserved in zebrafish. The observed differential expression patterns of the zebrafish ak2 isoforms may provide new insights into the function of AK2 in the development of the hematopoietic system, as well as other organs and offers prospects for the understanding of the molecular mechanisms involved in reticular dysgenesis. Disclosures:No relevant conflicts of interest to declare.

Muscle metabolic, enzymatic and transporter responses to a session of prolonged cycling

A single session of prolonged work was employed to investigate changes in selected metabolic, transporter and enzymatic properties in muscle. Ten active but untrained volunteers (weight=73.9±4.2kg) with a peak aerobic power [Formula: see text] of 2.95±0.27lmin(-1), cycled for 2h at 62±1.3% [Formula: see text] Tissue extraction from the vastus lateralis occurred prior to (E1-Pre) and following (E1-Post) exercise and on 3 consecutive days of recovery (R1, R2, R3). The exercise resulted in decreases (P<0.05) in ATP (-9.3%) and creatine phosphate (-49%) and increases in lactate (+100%), calculated free ADP (+253%) and free AMP (+1,207%), all of which recovered to E1-Pre by R1. Glycogen concentration, which was depressed (P<0.05) by 75% at E1-Post, did not recover until R3. Compared to E1-Pre, the cycling also resulted in decreases (P<0.05) in the activities of cytochrome c oxidase, phosphorylase, and hexokinase but not in citrate synthase (CS) or 3-hydroxy-CoA dehydrogenase at E1-Post. With the exception of CS, which was elevated (P<0.05) at R3, all enzyme activities were not different from E1-Pre during recovery. For the glucose (GLUT1, GLUT4) and monocarboxylate (MCT1, MCT4) transporters, changes in expression levels (P<0.05) were only observed for GLUT1 at R1 (+42%) and R3 (+33%). It is concluded that the metabolic stress produced by prolonged exercise is reversed by 1day of recovery. One day of exercise also resulted in a potential upregulation in the citric acid cycle and glucose transport capabilities, adaptations which are expressed at variable recovery durations.

Synthesis and characterisation of two novel proton transfer compounds and their inhibition studies on carbonic anhydrase isoenzymes

Two novel proton transfer compounds were prepared between 2,4-dichloro-5-sulphamoylbenzoic acid (lasamide) (Hsba) and ethylenediamine (en), namely ethane-1,2-diaminium 2,4-dichloro-5-sulphamoylbenzoate (1), and also between Hsba and 2-amino-3-methylpyridine (2-amino-3-picoline) (amp), namely 2-amino-3-methylpyridinium 2,4-dichloro-5-sulphamoylbenzoate (2). All these were characterised by elemental, spectral (IR and UV-vis), thermal analyses, and single crystal X-ray diffraction studies. Compounds 1 and 2 crystallised in the P-1 and P21/c space groups, respectively. Intermolecular non-covalent interactions, such as ion pairing, hydrogen bonding, and π-π stacking were observed for these ionic compounds. The free ligands Hsba, en and amp, the products 1 and 2, and acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on the human carbonic anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cells by affinity chromatography for their hydratase and esterase activities. The half maximal inhibitory concentration (IC50) values for products 1 and 2 with respect to hydratase activity are 0.15 and 0.32 µM for hCA I and 0.06 and 0.15 µM for hCA II, respectively. The IC50 values of the same inhibitors for esterase activity are 0.13 and 0.8 µM for hCA I and 0.14 and 0.1 µM for hCA II, respectively. In relation to esterase activities, the inhibition equilibrium constants (Ki) were also determined and found to be 0.137 and 0.99 µM on hCA I and 0.157 and 0.075 µM on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of the newly synthesised compounds 1 and 2 to the parent compounds Hsba and amp and also to AAZ indicated that 1 and 2 have an effective inhibitory activity on hCA I and II, and might be used as potential inhibitors.

Synthesis, characterization and antiglaucoma activity of a novel proton transfer compound and a mixed-ligand Zn(II) complex

A novel proton transfer compound, pyridin-2-ylmethanaminium 2,4-dichloro-5-sulfamoylbenzoate (1), and a mixed-ligand Zn(II) complex, bis(2,4-dichloro-5-sulfamoylbenzoate)(2-aminomethylpyridine)aquazinc(II) monohydrate (2), have been synthesized from the same free ligands, which are 2,4-dichloro-5-sulfamoylbenzoic acid (Hsba) and 2-aminomethylpyridine (amp). They have been characterized by elemental, spectral ((1)H NMR, IR and UV-vis.) and thermal analyses. Additionally, magnetic measurement and single crystal X-ray diffraction technique were applied to compound 2. In the complex, Zn(II) ion exhibits a distorted octahedral configuration coordinated by O1 and O1(i) atoms of two mono dentante sba anions and N1, N2, N2(i) atoms of bidentante amp anion and a water molecule (O1w). The free ligands Hsba and amp, and the products 1 and 2, and acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibitor effects on human Carbonic Anhydrase isoenzymes (hCA I and hCA II) purified from erythrocyte cell by affinity chromatography for their hydratase and esterase activities. The IC(50) values of products 1 and 2 for hydratase activity are 0.26 and 0.13microM for hCA I and 0.30 and 0.15microM for hCA II, respectively. The IC(50) values of the same inhibitors for esterase activity are 0.32 and 0.045microM for hCA I and 0.29 and 0.23microM for hCA II, respectively. In relation to esterase activities, the inhibition equilibrium constants (K(i)) were also determined and found 0.25 and 0.058microM on hCA I and 0.22 and 0.24microM on hCA II for 1 and 2, respectively. The comparison of the inhibition studies of newly synthesized compounds 1 and 2 to parent compounds Hsba and amp and to AAZ indicated that 1 and 2 have effective inhibitory activity on hCA I and II, and might be used potential inhibitors.

Time-dependent effects of short-term training on muscle metabolism during the early phase of exercise

In this study, we investigated the hypothesis that the metabolic adaptations observed during steady-state exercise soon after the onset of training would be displayed during the nonsteady period of moderate exercise and would occur in the absence of increases in peak aerobic power (Vo2peak) and in muscle oxidative potential. Nine untrained males [age = 20.8 +/- 0.70 (SE) yr] performed a cycle task at 62% Vo2peak before (Pre-T) and after (Post-T) training for 2 h/day for 5 days at task intensity. Tissue samples extracted from the vastus lateralis at 0 min (before exercise) and at 10, 60, and 180 s of exercise, indicated that at Pre-T, reductions (P < 0.05) in phosphocreatine and increases (P < 0.05) in creatine, inorganic phosphate, calculated free ADP, and free AMP occurred at 60 and 180 s but not at 10 s. At Post-T, the concentrations of all metabolites were blunted (P < 0.05) at 60 s. Training also reduced (P < 0.05) the increase in lactate and the lactate-to-pyruvate ratio observed during exercise at Pre-T. These adaptations occurred in the absence of change in Vo2peak (47.8 +/- 1.7 vs. 49.2 +/- 1.7 mlxkg(-1)xmin(-1)) and in the activities (molxkg protein(-1)xh(-1)) of succinic dehydrogenase (3.48 +/- 0.21 vs. 3.77 +/- 0.35) and citrate synthase (7.48 +/- 0.61 vs. 8.52 +/- 0.65) but not cytochrome oxidase (70.8 +/- 5.1 vs. 79.6 +/- 6.6 U/g protein; P < 0.05). It is concluded that the tighter metabolic control observed following short-term training is initially expressed during the nonsteady state, probably as a result of increases in oxidative phosphorylation that is not dependent on changes in Vo2peak while the role of oxidative potential remains uncertain.

Magnesium dependence of the measured equilibrium constants of aminoacyl-tRNA synthetases

The apparent equilibrium constants ( K′) for six reactions catalyzed by aminoacyl-tRNA synthetases from Escherichia coli were measured, the equations for the magnesium dependence of the equilibrium constants were derived, and best-fit analyses between the measured and calculated values were used. The K′ values at 1 mM Mg 2+ ranged from 0.49 to 1.13. The apparent equilibrium constants increased with increasing Mg 2+ concentrations. The values were 2–3 times higher at 20 mM Mg 2+ than at 1 mM Mg 2+, and the dependence was similar in the class I and class II synthetases. The main reason for the Mg 2+ dependence is the existence of PP i as two magnesium complexes, but only one of them is the real product. AMP exists either as free AMP or as MgAMP, and therefore also has some effect on the measured equilibrium constant. However, these dependences alone cannot explain the measured results. The measured dependence of the K′ on the Mg 2+ concentration is weaker than that caused by PP i and AMP. Different bindings of the Mg 2+ ions to the substrate tRNA and product aminoacyl-tRNA can explain this observation. The best-fit analysis suggests that tRNA reacts as a magnesium complex in the forward aminoacylation direction but this given Mg 2+ ion is not bound to aminoacyl-tRNA at the start of the reverse reaction. Thus Mg 2+ ions seem to have an active catalytic role, not only in the activation of the amino acid, but in the posttransfer steps of the aminoacyl-tRNA synthetase reaction, too.