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This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc 1 and b 6 f (Cyt bc 1 / b 6 f ) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cyts bc 1 / b 6 f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cyt bc 1 / b 6 f , combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cyts bc 1 / b 6 f . In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.

Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions., (© 2020 The Authors.)

Previously, we isolated 84 deletion mutants in Saccharomyces cerevisiae auxotrophic background that exhibited hypersensitive growth under high hydrostatic pressure and/or low temperature. Here, we observed that 24 deletion mutants were rescued by the introduction of four plasmids (LEU2, HIS3, LYS2, and URA3) together to grow at 25 MPa, thereby suggesting close links between the genes and nutrient uptake. Most of the highly ranked genes were poorly characterized, including MAY24/YPR153W. May24 appeared to be localized in the endoplasmic reticulum (ER) membrane. Therefore, we designated this gene as EHG (ER-associated high-pressure growth gene) 1. Deletion of EHG1 led to reduced nutrient transport rates and decreases in the nutrient permease levels at 25 MPa. These results suggest that Ehg1 is required for the stability and functionality of the permeases under high pressure. Ehg1 physically interacted with nutrient permeases Hip1, Bap2, and Fur4; however, alanine substitutions for Pro17, Phe19, and Pro20, which were highly conserved among Ehg1 homologues in various yeast species, eliminated interactions with the permeases as well as the high-pressure growth ability. By functioning as a novel chaperone that facilitated coping with high-pressure-induced perturbations, Ehg1 could exert a stabilizing effect on nutrient permeases when they are present in the ER.

The Transient Receptor Potential Vanilloid 2 (TRPV2) channel is a member of the temperature-sensing thermoTRPV family. Recent advances in cryo-electronmicroscopy (cryo-EM) and X-ray crystallography have provided many important insights into the gating mechanisms of thermoTRPV channels. Interestingly, crystallographic studies of ligand-dependent TRPV2 gating have shown that the TRPV2 channel adopts two-fold symmetric arrangements during the gating cycle. However, it was unclear if crystal packing forces played a role in stabilizing the two-fold symmetric arrangement of the channel. Here, we employ cryo-EM to elucidate the structure of full-length rabbit TRPV2 in complex with the agonist resiniferatoxin (RTx) in nanodiscs and amphipol. We show that RTx induces two-fold symmetric conformations of TRPV2 in both environments. However, the two-fold symmetry is more pronounced in the native-like lipid environment of the nanodiscs. Our data offers insights into a gating pathway in TRPV2 involving symmetry transitions., Competing Interests: LZ, AH, MB, SL No competing interests declared

Some effects of organophosphorus compounds (OPs) esters cannot be explained by action on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In previous studies, in membrane chicken brain fractions, four components (EPα, EPβ, EPγ and EPδ) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, mipafox, PMSF). EPγ is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPα PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPα is highly sensitive to mipafox and paraoxon, but is resistant to PMSF, and is spontaneously reactivated when inhibited with paraoxon. In this papers we shows that cholinesterase activities showed inhibition kinetic by PV, which does not fit with a competitive inhibition model when tested for the same experimental conditions used to discriminate the PVase components. Four enzymatic components (CP1, CP2, CP3 and CP4) were discriminated in cholinesterase activity in the membrane fraction according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA. Components CP1 and CP2 could be related to EPα as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of biological membranes and modulating critical signal transduction events. Omega-3 (n-3) long chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect against inflammatory diseases and enhance brain development and function. This had led to a marked increase in demand for fish and fish oils in human diets, supplements, and aquaculture and created a need for new, sustainable n-3 LC-PUFA sources. We have studied for the first time homogenous preparations of the membrane-type ω6 and ω3 fatty acid desaturases from the fungus Mortierella alpina, as a model system to produce PUFAs. These desaturases possess a di-iron metal center and are selective for 18:1 n-9 and 18:2 n-6 acyl-CoA substrates, respectively. Sequence alignments and membrane topology predictions support that these enzymes have unique cap regions that may include the rearrangement and repositioning of the active site, especially when compared to the mammalian stearoyl-coenzyme A desaturase-1 (SCD1) and the related sphingolipid α-hydroxylase (Scs7p) that act upon different substrates.

From bacteria to mammals, different phospholipid species are segregated between the inner and outer leaflets of the plasma membrane by ATP-dependent lipid transporters. Disruption of this asymmetry by ATP-independent phospholipid scrambling is important in cellular signaling, but its mechanism remains incompletely understood. Using MD simulations coupled with experimental assays, we show that the surface hydrophilic transmembrane cavity exposed to the lipid bilayer on the fungal scramblase nhTMEM16 serves as the pathway for both lipid translocation and ion conduction across the membrane. Ca 2+ binding stimulates its open conformation by altering the structure of transmembrane helices that line the cavity. We have identified key amino acids necessary for phospholipid scrambling and validated the idea that ions permeate TMEM16 Cl - channels via a structurally homologous pathway by showing that mutation of two residues in the pore region of the TMEM16A Ca 2+ -activated Cl - channel convert it into a robust scramblase.

Membrane-bound alcohol dehydrogenase (mADH) was overexpressed in Gluconobacter oxydans DSM 2003, and the effects on cell growth and glycolic acid production were investigated. The transcription levels of two terminal ubiquinol oxidases (bo 3 and bd) in the respiratory chain of the engineered strain G. oxydans-adhABS were up-regulated by 13.4- and 3.8-fold, respectively, which effectively enhanced the oxygen uptake rate, resulting in higher resistance to acid. The cell biomass of G. oxydans-adhABS could increase by 26%-33% when cultivated in a 7L bioreactor. The activities of other major membrane-bound dehydrogenases were also increased to some extent, particularly membrane-bound aldehyde dehydrogenase (mALDH), which is involved in the catalytic oxidation of aldehydes to the corresponding acids and was 1.26-fold higher. Relying on the advantages of the above, G. oxydans-adhABS could produce 73.3gl -1 glycolic acid after 45h of bioconversion with resting cells, with a molar yield 93.5% and a space-time yield of 1.63gl -1 h -1 . Glycolic acid production could be further improved by fed-batch fermentation. After 45h of culture, 113.8gl -1 glycolic acid was accumulated, with a molar yield of 92.9% and a space-time yield of 2.53gl -1 h -1 , which is the highest reported glycolic acid yield to date., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Acyl CoA:2-monoacylglycerol acyltransferase (MGAT)-2 has an important role in dietary fat absorption in the intestine. MGAT2 resides in the endoplasmic reticulum and catalyzes the synthesis of diacylglycerol which is then utilized as a substrate for triacylglycerol synthesis. This triacylglycerol is then incorporated into chylomicrons which are released into the circulation. In this study, we determined the membrane topology of human MGAT2. Protease protection experiments showed that the C-terminus is exposed to the cytosol, while the N-terminus is partially buried in the ER membrane. MGAT2, like murine DGAT2, was found to have two transmembrane domains. We also identified a region of MGAT2 associated with the ER membrane that contains the histidine-proline-histidine-glycine sequence present in all DGAT2 family members that is thought to comprise the active site. Proteolysis experiments demonstrated that digestion of total cellular membranes from cells expressing MGAT2 with trypsin abolished MGAT activity, indicating that domains that are important for catalysis face the cytosol. We also explored the role that the five cysteines residues present in MGAT2 have in catalysis. MGAT activity was sensitive to two thiol modifiers, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). Furthermore, mutation of four cysteines resulted in a reduction in MGAT activity. However, when the C-terminal cysteine (C334) was mutated, MGAT activity was actually higher than that of wild-type FL-MGAT2. Lastly, we determined that both transmembrane domains of MGAT2 are important for its ER localization, and that MGAT2 is present in mitochondrial-associated membranes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Scutia buxifolia is a tree native to South America and is used as a cardiotonic agent; however, this property has not been associated with a clear mechanism or a specific compound., Aim of the Study: Given the importance of Na(+),K(+)-ATPase as a drug target in the treatment of heart failure, this study aimed to investigate the possible inhibitory effect of S. buxifolia crude extract and fractions (dichloromethane, ethyl acetate, and butanolic fractions), and identified compounds with effects on the activity of Na(+),K(+)-ATPase in vitro., Materials and Methods: First, we characterized the crude extract and fractions by high-performance liquid chromatography, and then monitored their effects on the activity of Na(+),K(+)-ATPase obtained from heart muscle and brain membranes of adult male Wistar rats., Results: We identified gallic acid, chlorogenic acid, caffeic acid, rutin, quercitrin, quercetin, and ursolic acid in S. buxifolia stem bark and leaves; quercitrin and ursolic acid were the main compounds in the ethyl acetate and dichloromethane fractions from leaves and stem bark. The crude extract (3 and 30mg/ml), and the ethyl acetate and dichloromethane fractions (0.1 and 1mg/ml) of both the stem bark and leaves inhibited Na(+),K(+)-ATPase activity in heart and brain samples. We found that, of the identified compounds, only ursolic acid (0.1mg/ml) was able to diminish Na(+), K(+)-ATPase activity in heart and brain samples., Conclusions: These data indicated that the cardiotonic effects of S. buxifolia may be due to the inhibition of Na(+),K(+)-ATPase activity in heart muscle, supporting the popular use of this plant as a treatment for heart failure., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Oncogenic transformation is dependent on activated membrane-associated NADPH oxidase (NOX). However, the resultant extracellular superoxide anions are also driving the NO/peroxynitrite and the HOCl pathway, which eliminates NOX-expressing transformed cells through selective apoptosis induction. Tumor progression is dependent on dominant interference with intercellular apoptosis-inducing ROS signaling through membrane-associated catalase, which decomposes H2O2 and peroxynitrite and oxidizes NO. Particularly, the decomposition of extracellular peroxynitrite strictly requires membrane-associated catalase. We utilized small interfering RNA (siRNA)-mediated knockdown of catalase and neutralizing antibodies directed against the enzyme in combination with challenging H2O2 or peroxynitrite to determine activity and localization of catalase in cells from three distinct steps of multistage oncogenesis. Nontransformed cells did not generate extracellular superoxide anions and only showed intracellular catalase activity. Transformed cells showed superoxide anion-dependent intercellular apoptosis-inducing ROS signaling in the presence of suboptimal catalase activity in their membrane. Tumor cells exhibited tight control of intercellular apoptosis-inducing ROS signaling through a high local concentration of membrane-associated catalase. These data demonstrate that translocation of catalase to the outside of the cell membrane is already associated with the transformation step. A strong local increase in the concentration of membrane-associated catalase is achieved during tumor progression and is controlled by tumor cell-derived H2O2 and by transglutaminase.

The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Naturally occurring prenylated acylphloroglucinol derivatives are plant metabolites with diverse biological and pharmacological activities. Prenylation of acylphloroglucinols plays an important role in the formation of these intriguing natural products and is catalyzed in plants by membrane-bound enzymes. In this study, we demonstrate the prenylation of such compounds by a soluble fungal prenyltransferase AnaPT involved in the biosynthesis of prenylated indole alkaloids. The observed activities of AnaPT toward these substrates are much higher than that of a microsomal fraction containing an overproduced prenyltransferase from the plant hop.

The present study was undertaken to evaluate the cardioprotective role of (-)-epigallocatechin-gallate (EGCG) against Fluoride (F) induced oxidative stress mediated cardiotoxicity in rats. The animals exposed to F as sodium Fluoride (NaF) (25mg/kg BW) for 4 weeks exhibited a significant increase in the levels of cardiac troponins T and I (cTnT & I), cardiac serum markers, lipid peroxidative markers and plasma total cholesterol (TC), triglycerides (TG), phospholipids (PL), free fatty acids (FFA), low density lipoprotein cholesterol, very low density lipoprotein cholesterol as well as cardiac lipids profile (TC, TG and FFA) with the significant decrease of high density lipoprotein cholesterol and cardiac phospholipids. F intoxication also decreased the levels of mitochondrial enzymes such as ICDH, SDH, MDH, α-KGDH and NADH in the cardiac tissue of rats. The mitochondrial Ca(2+) ion level was also significantly reduced along with the significant decrease in the levels of enzymatic and non enzymatic antioxidants. Furthermore, F treatment significantly increased the DNA fragmentation, up regulate cardiac pro-apoptotic markers, inflammatory markers and down-regulate the anti-apoptotic markers in the cardiac tissue. Pre administration of EGCG (40mg/kg/bw) in F intoxicated rats remarkably recovered all these altered parameters to near normalcy through its antioxidant nature. Thus, results of the present study clearly demonstrated that treatment with EGCG prior to F intoxication has a significant role in protecting F-induced cardiotoxicity and dyslipidemia in rats., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

In contrast to soluble acyl-ACP desaturases from plants, little is known about the structure-guiding principle underlying substrate specificity and regioselectivity of membrane-bound acyl-CoA desaturases from animals, mainly due to lack of the three-dimensional structure information. Here we report identification of two homologous membrane-bound acyl-CoA Δ9 desaturases (ChDes9-1 and ChDes9-2) from the marine copepod Calanus hyperboreus that accumulates more than 90% of total storage lipids in the form of wax esters. ChDes9-2 is a common Δ9 desaturase with substrate specificity to long chain fatty acid 18:0, while ChDes9-1 is a new type of Δ9 desaturase introducing a Δ9 double bond into a wide range of very long chain fatty acids ranging from 20:0 to 26:0. Reciprocal domain swapping and site-directed mutagenesis guided by the membrane topology revealed that presence or absence of an amphipathic and bulky residue, tyrosine, in the middle of the second transmembrane domain was important in determining the substrate specificity of the two desaturases. To examine the mechanistic structure for the substrate specificity, tyrosine-scanning mutagenesis was employed to systematically substitute the residues in the transmembrane domain of the very long chain desaturase. The results showed that the transmembrane domain formed an α-helix structure probably involved in formation of the substrate-binding pocket and the corresponding residue of the tyrosine likely resided at the critical position within the pocket mediating the interaction with the substrates, thereby specifying the chain length of the substrates.

Background: P2Y receptor activation may link the effect of purines to increased maximal in vitro activity of the Na,K-ATPase in rat muscle. The hypothesis that a similar mechanism is present in human skeletal muscle was investigated with membranes from rat and human skeletal muscle., Results: Membranes purified from rat and human muscles were used in the Na,K-ATPase assay. Incubation with ADP, the stable ADP analogue MeS-ADP and UDP increased the Na+ dependent Na,K-ATPase activity in rat muscle membranes, whereas similar treatments of human muscle membranes lowered the Na,K-ATPase activity. UTP incubation resulted in unchanged Na,K-ATPase activity in humans, but pre-incubation with the antagonist suramin resulted in inhibition with UTP, suggesting that P2Y receptors are involved. The Na,K-ATPase in membranes from both rat and human could be stimulated by protein kinase A and C activation. Thus, protein kinase A and C activation can increase Na,K-ATPase activity in human muscle but not via P2Y receptor stimulation., Conclusion: The inhibitory effects of most purines (with the exception of UTP) in human muscle membranes are probably due to mass law inhibition of ATP hydrolysis. This inhibition could be blurred in rat due to receptor mediated activation of the Na,K-ATPase. The different effects could be related to a high density of ADP sensitive P2Y1 and P2Y13 receptors in rat, whereas the UTP sensitive P2Y11 could be more abundant in human. Alternatively, rat could possesses a mechanism for protein-protein interaction between P2Y receptors and the Na,K-ATPase, and this mechanism could be absent in human skeletal muscle (perhaps with the exception of the UTP sensitive P2Y11 receptor)., Perspective: Rat muscle is not a reliable model for purinergic effects on Na,K-ATPase in human skeletal muscle.

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity., (© 2013. Published by Elsevier B.V. All rights reserved.)

Intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca(2+)) channels, are most often studied through their reconstitution into planar lipid bilayers (also called black lipid membranes, or BLMs). General methods for making bilayers and for ion channel reconstitution into BLMs have been extensively detailed elsewhere; thus, here the focus is on specific details relevant for inositol(1,4,5)-trisphosphate receptor (InsP3R) recordings. These procedures describe how to perform single-channel recordings of native or recombinant InsP3Rs in BLMs. Similar procedures are used to study native or recombinant ryanodine receptors (RyanRs) in BLMs.

Proline utilization A (PutA) from Escherichia coli is a membrane-associated trifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate and moonlights as a transcriptional regulator. As a regulatory protein, PutA represses transcription of the put regulon, which contains the genes encoding PutA and the proline transporter PutP. The binding of proline to the proline dehydrogenase active site and the subsequent reduction of the flavin induce high affinity membrane association of PutA and relieve repression of the put regulon, thereby causing PutA to switch from its regulatory to its enzymatic role. Here, we present evidence suggesting that residues of the β3-α3 loop of the proline dehydrogenase domain (βα)8 barrel are involved in proline-mediated allosteric regulation of PutA-membrane binding. Mutation of the conserved residues Asp370 and Glu372 in the β3-α3 loop abrogates the ability of proline to induce functional membrane association. Both in vitro lipid/membrane binding assays and in vivo cell-based assays demonstrate that mutagenesis of Asp370 (D370N/A) or Glu372 (E372A) dramatically impedes PutA functional switching. The crystal structures of the proline dehydrogenase domain mutants PutA86-630D370N and PutA86-630D370A complexed with the proline analogue l-tetrahydro-2-furoic acid show that the mutations cause only minor perturbations to the active site but no major structural changes, suggesting that the lack of proline response is not due to a failure of the mutated active sites to correctly bind the substrate. Rather, these results suggest that the β3-α3 loop may be involved in transmitting the status of the proline dehydrogenase active site and flavin redox state to the distal membrane association domain.

The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265, was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.

The objectives of this study were to verify the presence of membranous vesicles (MV) in canine seminal plasma by mean of transmission electron microscopy (TEM), to describe the ultrastructural characteristics and to identify some enzymatic activity associated with them. Semen samples, collected by digital manipulation from dogs with proven fertility, were pooled and used for membrane vesicles preparation according to conventional procedures. TEM observations showed the existence of vesicular membranous structures of more or less spherical shape with different sizes. These vesicles were surrounded by a single-, double- or multiple-layered laminar membranes. The mean vesicle diameter was 117.6 ± 86.9 nm ranging from 24.4 to 716.6 nm. Enzyme activity determinations showed the presence of adenosine deaminase, 5'-nucleotidase, ADPase, ATPase, dipeptilpeptidase IV, alkaline phosphatase, total acid phosphatase and prostatic acid phosphatase, while the aminopeptidase activity was absent. In conclusion, results of this study, compatible with results from other mammals, showed for the first time the presence of MV, their ultrastructural and enzymatic characteristics in dog seminal plasma., (© 2012 Blackwell Verlag GmbH.)

Following conjugation with glutathione, xenobiotics are converted into cysteinylglycine conjugates, cysteine conjugates, and finally, mercapturic acids. The structural factors determining the activities of dipeptidases for the metabolism of toxicologically-relevant cysteinylglycine conjugates are not well understood. We purified porcine kidney cortex membrane dipeptidase (MDP) to homogeneity, via phosphatidylinositol-specific phospholipase C-mediated cleavage of the protein's membrane anchor and cilastatin affinity chromatography. The homodimeric structure of the MDP protein was confirmed by mass spectrometry. The cysteinylglycine conjugates of 1-(chloromethyl)naphthalene, 4-nitrobenzyl chloride, and 1-chloro-2,4-dinitrobenzene were synthesized and HPLC separation methods for their quantitation were developed. MDP catalyzed the hydrolysis of all three conjugates, but the rate of this activity was strongly dependent on the nature of the substituent on the cysteine sulfur atom.

Previous study has documented reduced alkaline phosphatase (ALP) activity in brush border membrane (BBM) isolated from renal cell carcinoma (RCC). Diminished activity of ALP is associated with alteration in both increased K(m) as well as decreased V(max) of enzyme suggests that there may be a change in the conformation of enzyme as well as decreased number of ALP active molecules. The present study was conducted to find out any role of BBM lipid composition and its fluidity in diminished activity of alkaline phosphatase in renal cell carcinoma. Total phospholipids and glycolipids were significantly augmented in BBM from RCC as compared to control. Fractional analysis of total phospholipids revealed significantly increased phosphatidylethanolamine. Decreased fractions of sphingomyelin and phosphatidylinositol were observed. Cholesterol-to-total phospholipid molar ratios in tumor BBM was a significantly lower in tumor BBM. A significant reduction in polarization and microviscosity was found in BBM from RCC. Therefore, we conclude that alteration in membrane lipid composition and fluidity may play a substantial role in reduced activity of ALP in RCC.

The rhizomes of Gastrodia elata have been used for the treatment of insomnia in oriental countries. N⁶-(4-hydroxybenzyl) adenine riboside (NHBA) was originally isolated from G. elata. For the first time we report a detailed study on the effects and mechanisms of NHBA on its sedative and hypnotic activity. Adenosine, an endogenous sleep factor, regulates sleep-wake cycle via interacting with adenosine A₁/A(2A) receptors. Using radioligand binding studies and cAMP accumulation assays, our results show that NHBA may be a functional ligand for the adenosine A₁ and A(2A) receptors. NHBA significantly decreases spontaneous locomotor activity and potentiates the hypnotic effect of sodium pentobarbital in mice. Sleep architecture analyses reveal that NHBA significantly decreases wakefulness time and increases NREM sleep times. However, NHBA does not affect the amount of REM sleep. Pretreatment with the adenosine A₁ receptor antagonist DPCPX or the A(2A) receptor antagonist SCH 58261 significantly reverses the increase in sleeping time induced by NHBA in sodium pentobarbital treated mice. Immunohistochemical studies show that NHBA increases c-Fos expression in GABAergic neurons of the ventrolateral preoptic area (VLPO), which suggests that NHBA activates the sleep center in the anterior hypothalamus. Altogether, these results indicate that NHBA produces significant sedative and hypnotic effects. Such effects might be mediated by the activation of adenosine A₁/A(2A) receptors and stimulation of the sleep center VLPO., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Multidrug resistance has for many years attracted attention of numerous investigators. Attempts have also been made to increase efficiency of anti-neoplastic therapy. For this reason, most of efforts have been devoted to analysing proteins engaged in the mechanism of multidrug resistance such as the N-glycosylated membrane protein glycoprotein P. Interestingly, glycosylation probably plays a significant role in the intracellular location and activity of modified proteins. Inhibitors of glycosylation have been demonstrated to alter the activity of glycoprotein P in various ways, depending on the cell line examined. These inhibitors markedly reduce multidrug resistance of cancer cells, thus promoting success of anti-neoplastic therapy. Here, we review the basic knowledge on N-glycosylation inhibitors, their effect on glycoprotein P and their therapeutic potential.

Particle-wear-induced inflammatory osteolysis remains a major problem for the long-term success of total joint arthroplasty. Previous studies have demonstrated that cyclooxygenase-2 (COX-2) is expressed abundantly in the tissue around a failed implant. However, the role of COX-2 in the development of particle-wear-induced osteoclastogenesis remains unclear. The aim of the study was to test the hypothesis that Dynastat, a COX-2 inhibitor, ameliorates particle-wear-induced inflammatory osteoclastogenesis through the down-regulation of the receptor activators of nuclear factor-κB (RANK) and nuclear factor-κB ligand (RANKL) expression in a murine osteolysis model. Titanium (Ti) particles were introduced into established air pouches in BALB/c mice, followed by the implantation of calvaria bone from syngeneic littermates. Dynastat was given to mice intraperitoneally 2 days before the introduction of Ti particles and maintained until the mice were sacrificed. Pouch tissues were collected 14 days after Ti inoculation for molecular and histological analysis. The results showed that Dynastat has more impact on Ti-particle-induced prostaglandin E(2) expression and less on the expression of interleukin-1β and tumor necrosis factor-α. Dynastat inhibited Ti-particle-induced osteoclastogenesis by reducing the gene activation of RANK and RANKL, and diminishing the RANKL expression in Ti-particle-charged pouches. Dynastat markedly reduced the number of tartrate-resistant acid-phosphatase-positive cells in pouch tissues stimulated by Ti particles. In conclusion, this study provides evidence that Dynastat can markedly inhibit Ti-particle-induced osteoclastogenesis by the down-regulation of RANK/RANKL in a murine air pouch model, and is a promising therapeutic candidate for the treatment of inflammatory osteolysis induced by wear particles., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

L-Dopa Decarboxylase is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of L-Dopa to dopamine. In this study, we investigated the cellular topology of the active human enzyme. Fractionation of membranes from human cell lines, of neural and non-neural origin, by temperature-induced phase separation in Triton X-114 resulted in the detection of DDC molecules in all separation phases. Solubilization of membrane-associated DDC was observed in a pH and time-dependent manner and was affected by divalent cations and protease inhibitors, suggesting the involvement of a possible release mechanism. The study of the biological properties and function of the solubilization phenomenon described here, as well as, the study of the membrane-associated enzyme could provide us with new information about the participation of the human L-Dopa decarboxylase in physiological and aberrant processes.

Ingestion of Cleistanthus collinus, a shrub native to South India, either intentionally or accidentally, is a common cause of death in the area. Consumption of a boiled decoction of leaves is highly toxic, but medical management of patients is mainly supportive because the molecular mechanisms of toxin action are unknown. Distal renal tubular acidosis is one of the symptoms of poisoning in patients and adenosine triphosphate (ATP) requiring proton pumps is important for acid secretion in the kidney. Hence, we hypothesized that these may be putative targets for C. collinus action and we tested this by exposing rat renal brush border membrane (BBM) as well as cultured kidney cells to a boiled decoction of C. collinus. Exposure to the C. collinus decoction resulted in significant inhibition of vacuolar type H(+)-ATPase (V-ATPase) activity in renal BBM as well as blocking of the proton pump in renal BBM vesicles. C. collinus decoction was also found to inhibit acidification of intracellular organelles in cells in culture, similar to the effect seen with either bafilomycin or concanamycin - specific inhibitors of the V-ATPase. This was accompanied by a decrease in V-ATPase activity, but an increase in protein levels. These results demonstrate that the V-ATPase in renal cells is a putative target for the toxins in C. collinus and the inhibition of this important proton pump probably plays a role in the development of distal renal tubular acidosis and subsequent renal failure seen in poisoned patients.

Dipeptidyl peptidase IV is an ectopeptidase with multiple physiological roles including the degradation of incretins, and a target of therapies for type 2 diabetes mellitus. Divalent cations can inhibit its activity, but there has been little effort to understand how they act. The intact membrane-bound form of porcine kidney dipeptidyl peptidase IV was purified by a simple and fast procedure. The purified enzyme hydrolyzed Gly-Pro-p-nitroanilide with an average V(max) of 1.397±0.003 μmol min(-1) mL(-1), k(cat) of 145.0±1.2 s(-1), K(M) of 0.138±0.005 mM and k(cat)/K(M) of 1050 mM(-1) s(-1). The enzyme was inhibited by bacitracin, tosyl-L-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor L-threo-Ile-thiazolidide (K(i) 70 nM). The enzyme was inhibited by the divalent ions Ca(2+), Co(2+), Cd(2+), Hg(2+) and Zn(2+), following kinetic mechanisms of mixed inhibition, with K(i) values of 2.04×10(-1), 2.28×10(-2), 4.21×10(-4), 8.00×10(-5) and 2.95×10(-5) M, respectively. According to bioinformatic tools, Ca(2+) ions preferentially bound to the β-propeller domain of the porcine enzyme, while Zn(2+) ions to the α-β hydrolase domain; the binding sites were strikingly conserved in the human enzyme and other homologues. The functional characterization indicates that porcine and human homologues have very similar functional properties. Knowledge about the mechanisms of action of divalent cations may facilitate the design of new inhibitors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Blood hemostasis is accomplished by a complex network of (anti-)coagulatory and fibrinolytic processes. These physiological processes are implemented by the assembly of multiprotein complexes involving both humoral and cellular components. Coagulation factor X, and particularly, factor IX, exemplify the dramatic enhancement that is obtained by the synergistic interaction of cell surface, inorganic and protein cofactors, protease, and substrate. With a focus on structure-function relationship, we review the current knowledge of activity modulation principles in the coagulation proteases factors IX and X and indicate future challenges for hemostasis research. This chapter is organized by describing the principles of hierarchical activation of blood coagulation proteases, including endogenous and exogenous protease activators, cofactor binding, substrate specificities, and protein inhibitors. We conclude by outlining pharmaceutical opportunities for unmet needs in hemophilia and thrombosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The significance of ErbB4 in tumor biology is poorly understood. The ERBB4 gene is alternatively spliced producing juxtamembrane (JM-a and JM-b) and cytoplasmic (CYT-1 and CYT-2) isoforms. Here, signaling via the two alternative ErbB4 JM isoforms (JM-a CYT-2 and JM-b CYT-2) was compared. Fibroblasts expressing ErbB4 JM-a demonstrated enhanced ErbB4 autophosphorylation, growth, and survival. In contrast, cells overexpressing ErbB4 JM-b underwent starvation-induced death. Both pro- and antisurvival responses to the two ErbB4 isoforms were sensitive to an ErbB kinase inhibitor. Platelet-derived growth factor receptor-alpha (PDGFRA) was identified as an ErbB4 target gene that was differentially regulated by the two ErbB4 isoforms. The soluble intracellular domain of ErbB4, released from the JM-a but not from the JM-b isoform, associated with the transcription factor AP-2 and promoted its potential to enhance PDGFRA transcription. Survival of cells expressing JM-a was suppressed by targeting either PDGFR-α or AP-2, whereas cells expressing JM-b were rescued from cell death by the PDGFR-α agonist, PDGF-BB. These findings indicate that two alternative ErbB4 isoforms may promote antagonistic cellular responses and suggest that pharmacological inhibition of ErbB4 kinase activity may lead to either suppression or promotion of cellular growth.

An optical biosensor based on the eggshell membrane immobilized tyrosinase is described for the detection of L-tyrosine (L-Tyr). The detection scheme was based on the measurement of absorption value of color adduct resulting from the reaction of 3-methyl-2-benzothiazolinone hydrazone and dopa-quinone produced from the enzymatic oxidation of L-Tyr. The prepared biosensor demonstrated optimum activity at pH 7, optimum temperature range of 20-40 degrees C and a linear response for the L-Tyr concentration in range of 5-200 microM. It also showed good operation stability for repeated measurements (over 300 times) and storage stability after it had been kept at 4 degrees C for 3 months.

The cyclization of lycopene generates provitamin A carotenoids such as beta-carotene and paves the way toward the formation of cyclic xanthophylls playing distinct roles in photosynthesis and as precursors for regulatory molecules in plants and animals. The biochemistry of lycopene cyclization has been enigmatic, as the previously proposed acid-base catalysis conflicted with the possibility of redox catalysis as predicted by the presence of a dinucleotide binding site. We show that reduced FAD is the essential lycopene cyclase (CrtY) cofactor. Using flavin analogs, mass spectrometry, and mutagenesis, evidence was obtained based on which a catalytic mechanism relying on cryptic (net) electron transfer can be refuted. The role of reduced FAD is proposed to reside in the stabilization of a transition state carrying a (partial) positive charge or of a positively charged intermediate via a charge transfer interaction, acid-base catalysis serving as the underlying catalytic principle. Lycopene cyclase, thus, ranks among the novel class of non-redox flavoproteins, such as isopentenyl diphosphate:dimethylallyl diphosphate isomerase type 2 (IDI-2) that requires the reduced form of the cofactor.

Aims: P-glycoprotein (P-gp), one of the important drug-efflux pumps, is known to be affected by pathological conditions such as inflammation or infection. Recently, it is reported that high glucose or hyperglycemia can alternate P-gp expression levels at the blood-brain barrier or in the kidney, although the details are still unknown. Here, we analyzed the alteration of intestinal P-gp expression and function in the development of diabetes and elucidated the mechanisms., Main Methods: Type 1 diabetes was induced in male ddY mice by an i.p. injection of streptozotocin (STZ) (230 mg/kg). We analyzed ileal P-gp expression and function using Western blot analysis and an in situ closed loop method, respectively., Key Findings: A significant reduction of P-gp expression level in ileum was found 9 days after STZ administration. In contrast, a remarkable decrease in P-gp function was observed on the 3rd and 9th days. Interestingly, nitric oxide synthase (NOS) activity in ilea was significantly increased on the 9th day. The decrease of P-gp expression levels observed on the 9th day was completely suppressed by L-N(G)-nitroarginine methyl ester (L-NAME), a broad range NOS inhibitor, or aminoguanidine, a specific inducible NOS (iNOS) inhibitor., Significance: These results indicate the possibility that nitric oxide (NO), produced by iNOS in the ileum, is involved in the reduction of ileal P-gp expression under STZ-induced diabetic conditions., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Detergent-resistant membranes (DRMs) are a class of specialized microdomains that compartmentalize several signal transduction processes. In this work, DRMs were isolated from cerebral cortex synaptic endings (Syn) on the basis of their relative insolubility in cold Triton X-100 (1%). The lipid composition and marker protein content were analyzed in DRMs obtained from adult and aged animals. Both DRM preparations were enriched in Caveolin, Flotillin-1 and c-Src and also presented significantly higher sphingomyelin (SM) and cholesterol content than purified Syn. Total phospholipid-fatty acid composition presented an increase in 16:0 (35%), and a decrease in 20:4n-6 (67%) and 22:6n-3 (68%) content in DRM from adults when compared to entire synaptic endings. A more dramatic decrease was observed in the 20:4n-6 and 22:6n-3 content in DRMs from aged animals (80%) with respect to the results found in adults. The coexistence of phosphatidylcholine-specific-phospholipase C (PC-PLC) and phospholipase D (PLD) in Syn was previously reported. The presence of these signaling pathways was also investigated in DRMs isolated from adult and aged rats. Both PC-PLC and PLD pathways generate the lipid messenger diacylglycerol (DAG) by catalyzing PC hydrolysis. PC-PLC and PLD1 localization were increased in the DRM fraction. The increase in DAG generation (60%) in the presence of ethanol, confirmed that PC-PLC was also activated when compartmentalized in DRMs. Conversely, PLD2 was excluded from the DRM fraction. Our results show an age-related differential fatty acid composition and a selective localization of PC-derived signaling in synaptic DRMs obtained from adult and aged rats., (Copyright 2010 Elsevier B.V. All rights reserved.)

Cardiovascular diseases are emerging as a major public health problem in most parts of the world even in developing countries still afflicted by infectious diseases, undernutrition, and other illnesses related to poverty. In the present study, we investigated the protective effect of betaine, a potent lipotropic molecule, on changes in the levels of membrane-bound ATPase activities, lipid peroxidation, sulfhydryl activities, and mineral status in isoprenaline-induced myocardial infarction in Wistar rats, an animal model of myocardial infarction in man. Oral administration of betaine (250 mg/kg body weight/day for a period of 30 days) significantly (p < 0.05) reduced the isoprenaline-induced abnormalities noted in the levels of sodium, potassium, and calcium in plasma and heart tissue. Pretreatment with betaine significantly attenuated isoprenaline-induced membrane-bound ATPase depletion in the heart tissue and preserved the myocardial membrane-bound ATPase activities at levels comparable to that of control rats. Oral administration of betaine significantly attenuated the isoprenaline-altered sulfhydryl groups in the heart tissue and preserved the myocardial sulfhydryl activities at levels comparable to that of control rats. It also significantly counteracted the isoprenaline-mediated lipid peroxidation and maintained the level at near normal. In the results of the present study, betaine administration significantly prevented the isoprenaline-induced alterations in the activities of membrane-bound ATPases, lipid peroxides, myocardial sulfhydryl levels, and maintained the mineral status at near normal.

We investigated a change in tissue fluid osmolality and developmental sequences of mitochondria-rich (MR) cells during embryonic and larval stages of Mozambique tilapia, Oreochromis mossambicus, developing in freshwater. Tissue osmolality, representing body fluid osmolality, ranged from 300 to 370 mOsm/kg during embryonic and larval stages. This suggests that tilapia embryos and larvae are also able to regulate body fluid osmolality to some extent, although the levels are somewhat higher and fluctuate more greatly in embryos and larvae than in adults. Na(+)/K(+)-ATPase-immunoreactive MR cells were first detected in the yolk-sac membrane 3 days before hatching (day -3), followed by their appearance in the body skin on day -2. Subsequently, MR cells in both the yolk-sac membrane and body skin increased in number, and most densely observed on days -1 and 0. Whereas yolk-sac and skin MR cells decreased after hatching, MR cells in turn started developing in the gills after hatching. Thus, the principal site for MR cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gills during larval stages, and tilapia could maintain continuously their ion balance through those MR cells during early life stages.

Chloroperoxidase (CPO) from Caldariomyces fumago was optimally covalently immobilized on chitosan membranes pretreated with 0.8 M glutaraldehyde at pH 3.5 to give 3.18 mg CPO g(-1) support. Using monochlorodimedone (MCD) as assay substrate, the immobilized-CPO retained 40% activity at 50 degrees C after 40 min whereas free CPO retained only 0.02%. The residual activity for immobilized-CPO was 99 and 58% compared with 68 and 43% for free CPO in the presence of 1.5 M urea and 300 microM H(2)O(2), respectively, after 20 h.

A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.

Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.

Purpose: The objective of this study was to investigate the expression of cyclooxygenase (COX)-2 in human choroidal neovascular membranes., Methods: Paraffin-embedded sections of choroidal neovascular membranes excised from 16 patients with wet age-related macular degeneration were used for this study. Sections were subjected to immunohistochemistry using a monoclonal mouse antihuman COX-2 antibody. Staining was classified as either negative or positive in retinal pigment epithelial cells, vascular endothelial cells, and fibroblasts. Serial sections were stained for vimentin expression to confirm tissue antigenicity., Results: Eleven of 16 (69%) choroidal neovascular membranes stained positive for COX-2 in retinal pigment epithelial cells, with 6 (38%) of these also expressing COX-2 in vascular endothelial cells and 6 (38%) in fibroblasts. None of the sections that were negative for COX-2 in the retinal pigment epithelial cells showed COX-2 expression in the other cell types assessed. There was a statistically significant difference (P = 0.0097) in the mean ages between the COX-2 positive group (65.6 years) and COX-2 negative group (76.8 years) as determined by a two-tailed, unpaired Student's t-test., Conclusion: The expression of COX-2 in human choroidal neovascular membranes suggests a possible role for this modulator in age-related macular degeneration pathogenesis. The age-dependent expression observed is novel and warrants further investigation.

The significance of the coexistence of 2 mannose-6-phosphate receptors (MPRs) in most cell types still remains poorly understood. In this study, expression of the cation-dependent MPR (CD-MPR) and the cation-independent MPR (CI-MPR) was measured by Western blot in rat organs at 3 ages, i.e. in newborn and 10- and 90-day-old rats. It was observed that expression of the CI-MPR tends to diminish from newborns to adults in 5 of the 6 organs studied, whereas the CD-MPR did not show a clear tendency over time. In pancreas, conversely, both MPRs increased progressively from newborns to adults. The activity of 2 acid hydrolases was also measured at the different ages, and a low correlation was found with the expression of the 2 MPRs. With the exception of the pancreas, it is possible that the CI-MPR is mostly occupied with the clearance of insulin-like growth factor-II at early stages of development, and that later both MPRs may participate in the maturation of the lysosomal apparatus. We propose that in the pancreas, both receptors may be involved in increasing the proteolytic activity of this exocrine gland during postnatal development., ((c) 2008 S. Karger AG, Basel.)

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn(2+). Inhibition of liposome-bound bovine cytochrome oxidase by external Zn(2+) titrates with a K(i) of 1+/-0.3 microM. Presumably, the Zn(2+)-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu(B) is tentatively proposed to be responsible for the effect.

Inhibition of cytochrome P450s (CYPs) is a major cause of adverse drug-drug interactions. Alternatively, inhibition of glutathione S-transferases (GSTs) may increase harmful effects of electrophilic compounds or metabolites. In the present study, aqueous extracts of seven Ghanaian medicinal plants were investigated for their inhibitory potential towards recombinant human CYP1A2, CYP2C9, CYP2D6 and CYP3A4, heterologously expressed in Escherichia coli. Effects of these extracts on recombinant human GSTA1-1, GSTM1-1, GSTP1-1, human and rat cytosolic GSTs were also investigated. Seven extracts, including Phyllanthus amarus whole plant, leaf, stem and root, Cassia siamea and Momordica charantia, inhibited CYP1A2 and CYP2C9 with IC50 values ranging between 28.3-134.3microg/ml and between 63.4-425.9microg/ml, respectively. Similarly, both CYP2D6 and CYP3A4 were inhibited by five extracts including Phyllanthus amarus whole plant, leaf, stem and root and Cassia alata, with IC50 values ranging between 45.8-182.0microg/ml and between 79.2-158.8microg/ml respectively. Human and rat liver cytosolic GSTs were inhibited with IC50 values ranging between 25.2-95.5microg/ml and between 8.5-139.4microg/ml, respectively. GSTM1-1 was most susceptible to the inhibition by the extracts, with IC50 values ranging between 3.6-50.0microg/ml, whilst IC50 values of 8.9-159.0microg/ml and 68.6-157.0microg/ml were obtained for GSTA1-1 and GSTP1-1, respectively. These findings show a significant potential both for CYP- and GST-mediated herb-drug interactions of the Ghanaian medicinal plants investigated.

Out of six classes of adenylyl cyclases all class III enzymes convert ATP to cAMP in a catalytic centre moulded into an interface of bacterial homodimers and eukaryotic (pseudo)heterodimers. Formation of the catalytic centre, therefore, requires meticulous coordination of catalytic amino acids. Regulation of adenylyl cyclase activity via subtle or profound reorientation processes within the dimer interface is demonstrated on the basis of four class III adenylyl cyclase structures, a mammalian heterodimer, a mycobacterial holoenzyme that is pH regulated, another mycobacterial isoform that reorients upon substrate binding and a cyanobacterial cyclase activated by bicarbonate. Thus the interface of class III adenylyl cyclases is a like scaffold used in the regulation of activity by intrinsic and extrinsic signaling modules.

Endogenous polyamines mediate acute metabolic effects and cardiac hypertrophy associated to beta-adrenoceptor stimulation. The aim of this study is to characterize the role of polyamines on beta-adrenoceptor system mediated responses. To this end, the functional interaction of polyamine modifying drugs on isoproterenol-elicited cardiotonic effect, in isolated left atria of male Wistar rats, and their effects on [(3)H]dihydroalprenolol (DHA) binding on beta-adrenoceptors and on adenylyl cyclase activity of membrane heart were studied. Polyamines interact with beta-adrenoceptors in rat heart, as shown by the displacement of [(3)H]DHA binding. Furthermore, putrescine (but not spermidine or spermine) increased adenylyl cyclase activity, elicited a positive inotropism and increased intracellular cAMP. The putrescine effect on adenylyl cyclase was not antagonized by the beta-adrenoceptors blockers, alprenolol and ICI-118,551, and facilitated the isoproterenol effect. Neither alprenolol, atenolol nor ICI-118,551 antagonized putrescine-elicited positive inotropism. However, the effect was abolished in preparations with desensitized beta-adrenoceptors. alpha-Difluoromethylornithine, an inhibitor of ornithine decarboxylase, antagonized the effect of isoproterenol on inotropism and cAMP increase. In addition, putrescine might elicit effects by mechanisms independent of beta-adrenoceptor system, since in left atria with functional desensitized receptors an interaction with ouabain-elicited cardiotonic effect was observed. These results suggest that putrescine may act as a low affinity agonist on beta-adrenoceptors and modulate acute responses mediated by beta-adrenoceptors. These findings may be of importance in the physiology and in diseases involving cardiac beta-adrenoceptors.

Angiotensin-converting enzyme 2 (ACE2), a homologue of angiotensin-converting enzyme (ACE), converts angiotensin (Ang) I to Ang(1-9) and Ang II to Ang(1-7), but does not directly process Ang I to Ang II. Cardiac function is compromised in ACE2 null mice; however, the importance of ACE2 in the processing of angiotensin peptides within the murine heart is not known. We determined the metabolism of angiotensins in wild-type (WT), ACE (ACE(-/-)) and ACE2 null mice (ACE2(-/-)). Angiotensin II was converted almost exclusively to Ang(1-7) in the cardiac membranes of WT and ACE(-/-) strains, although generation of Ang(1-7) was greater in the ACE(-/-) mice (27.4 +/- 4.1 versus 17.5 +/- 3.2 nmol(-1) mg h(-1) for WT). The ACE2 inhibitor MLN4760 significantly attenuated Ang II metabolism and the subsequent formation of Ang(1-7) in both strains. In the ACE2(-/-) hearts, Ang II metabolism and the generation of Ang(1-7) were significantly attenuated; however, the ACE2 inhibitor reduced the residual Ang(1-7)-forming activity in this strain. Angiotensin I was primarily converted to Ang(1-9) (WT, 28.9 +/- 3.1 nmol(-1) mg h(-1); ACE(-/-), 49.8 +/- 5.3 nmol(-1) mg h(-1); and ACE2(-/-), 35.9 +/- 5.4 nmol(-1) mg h(-1)) and to smaller quantities of Ang(1-7) and Ang II. Although the ACE2 inhibitor had no effect on Ang(1-9) formation, the carboxypeptidase A inhibitor benzylsuccinate essentially abolished the formation of Ang(1-9) and increased the levels of Ang I in cardiac membranes. In conclusion, our studies in the murine heart suggest that ACE2 is the primary pathway for the metabolism of Ang II and the subsequent formation of Ang(1-7), a peptide that, in contrast to Ang II, exhibits both antifibrotic and antiproliferative actions.

A novel sesquiterpene furan compound CJ-01 was isolated from the methanol extract of the whole plant of Chloranthus japonicus SIEB. by monitoring the inhibitory activity of chitin synthase 2 from Saccharomyces cerevisiae. Based on spectroscopic analysis, the structure of compound CJ-01 was determined as 3,4,8a-trimethyl-4a,7,8,8a-tetrahydro-4a-naphto[2,3-b]furan-9-one. The compound inhibited chitin synthase 2 of Saccharomyces cerevisiae in a dose-dependent manner with an IC50 of 39.6 microg/ml, whereas it exhibited no inhibitory activities against chitin synthase 1 and 3 of S. cerevisiae up to 280 microg/ml. CJ-01 has 1.7-fold stronger inhibitory activity than polyoxin D (IC50=70 microg/ml), a well-known chitin synthase inhibitor. These results indicate that the compound is a specific inhibitor of chitin synthase 2 from S. cerevisiae. In addition, CJ-01 showed antifungal activities against various human and phytopathogenic fungi. Therefore, the compound might be an interesting lead to develop effective antifungal agents.

We identified and characterized a novel RING finger gene, Rines/RNF180, which is well conserved among vertebrates. Putative Rines gene product (Rines) contains a RING finger domain, a basic coiled-coil domain, a novel conserved domain (DSPRC) and a C-terminal hydrophobic region that is predicted to be a transmembrane domain. N-terminally epitope tagged-Rines (Nt-Rines) was detected in the endoplasmic reticulum membrane/nuclear envelope in cultured mammalian cells. Nt-Rines was not extracted by high salt or alkaline buffers and was degraded in intact endoplasmic reticulum treated with proteinase K, indicating that Nt-Rines is an integral membrane protein with most of its N-terminal regions in the cytoplasm. Rines was expressed in brain, kidney, testis and uterus of adult mice, and in developing lens and brain, particularly in the ventricular layer of the cerebral cortex at embryonic stages. In cultured cells, Nt-Rines can bind another protein and promoted its degradation. The degradation was inhibited by proteasomal inhibitors. In addition, Nt-Rines itself was heavily ubiquitinated and degraded by proteasome. The involvement of Rines in the ubiquitin-proteasome pathway was further supported by its binding to the UbcH6 ubiquitin-conjugating enzyme and by its trans-ubiquitination enhancing activities. These results suggest that Rines is a membrane-bound E3 ubiquitin ligase.

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.