The membrane form of acetylcholinesterase from rat brain contains a 20 kDa hydrophobic anchor

Rat brain acetylcholinesterase (AChE, EC 3.1.1.7) consists of about 80% amphiphilic detergent-soluble (DS-) AChE and 20% hydrophilic salt-soluble (SS-) AChE. DS-AChE contains about 65% tetrameric, 20% dimeric and 10% monomeric, SS-AChE about 40% tetrameric and 60% monomeric forms. N-terminal sequencing of DS- and SS-AChE gave identical N-termini corresponding to the published cDNA sequence of the mature enzyme. The band pattern on SDS-gels is similar to that of AChE from human and bovine brain. SDS-PAGE of hydrophobically labeled DS-AChE revealed the presence of a disulfide bonded hydrophobic membrane anchor of about 20 kDa. Monoclonal antibodies (mAbs) recognizing the anchor-containing subunits of mammalian brain DS-AChE, crossreacted with rat brain DS-AChE but not with SS-AChE. DS- and SS-AChE also reacted with antibodies raised against a peptide comprising the last 10 amino acids of the sequence of bovine brain AChE. Our results led us to conclude that both DS- and SS-AChE from rat brain contain T-type catalytic subunits, and DS-AChE in addition a P-type hydrophobic anchor similar to other mammalian brain DS-AChE.     

Estimating the distribution of the G(2) phase duration from flow cytometric histograms

A mathematical model, based on branching processes, is proposed to interpret BrdUrd DNA FCM-derived data. Our main interest is in determining the distribution of the G(2) phase duration. Two different model classes involving different assumptions on the distribution of the G(2) phase duration are considered. Different assumptions of the G(2) phase duration result in very similar distributions of the S phase duration and the estimated means and standard deviations of the G(2) phase duration are all in the same range.     

NIS/H2SO4-Silica: a mild and efficient reagent system for the hydrolysis of thioglycosides

Chemoselective hydrolysis of a variety of thioglycosides in the presence of a wide range of protecting groups has been achieved by using N-iodosuccinimide and H(2)SO(4) immobilized on silica in good to excellent yields.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein

We produced human leukotriene B(4) (LTB(4)) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB(4): (i) a low-affinity state (K(a)=7.8x10(8)M(-1)) that involves a receptor homodimer (BLT1.LTB(4))(2); we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (K(a)=1.3x10(10)M(-1)) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2). Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB(4), association with a G-protein and activation of Galpha. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1.LTB(4))(2):Galpha(i2)beta(1)gamma(2) pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB(4)-induced signal.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Fluxes of CO2, CH4 and N2O from a Welsh peatland following simulation of water table draw-down: Potential feedback to climatic change

A potential effect of climatic change was simulated by manipulating the water table height within intact peat monoliths. The treatment decreased methane flux (maximum –80%) and increased both carbon dioxide flux (maximum 146%) and nitrous oxide flux maximum 936%). Returning the water table height to its original level caused both nitrous oxide and carbon dioxide flux to rapidly return to control levels. However, methane flux remained at its experimentally induced low levels.     

Synthesis and SAR of a novel metabotropic glutamate receptor 4 (mGlu4) antagonist: unexpected 'molecular switch' from a closely related mGlu4 positive allosteric modulator

Herein we report the discovery and SAR of a novel antagonist of metabotropic glutamate receptor 4 (mGlu(4)). The antagonist was discovered via a molecular switch from a closely related mGlu(4) positive allosteric modulator (PAM). This antagonist (VU0448383) displays an IC(50) value of 8.2±0.4 μM and inhibits an EC(80) glutamate response by 63.1±6.6%.     

Characterization of the interaction of P1,P4-diadenosine 5'-tetraphosphate with luciferase

Adenylated dinucleotides (Ap(n)A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap(4)A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap(4)A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap(4)A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap(4)A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P(2) receptors, Ap(4)A also has a dual interaction with luciferase. Other Ap(n)As (n = 2, 5, and 6) also inhibited the ATP-luciferase interaction. Since Ap(n)As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap(n)A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles.     

Crystal structure of the platelet activator convulxin, a disulfide-linked alpha4beta4 cyclic tetramer from the venom of Crotalus durissus terrificus

Convulxin (CVX), a C-type lectin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, causes cardiovascular and respiratory disturbances and is a potent platelet activator which binds to platelet glycoprotein GPVI. The structure of CVX has been solved at 2.4A resolution to a crystallographic residual of 18.6% (R(free)=26.4%). CVX is a disulfide linked heterodimer consisting of homologous alpha and beta chains. The heterodimers are additionally linked by disulfide bridges to form cyclic alpha(4)beta(4)heterotetramers. These domains exhibit significant homology to the carbohydrate-binding domains of C-type lectins, to the factor IX-binding protein (IX-bp), and to flavocetin-A (Fl-A) but sequence and structural differences are observed in both the domains in the putative Ca(2+)and carbohydrate binding regions.     

Purification and characterization of a cytosolic Ca<Superscript>2+</Superscript>-independent phospholipase A<Subscript>2</Subscript> from bovine brain

The Ca²⁺-independent phospholipase A₂ (iPLA₂) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA₂ from bovine brain. iPLA₂ was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA₂ activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on twodimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF₃), Triton X-100, iron, and Ca²⁺. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA₂, and adenosine triphosphate (ATP). The spot with the iPLA₂ activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA₂.     

Evolution of C4 plants: a new hypothesis for an interaction of CO2 and water relations mediated by plant hydraulics

C(4) photosynthesis has evolved more than 60 times as a carbon-concentrating mechanism to augment the ancestral C(3) photosynthetic pathway. The rate and the efficiency of photosynthesis are greater in the C(4) than C(3) type under atmospheric CO(2) depletion, high light and temperature, suggesting these factors as important selective agents. This hypothesis is consistent with comparative analyses of grasses, which indicate repeated evolutionary transitions from shaded forest to open habitats. However, such environmental transitions also impact strongly on plant-water relations. We hypothesize that excessive demand for water transport associated with low CO(2), high light and temperature would have selected for C(4) photosynthesis not only to increase the efficiency and rate of photosynthesis, but also as a water-conserving mechanism. Our proposal is supported by evidence from the literature and physiological models. The C(4) pathway allows high rates of photosynthesis at low stomatal conductance, even given low atmospheric CO(2). The resultant decrease in transpiration protects the hydraulic system, allowing stomata to remain open and photosynthesis to be sustained for longer under drying atmospheric and soil conditions. The evolution of C(4) photosynthesis therefore simultaneously improved plant carbon and water relations, conferring strong benefits as atmospheric CO(2) declined and ecological demand for water rose.     

The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1-octylquinaldinium

The bovine heart mitochondrial F₁-ATPase (MF₁) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I_0.5 value of 48 μM. When irradiated in the presence of AOQ, MF₁ is photoinactivated with an apparent K_d of 12 μM. About 1.1 mol of [³H]AOQ were incorporated per mol of MF₁ on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF₁ photolabeled with [³H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with³H. Sequence analysis of the labeled peptides revealed that one contained residues 423–441 of the β subunit. A gap in position 2 of the sequence indicates that βPhe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342–358 of the β subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains βTyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that βTyr345 is labeled in this peptide.     

Rate of accumulation and emission of N2, N2O and CH4 from a flooded rice soil

In a field experiment using microplots, a flooded Crowley silt loam (Typic Albaqualfs) rice soil was fertilized with ¹⁵N labelled (60–74 atom %) urea and KNO₃. Emission of N₂, N₂O and CH₄ and accumulation in soil were measured for 21 d after fertilizer application.Emission of ¹⁵N₂-N measured from the urea and KNO₃ treated plots ranged from <15 to 570 and from 330 to 3,420 g ha^–1 d^–1, respectively. Entrapped ¹⁵N₂-N in the urea treated microplots was significantly lower (<15 g to 2.1 kg ha^–1) on all sampling dates compared to the ¹⁵N₂-N gas accumulation in the KNO₃ treated plots (6.4 to 31.5 kg ha^–1). Emissions of N₂O-N were low and did not exceed 4 g ha^–1 d^–1. Fluxes of CH₄ from the fertilizer and control plots were low and never exceeded 33 g ha^–1 d^–1. Maximum accumulation of CH₄ in the flooded soil measured 460 and 195 g ha^–1 for the urea and KNO₃ treatments, respectively.     

Photoaffinity labeling of the multidrug resistance protein 2 (ABCC2/cMOAT) with a photoreactive analog of LTC4

Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC₄); however direct binding of the LTC₄ to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC₄ (IAALTC₄) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC₄ in plasma membranes from MDCKII^MRP2 cells. The photoaffinity labeling signal of MRP2 with IAALTC₄ was much lower than that of MRP1, consistent with previous studies whereby the measured K_m values of MRP1 and MRP2 for LTC₄ were 1 μM and 0.1 μM LTC₄, respectively. Competition of IAALTC₄ photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 μM excess MK571. Interestingly, unmodified LTC₄ enhanced the photoaffinity labeling of IAALTC₄ to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC₄ binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC₄ binding to MRP2.     

Sequestering of As(III) and As(V) from wastewater using a novel neem leaves/MnFe2O4 composite biosorbent

An arsenic biosorbent comprising neem leaves (NL) and MnFe₂O₄ particles was developed and its removal potential was investigated. Physicochemical analysis of the NL/MnFe₂O₄ composite (MNL) was performed for the Brunauer, Emmett and Teller surface area, Fourier transform infrared spectra (FT-IR), and scanning electron microscopy–Energy-dispersive X-ray (EDX). The following parameters were optimized: pH, biosorbent dose, contact time, temperature, and initial arsenic concentration. The optimum pH values achieved for biosorption of As(III) and As(V) were 7.0 and 4.0, respectively, when the equilibrium time was 110 minutes for both. MNL was found to be efficient with 85.217% and 88.154% biosorption efficiency at a concentration of 50 mg/L of As(III) or As(V) solution, respectively. This was also proved by the FT-IR study of arsenic-loaded biosorbent. For establishing the best suitable correlation for the equilibrium curves exploiting the procedure of the nonlinear regression for curve fitting analysis, isotherm studies were conducted for As(III) and As(V) using 30 isotherm models. The pattern of biosorption fitted well with Brouers–Sotolongo isotherm model for As(III) and Langmuir–Freundlich as well as Sips isotherm models for As(V). Dubinin–Radushkevich (D-R) isotherm studies specified that ion exchange might play a significant role. The influence of various co-existing ions at different concentrations was examined. Desorption study was performed using various concentrations of NaOH solution.     

Membrane solubilization with and reconstitution from surfactant solutions: a comparison of phosphatidylserine and phosphatidylcholine interactions with octyl glucoside

Summary The acute effects of carbon monoxide (CO) on cardiac metabolism at the mitochondrial level were investigated. Rats were exposed to 4% CO for 4 minutes in a closed chamber. Immediately after exposure, hearts were removed and frozen with a precooled clamp. Blood from the thoracic cavity was sampled for analysis. Gas analysis of arterial blood taken from the abdominal aorta demonstrated severe hypoxia with oxygen partial pressure less than 20 mmHg, metabolic acidosis and reduced pH value. There were no significant changes in the plasma level of glucose and non esterified fatty acids (NEFA). In the myocardium, ATP levels decreased significantly, and concomitantly, a significant increase in the plasma uric acid level was observed. Although no significant change was observed in short chain acyl carnitine, free carnitine levels decreased to one fourth of the control value. Long chain acylcarnitine increased 11-fold. Coenzyme Q₉ (CoQ₉) level decreased significantly, but there was no significant change in Coenzyme Q₁₀ (CoQ₁₀).