δ(13) C of leaf-respired CO(2) reflects intrinsic water-use efficiency in barley

Leaf intrinsic water-use efficiency (WUE), the ratio of photosynthetic rate to stomatal conductance (A/g(s) ), is a key plant trait linking terrestrial carbon and water cycles. A rapid, integrative proxy for A/g(s) is of benefit to crop breeding programmes aiming to improve WUE, but also for ecologists interested in plant carbon-water balance in natural systems. We hypothesize that the carbon isotope composition of leaf-respired CO(2) (δ(13) C(Rl) ), two hours after leaves are transferred to the dark, records photosynthetic carbon isotope discrimination and so provides a proxy for A/g(s) . To test this hypothesis, δ(13) C(Rl) was measured in four barley cultivars grown in the field at two levels of water availability and compared to leaf-level gas exchange (the ratio of leaf intercellular to ambient CO(2) partial pressure, C(i) /C(a) , and A/g(s) ). Leaf-respired CO(2) was more (13) C-depleted in plants grown at higher water availability, varied between days as environmental conditions changed, and was significantly different between cultivars. A strong relationship between δ(13) C(Rl) and δ(13) C of sucrose was observed. δ(13) C(Rl) was converted into apparent photosynthetic discrimination (Δ(13) C(Rl) ) revealing strong relationships between Δ(13) C(Rl) and C(i) /C(a) and A/g(s) during the vegetative stage of growth. We therefore conclude that δ(13) C(Rl) may provide a rapid, integrative proxy for A/g(s) in barley.     

Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor alpha

A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the covalent attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile covalent attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The covalent attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the covalent attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).     

Effects of cultivation practice on floristic and flowering diversity of spontaneously growing plant species on arable fields

In the past, the floristic diversity of arable fields has been described in terms of species diversity (SD) and their degree of coverage (C), but never in combination with the recording of the actually flowered species (FS) and their flowering intensity (FI) to striking differences in the cultivation methods on arable land. In relation to SD and C, however, FS and FI may provide important additional information on the functional biodiversity of fields. The aim was therefore to investigate the effects of (a) conventional, (b) organic, and (c) smallholder (never application of herbicides) on the floristic diversity. Using a region in Germany, we investigated SD, C, FS, and FI synchronously in (a), (b), and (c), by 356 vegetation surveys (5 × 5 m plots) conducted in spring and summer in 2019 in winter cereals. Statistical tests were used to analyze the differences between (a), (b), and (c). The medians were used to compare the floristic diversity of (a), (b), and (c) and finally relationships of FS and FI to SD were analyzed in relation to the cultivation methods. Significant differences in SD, C, FS, and FI were found between the (a), (b), and (c) in spring and summer characterized by sharp declines from (c) to (b) to (a). A drastic reduction in floristic diversity from (c) 100 to (b) 52 to (a) 3 was determined. Plants in flower (FS, FI) were very poorly in (a), moderately well to well in (b), and well to very well represented in (c). (C) to (a) was characterized by a sharp decline and from (a) to (b) by sharp increase in floristic diversity. With current acreage proportions of (a) in mind, this would affect, about one third of land area in Germany, associated with a drastic reduction in functional biodiversity for insects.     

A complete set of novel 2D correlation NMR experiments based on heteronuclear J-cross polarization

A suite of multiple-purpose sensitivity-enhanced 2D correlation NMR experiments based on heteronuclear J-cross polarization (HCP) techniques are introduced for isotropic liquid samples. Several pulse sequences using an adaptable heteronuclear TOCSY mixing building block are proposed for different types of effective coherence-order-selective (COS) heteronuclear coherence-transfer mechanisms. They are based on the anisotropic behaviour of the involved HCP process that is easily described and analysed in terms of cartesian product-operator formalism. A number of different versions are given for in-phase to in-phase (II-COS: S (-) \\ to I (-)), in-phase to anti-phase (IA-COS: S (-) \\ to 2 I (-) S (z)), in-phase to spin-state-selective (IS(3)-COS: S (-) \\ to 2 I (-) S (alpha /beta)), anti-phase to in-phase (AI-COS: 2 I (z) S (-) \\ to I (-)), anti-phase to anti-phase (AA-COS: 2 I (z) S (-) \\ to 2 I (-) S (z)), anti-phase to spin-state-selective (AS(3)-COS: 2 I (z) S (-) \\ to 2 I (-) S (alpha /beta)) and spin-state-selective to spin-state-selective (S(3)S(3)-COS: 2 I (alpha /beta) S (-) \\ to 2 I (-) S (alpha /beta )) coherence transfers. The combination of the echo/anti-echo approach, heteronuclear gradient echoes and the preservation of equivalent pathways (PEP) methodology affords a general approach to obtain sensitivity-enhanced pure-absorption 2D spectra that can be used as interesting alternatives to conventional pulse-interrupted free-precession INEPT-based pulse schemes, such as HSQC-type and TROSY-type experiments.     

Molecular mass and chain conformation of carboxymethylated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Seven water-insoluble (1 --> 3)-beta-D-glucan fractions TM8-1 to TM8-7 with weight-average molecular mass M(w) ranged from 2.22 to 77.4 x 10(4) obtained from the sclerotia of Pleurotus tuber-regium were carboxymethylated to produce the water-soluble fractions CTM8-1 to CTM8-7 with M(w) ranged from 3.87 to 87.8 x 10(4). The degree of substitution (DS) of CTM8 fractions was analyzed by ir and elemental analysis (EA) to be 0.3-0.68. The M(w) and the intrinsic viscosity [eta] of the CTM8 fractions were measured by size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), MALLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependencies of [eta] and radius of gyration (z) (1/2) on M(w) for the CTM8 samples were found to be [eta] = (8.82 +/- 0.03) x 10(-3) M(w)(0.78 +/- 0.04) (cm(3) g(-1)) and (z) (1/2) = (3.09 +/- 0.05) x 10(-3) M(w)(0.75 +/- 0.06) (nm) in the M(w) range from 3.87 x 10(4) to 53.2 x 10(4). Based on current theories for wormlike chain model, the conformational parameters of the CTM8 were obtained to be 790 (nm(-1)) for M(L), 9.6 (nm) for q, which were higher than those of the native TM8 fractions, suggesting a more extended flexible chain of CTM8 in PBS. On the whole, the CTM8 fractions showed higher antitumor activity than their corresponding TM8 fractions. In view of data from molecular parameters and bioactivity, the antitumor activity of the CTM8 fractions may be correlated to its water solubility and relatively extended chain.     

激活δ阿片受体可抑制大鼠心室肌细胞L-型钙电流和瞬时外向钾电流

本研究采用全细胞膜片钳技术观察了SNCl62(一种选择性δ阿片受体激动剂)对人鼠心室肌细胞L型钙电流(L-type Ca2 current,ICa-L)和瞬时外向钾电流(transient outward K current,Ito)的影响.结果显示,SNCl62明显抑制大鼠心室肌细胞,Ica L和Ica L,对Ica L.和k的最大抑制率分别为(46.13±4.12)%和(36.53±10.57)%.1x10-4mol/L SNCl62使,Ica L的甲均电流密度从(8.98±0.40)pA/pF下降到(4.84±0.44)pA/pF(P<0.01,n=5),Ito的平均电流密度从(18.69±2.42)pA/pF降低到(11.73±1.67)pA/pF(P<0.01,n=5).单独应用naltrindole(一种选择性δ阿片受体拮抗剂)对大鼠心室肌细胞Ica L和Ito无显著作用,但预先应用naltrindole可以消除SNCl62对Ica L和Ito的抑制作用.结果表明,通过δ阿片受体,SNCl62(1x10-6～1x10-4mol/L)浓度依赖性地抑制人鼠心室肌细胞Ica L和Ito这可能是激动δ阿片受体产生抗心律失常效应的重要机制.     

Maintenance and growth requirements in the metabolism of Debaryomyces hansenii performing xylose-to-xylitol bioconversion in corncob hemicellulose hydrolyzate

In order to improve the biotechnological production of xylitol, the metabolism of Debaryomyces hansenii NRRL Y-7426 in corncob hemicellulose hydrolyzate has been investigated under different conditions, where either maintenance or growth requirements predominated. For this purpose, the experimental results of two sets of batch bioconversions carried out alternatively varying the starting xylose concentration in the hydrolyzate (65.6 < or = S(0) < or = 154.7 g L(-1)) or the initial biomass level (3.0 < or = X(0) < or = 54.6 g(DM) L(-1)) were used to fit a metabolic model consisting of carbon material and ATP balances based on five main activities, namely fermentative assimilation of pentoses, semi-aerobic pentose-to-pentitol bioconversion, biomass growth on pentoses, catabolic oxidation of pentoses, and acetic acid and NADH regeneration by the electron transport system. Such an approach allowed separately evaluating the main bioenergetic constants of this microbial system, that is, the specific rates of ATP and xylose consumption due to maintenance (m(ATP) = 21.0 mmol(ATP) C-mol(DM) (-1)h(-1); m(Xyl) = 6.5 C-mmol(Xyl) C-mol(DM) (-1)h(-1)) and the true yields of biomass on ATP (Y(ATP) (max) = 0.83 C-mol(DM) mol(ATP) (-1)) and on xylose (Y(Xyl) (max) = 0.93 C-mol(DM) C-mol(Xyl) (-1)). The results of this study highlighted that the system, at very high S(0) and X(0) values, dramatically increased its energy requirements for cell maintenance, owing to the occurrence of stressing conditions. In particular, for S(0) > 130 g L(-1), these activities required an ATP consumption of about 2.1 mol(ATP) L(-1), that is, a value about seven- to eightfold that observed at low substrate concentration. Such a condition led to an increase in the fraction of ATP addressed to cell maintenance from 47% to 81%. On the other hand, the very high percentage of ATP addressed to maintenance (> 96%) at very high cell concentration (X(0) > or = 25 g(DM) L(-1)) was likely due to the insufficient substrate to sustain the growth.     

Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions

The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO(2) (-), SeO(3) (2-), TeO(3) (2-)) and three metal cations (Cd(2+), Ni(2+), Al(3+)) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria-Bertani) medium were thicker (23 mum) than biofilms grown in minimal (SA saccarose-arginine) medium (13 mum). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50-100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO(3) (2-) to elemental Te(0) or SeO(3) (2-) to elemental Se(0) was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO(3) (2-) and SeO(3) (2-) is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.     

Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.     

Modulation of zinc- and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36

Cysteine-rich Zn(II)-binding sites in proteins serve two distinct functions: to template or stabilize specific protein folds, and to facilitate chemical reactions such as alkyl transfers. We are interested how the protein environment controls metal site properties, specifically, how naturally occurring tetrahedral Zn(II) sites are affected by the surrounding protein. We have studied the Co(II)- and Zn(II)-binding of a series of derivatives of L36, a small zinc ribbon protein containing a (Cys)(3)His metal coordination site. UV-vis spectroscopy was used to monitor metal binding by peptides at pH 6.0. For all derivatives, the following trends were observed: (1) Zn(II) binds tighter than Co(II), with an average K (A) (Zn) /K (A) (Co) of 2.8(+/-2.0)x10(3); (2) mutation of the metal-binding ligand His32 to Cys decreases the affinity of L36 derivatives for both metals; (3) a Tyr24 to Trp mutation in the beta-sheet hydrophobic cluster increases K (A) (Zn) and K (A) (Co) ; (4) mutation in the beta-hairpin turn, His20 to Asn generating an Asn-Gly turn, also increases K (A) (Zn) and K (A) (Co) ; (5) the combination of His20 to Asn and Tyr24 to Trp mutations also increases K (A) (Zn) and K (A) (Co) , but the increments versus C(3)H are less than those of the single mutations. Furthermore, circular dichroism, size-exclusion chromatography, and 1D and 2D (1)H NMR experiments show that the mutations do not change the overall fold or association state of the proteins. L36, displaying Co(II)- and Zn(II)-binding sensitivity to various sequence mutations without undergoing a change in protein structure, can therefore serve as a useful model system for future structure/reactivity studies.     

Identification of a yolk sac cell population with hematopoietic activity in view of CD45/c-Kit expression

During murine embryonic development, primitive hematopoiesis occurs in the yolk sac (YS). Recent studies have shown that the YS also harbors definitive hematopoietic activity. However, the population of YS cells contributing to definitive hematopoiesis has not been identified. In this study, we characterized the hematopoietic cell populations in the YS of mouse embryos from E9.5 to E14.5 in view of the expression profiles of CD45 and c-Kit. The YS cells from E9.5 to E11.5 could be divided into six populations: CD45(-) c-Kit(-) , CD45(-) c-Kit(low) , CD45(-) c-Kit(high) , CD45(low) c-Kit(high) , CD45(high) c-Kit(high) and CD45(high) c-Kit(very low) . Among these populations, CD45(low) c-Kit(high) cells showed the highest multilineage hematopoietic colony-forming activity. Later in development, the YS cells from E12.5 to E14.5 lost the second and fourth populations (i.e., they retained CD45(-) c-Kit(-) , CD45(-) c-Kit(high) , CD45(high) c-Kit(high) and CD45(high) c-Kit(very low) cells), and concurrently with the disappearance of the CD45(low) c-Kit(high) population, no significant hematopoietic activity was found in any of the populations on and after E12.5. CD45(low) c-Kit(high) YS cells, which had a round morphology with a large nucleus, possessed the ability to differentiate into myeloid and B lymphoid cells when cultured with stromal cells. These findings suggest that CD45(low) c-Kit(high) YS cells include more undifferentiated cells than the other YS cell populations and possess in vitro potency to differentiate into multilineage hematopoietic cells. Furthermore, this cell population disappears from the YS at around E12.5, when the site of hematopoiesis has already shifted to the fetal liver and the placenta.     

Effects of carvedilol on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

Carvedilol is a beta- and alpha(1)-adrenoceptor antagonist. It is widely used in the treatment of cardiovascular diseases including atrial arrhythmias. However, it is unclear whether carvedilol may affect the repolarization currents, transient outward K(+) current (I(to)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in the human atrium. The present study evaluated effects of carvedilol on I(to) and I(Kur) in isolated human atrial myocytes by whole-cell patch-clamp recording technique. We found that carvedilol reversibly inhibited I(to) and I(Kur) in a concentration-dependent manner. Carvedilol (0.3 microM) suppressed I(to) from 9.2+/-0.5 pA/pF to 4.8+/-0.5 pA/pF (P<0.01) and I(Kur) from 3.6+/-0.5 pA/pF to 1.9+/-0.3 pA/pF (P<0.01) at +50 mV. I(to) was inhibited in a voltage-dependent manner, being significantly attenuated at test potentials from +10 to +50 mV, whereas the inhibition of I(Kur) was independent. The concentration giving a 50% inhibition was 0.50 microM for I(to) and 0.39 microM for I(Kur). Voltage-dependence of activation, inactivation and time-dependent recovery from inactivation of I(to) were not altered by carvedilol. However, time to peak and time-dependent inactivation of I(to) were significantly accelerated, indicating an open channel blocking action. The findings indicate that carvedilol significantly inhibits the major repolarization K(+) currents I(to) and I(Kur) in human atrial myocytes.     

Transport of Cu(II) from an albumin mimic peptide, GlyGlyHisGly, to histidine and penicillamine

Cu in blood has been believed to transport into cell via albumin and some amino acids. To shed light on the Cu transport process we studied the reaction of the Cu(II)-peptide with the amino acid by absorption and CD spectra. Albumin mimic peptides GlyGly-L-HisGly (GGHG) and penta-Gly(G5) formed stable 4N coordinated Cu(II) complexes, but in the reaction with histidine (His) and penicillamine (Pes) the ternary Cu(II) complex formations were observed different by the kinetic study. Cu(II)-G5 complexes reacted with Pes to form the ternary complex Cu(H(-1)G5)(Pes(-)) which was subsequently transformed to the binary complex Cu(Pes(-))(2). In the system with GGHG the Cu(II) was also transported from GGHG to Pes, but the ternary Cu(H(-1)GGHG)(Pes(-)) complex as the intermediate was detected a trace. The ternary complex would be spontaneously transformed to Cu(Pes(-))(2) upon forming, because the rate constant of the ternary complex formation k(1+)= approximately 2M(-1)s(-1) was less than k(2+)= approximately 5 x 10(2)M(-1)s(-1) for the Cu(Pes(-))(2) formation at physiological pH. In the Cu(II)-GGHG-His system the ternary Cu(H(-1)GGHG)(His) complex was also hardly identified because the formation constant K(1) and k(1+) were very small and the equilibrium existed between Cu(H(-2)GGHG) and Cu(His)(2) and its overall equilibrium constant beta(2) for Cu(His)(2) was very small to be 1.00+/-0.05 M(-1) at pH 9.0. These results indicated that the ternary complex is formed in the Cu transport process from the albumin to the amino acid, but His imidazole nitrogen in the fourth-binding site of Cu(II) strongly resists the replacement by the incoming ligand.     

Copper-specific damage in human erythrocytes exposed to oxidative stress

Ascorbate and complexes of Cu(II) and Fe(III) are capable of generating significant levels of oxygen free radicals. Exposure of erythrocytes to such oxidative stress leads to increased levels of methemoglobin and extensive changes in cell morphology. Cu(II) per mole is much more effective than Fe(III). However, isolated hemoglobin is oxidized more rapidly and completely by Fe(III)- than by Cu(II)-complexes. Both Fe(III) and Cu(II) are capable of inhibiting a number of the key enzymes of erythrocyte metabolism. The mechanism for the enhanced activity of Cu(II) has not been previously established. Using intact erythrocytes and hemolysates we demonstrate that Cu(II)-, but not Fe(III)-complexes in the presence of ascorbate block NADH-methemoglobin reductase. Complexes of Cu(II) alone are not inhibitory. The relative inability of Fe(III)-complexes and ascorbate to cause methemoglobin accumulation is not owing to Fe(III) association with the membrane, or its failure to enter the erythrocytes. The toxicity of Cu(II) and ascorbate appears to be a result of site-specific oxidative damage of erythrocyte NADH-methemoglobin reductase and the enzyme's subsequent inability to reduce the oxidized hemoglobin.     

Pollenkitt – its composition, forms and functions

Two types of sticky pollen coat material exist in angiosperms, both produced by the anther tapetum. Pollenkitt is the most common adhesive material present around pollen grains of almost all angiosperms pollinated by animals, whereas tryphine seems to be restricted only to Brassicaceae. Tapetal cell protoplasts have different patterns of development according to the products formed during their development and degeneration. If tryphine is formed, the tapetal cell protoplasts lose their individuality at the microspore stage. If pollenkitt is formed, their contents degenerate at later stages. Cell content is totally reabsorbed, when ripe pollen is not surrounded by any gluing material. Current knowledge of pollenkitt formation, deposition on pollen grains and chemical composition are reviewed and discussed. Methods for detecting this viscous fluid are also presented. The many functions of pollenkitt, deduced from personal observations and the literature, act in the period between anther opening and pollen hydration on the stigma; they are: (1) to hold pollen in the anther until dispersal; (2) to enable secondary pollen presentation; (3) to facilitate pollen dispersal; (4) to protect pollen from water loss; (5) to protect pollen from ultra-violet radiation; (6) to maintain sporophytic proteins responsible for pollen–stigma recognition inside exine cavities; (7) to protect pollen protoplasts from fungi and bacteria; (8) to keep together pollen grains during transport; (9) to protect pollen from hydrolysis and exocellular enzymes; (10) to render pollen attractive to animals; (11) to render pollen visible to animal eyes; (12) to hide pollen from animal eyes; (13) to avoid predation of pollen through smell; (14) to enable adhesion to insect bodies; (15) to enable pollen packaging by bees and to form corbicules; (16) to provide a digestible reward for pollinators; (17) to enable pollen clumps to reach the stigma; (18) to allow self-pollination; (19) to facilitate adhesion to the stigma; (20) to facilitate pollen rehydration. Depending on the developmental program of the species, these functions may act during pollen presentation, in relation to pollinators, during pollen dispersal and when pollen reaches the stigma.     

Enantioselective plasma protein binding of bimoclomol

The binding of bimoclomol enantiomers to human plasma, its components, as well as to plasma from monkey, dog, rat, and mouse was investigated by ultrafiltration and equilibrium dialysis. The considerably stronger binding of the (-)-(S)-enantiomer found in human plasma is due to the alpha(1)-acid glycoprotein (AAG) component. The binding parameters for AAG (n(R)K(R) = 1.3 x 10(4) M(-1) and n(S)K(S) = 1.0 x 10(5) M(-1)) revealed high enantioselectivity, while the binding to human serum albumin was found to be weak (nK = 5 x 10(3) M(-1)) and not stereoselective. (-)-(S)-Bimoclomol was extensively displaced in the presence of specific marker ligands for the "FIS" subfraction of human AAG. Comparative binding studies indicated considerable differences between plasma of the five species investigated.     

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.     

Tetraspanin CD151 Stimulates Adhesion-dependent Activation of Ras, Rac, and Cdc42 by Facilitating Molecular Association between β1 Integrins and Small GTPases

Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.     

ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678

ATP binds to sarcoplasmic reticulum Ca(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca(2)E(1)P --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg(678) in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E(2)P and E(2) intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E(1) form and ADP binding to Ca(2)E(1)P, as well as ATP/MgATP binding in modulatory modes to E(2)P and E(2), whereas the effects on ATP/MgATP acceleration of the Ca(2)E(1)P --> E(2)P transition were small, suggesting that the nucleotide that accelerates Ca(2)E(1)P --> E(2)P binds differently from that modulating the E(2)P --> E(2) and E(2) --> Ca(2)E(1) reactions. Mutation of Glu(439) hardly affected nucleotide binding to E(1), Ca(2)E(1)P, and E(2), but it led to disruption of the modulatory effect of ATP on E(2)P --> E(2) and acceleration of the latter reaction, indicating that ATP normally modulates E(2)P --> E(2) by interfering with the interaction between Glu(439) and Ser(186). Gly(438) seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu(439) and Thr(441).     

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.