Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients

It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-beta(93) (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNO(RBC)), preserving Hb allostery. In room air, the ratio of SNO(RBC) to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNO(RBC) with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNO(RBC) stability is uncoupled from O(2) tension when Hb is locked in the R state by CO pretreatment. Also, SNO(RBC) is increased approximately 20-fold in human septic shock (P = 0.002) and the O(2)-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O(2) saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.     

The Yeast Phenylalanyl-Transfer RNA Synthetase Recognition Site: The Region Adjacent to the Dihydrouridine Loop

Purified yeast phenylalanyl-tRNA synthetase can aminoacylate (yeast) tRNA(Phe), (wheat) tRNA(Phe), and (Escherichia coli) tRNA(1) (Val) (1, 2). We now report that this synthetase can also aminoacylate (E. coli) tRNA(Phe) and (E. coli) tRNA(1) (Ala). Highly purified (E. coli) tRNA(Phe) is heterologously aminoacylated to approximately 90% of the extent achieved with the homologous enzyme (crude E. coli phenylalanyl-tRNA synthetase). Pure (E. coli) tRNA(1) (Ala) (the major species) is heterologously aminoacylated to 70% of the extent achieved with the homologous synthetase (crude E. coli alanyl-tRNA synthetase).(E. coli) tRNA(Phe) is the fourth purified transfer RNA of known sequence to be shown to be an acceptable substrate for purified yeast phenylalanyl-tRNA synthetase. A comparison of these sequences shows that only one region is extremely similar in all four tRNAs. This region is located adjacent to the dihydrouridine loop, and consists of the nucleotides [Formula: see text] We conclude that this is the synthetase recognition site for yeast phenylalanyl-tRNA synthetase.This conclusion is further supported by partial fragment analysis of (E. coli) tRNA(1) (Ala).     

Biregular classification of Fano 3-folds and Fano manifolds of coindex 3

The Fano 3-folds and their higher dimensional analogues are classified over an arbitrary field k [unk] C by applying the theory of vector bundles (in the case B(2) = 1) and the theory of extremal rays (in the case B(2) >/= 2). An n-dimensional smooth projective variety X over k is a Fano manifold if its first Chern class c(1)(X) epsilon H(2)(X, Z) is positive in the sense of Kodaira [Kodaira, K. (1954) Ann. Math. 60, 28-48] (or ample). If n = 3 and c(1)(X) generates H(2)(X, Z), then either (i) X is a complete intersection in a Grassmann variety G with respect to a homogeneous vector bundle E on G: the rank of E is equal to codim(G)X and X is isomorphic to the zero locus of a global section of E, (ii) X is a linear section of a 10-dimensional spinor variety X(12) (10) [unk] P(k) (15), or (iii) X is isomorphic to a double cover of P(k) (3), a 3-dimensional quadric Q(k) (3), or a quintic del Pezzo 3-fold V(5) [unk] P(k) (6). If n = 4 and c(1)(X) is divisible by 2, then X [unk] C is isomorphic to (a) a complete intersection in a homogeneous space or its double cover, (b) a product of P(1) and a Fano 3-fold, (c) the blow-up of Q(4) [unk] P(5) along a line or along a conic, or (d) a P(1)-bundle compactifying a line bundle on P(3) or on Q(3) [unk] P(4).     

Lipoprotein(a): an elusive cardiovascular risk factor

Lipoprotein (a) [Lp(a)], is present only in humans, Old World nonhuman primates, and the European hedgehog. Lp(a) has many properties in common with low-density lipoprotein (LDL) but contains a unique protein, apo(a), which is structurally different from other apolipoproteins. The size of the apo(a) gene is highly variable, resulting in the protein molecular weight ranging from 300 to 800 kDa; this large variation may be caused by neutral evolution in the absence of any selection advantage. Apo(a) influences to a major extent metabolic and physicochemical properties of Lp(a), and the size polymorphism of the apo(a) gene contributes to the pronounced heterogeneity of Lp(a). There is an inverse relationship between apo(a) size and Lp(a) levels; however, this pattern is complex. For a given apo(a) size, there is a considerable variation in Lp(a) levels across individuals, underscoring the importance to assess allele-specific Lp(a) levels. Further, Lp(a) levels differ between populations, and blacks have generally higher levels than Asians and whites, adjusting for apo(a) sizes. In addition to the apo(a) size polymorphism, an upstream pentanucleotide repeat (TTTTA(n)) affects Lp(a) levels. Several meta-analyses have provided support for an association between Lp(a) and coronary artery disease, and the levels of Lp(a) carried in particles with smaller size apo(a) isoforms are associated with cardiovascular disease or with preclinical vascular changes. Further, there is an interaction between Lp(a) and other risk factors for cardiovascular disease. The physiological role of Lp(a) is unknown, although a majority of studies implicate Lp(a) as a risk factor.     

Protein kinase G phosphorylates Cav1.2 alpha1c and beta2 subunits

Voltage-dependent Ca(2+) channel function (Ca(v)1.2, L-type Ca(2+) channel) is required for cardiac excitation-contraction (E-C) coupling. Ca(v)1.2 plays a key role in modulating cardiac function in response to classic signaling pathways, such as the renin-angiotensin system and sympathetic nervous system. Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with Ca(v)1.2 current through the actions of cAMP and cGMP. Cardiac cGMP, which activates protein kinase G (PKG), is regulated by nitric oxide (NO), and natriuretic peptides. Although PKG has been reported to activate or inhibit Ca(v)1.2 function, it is still unclear whether Ca(v)1.2 subunits are PKG substrates. We have identified phosphorylation sites within the alpha(1c) and beta(2a) subunits that are phosphorylated by PKGIalpha in vitro. We demonstrate that a subset of these phosphorylation sites is modulated, in a cGMP-PKG-specific manner, in intact HEK cells heterologously expressing alpha(1c) and beta(2a) subunits. Using phospho-epitope-specific antibodies, we show that the phosphorylation of these residues is enhanced by PKG in intact cardiac myocytes. Activation of PKG in HEK cells transfected with alpha(1c) and beta(2a) subunits caused an inhibition of Ca(v)1.2 whole-cell current. PKG-mediated inhibition of Ca(v)1.2 current was significantly reduced by coexpression of an alanine-substituted Ca(v)1.2 beta(2a) subunit (Ser(496)). Our results identify a molecular mechanism by which cGMP-PKG regulates Ca(v)1.2 phosphorylation and function.     

Positioning of the alpha-subunit isoforms confers a functional signature to gamma-aminobutyric acid type A receptors

Fast synaptic inhibitory transmission in the CNS is mediated by gamma-aminobutyric acid type A (GABA(A)) receptors. They belong to the ligand-gated ion channel receptor superfamily, and are constituted of five subunits surrounding a chloride channel. Their clinical interest is highlighted by the number of therapeutic drugs that act on them. It is well established that the subunit composition of a receptor subtype determines its pharmacological properties. We have investigated positional effects of two different alpha-subunit isoforms, alpha(1) and alpha(6), in a single pentamer. For this purpose, we used concatenated subunit receptors in which subunit arrangement is predefined. The resulting receptors were expressed in Xenopus oocytes and analyzed by using the two-electrode voltage-clamp technique. Thus, we have characterized gamma(2)beta(2)alpha(1)beta(2)alpha(1), gamma(2)beta(2)alpha(6)beta(2)alpha(6), gamma(2)beta(2)alpha(1)beta(2)alpha(6), and gamma(2)beta(2)alpha(6)beta(2)alpha(1) GABA(A) receptors. We investigated their response to the agonist GABA, to the partial agonist piperidine-4-sulfonic acid, to the noncompetitive inhibitor furosemide and to the positive allosteric modulator diazepam. Each receptor isoform is characterized by a specific set of properties. In this case, subunit positioning provides a functional signature to the receptor. We furthermore show that a single alpha(6)-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the alpha-subunit neighboring the gamma(2)-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain alpha(1)- and alpha(6)-subunits. This method may also be applied to the study of other ion channels.     

Juxtaposition of C(2)M and the transverse filament protein C(3)G within the central region of Drosophila synaptonemal complex

The synaptonemal complex (SC) is intimately involved in the process of meiotic recombination in most organisms, but its exact role remains enigmatic. One reason for this uncertainty is that the overall structure of the SC is evolutionarily conserved, but many SC proteins are not. Two putative SC proteins have been identified in Drosophila: C(3)G and C(2)M. Mutations in either gene cause defects in SC structure and meiotic recombination. Although neither gene is well conserved at the amino acid level, the predicted secondary structure of C(3)G is similar to that of transversefilament proteins, and C(2)M is a distantly related member of the alpha-kleisin family that includes Rec8, a meiosis-specific cohesin protein. Here, we use immunogold labeling of SCs in Drosophila ovaries to localize C(3)G and C(2)M at the EM level. We show that both C(3)G and C(2)M are components of the SC, that the orientation of C(3)G within the SC is similar to other transverse-filament proteins, and that the N terminus of C(2)M is located in the central region adjacent to the lateral elements (LEs). Based on our data and the known phenotypes of C(2)M and C(3)G mutants, we propose a model of SC structure in which C(2)M links C(3)G to the LEs.     

Strongly nonlinear integral equations of hammerstein type

This paper studies the solution of the nonlinear Hammerstein equation u(x) + k(x,y)f[y,u(y)]mu(dy) = h(x) in the singular case, i.e., where the linear operator K with kernel k(x,y) is not defined for all the range of the nonlinear mapping F given by Fu(y) = f[y,u(y)] over the whole class X of functions u which are potential solutions of the equation. An existence theorem is derived under relatively minimal assumptions upon k and f, namely that (Ku,u) >/= 0, that K maps L(1) into L(1) (loc) and is compact from L(1) [unk] L(infinity) into L(1) (loc), that f(y,s) has the same sign as s for s >/= R, and that for each constant r > 0, f(y,s) 相似文献   

Standard energy metabolism of a desert harvester ant, Pogonomyrmex rugosus: Effects of temperature, body mass, group size, and humidity

Pogonomyrmex rugosus (Hymenoptera: Formicidae) is an important seed predator in the Mojave Desert of the southwestern United States. Its standard rate of O(2) consumption ( Vo(2)) varied significantly with temperature ( Vo(2) = 10((-1.588 + 0.0315T)), where Vo(2) is ml.g(-1).hr(-1) and T is body temperature in degrees C). The ratio of the Vo(2) values at 10 degrees C increments in body temperature, Q(10), also varied with temperature; methods of calculating Vo(2) from temperature with a shifting Q(10) are described. Vo(2) also varied with body mass ( Vo(2) = 0.0462M(0.669), where Vo(2) is ml.hr(-1) and M is body mass in g). Vo(2) was inversely related to relative humidity and was independent of group size. The rise in Vo(2) at low relative humidities was caused by increased activity and resulted in higher rates of net water loss. The primary metabolic adaptation to xeric conditions in P. rugosus appears to be a lower-than-predicted metabolic rate.     

脂蛋白(a)与心血管疾病的关系

脂蛋白(a)[Lp(a)]结构类似于低密度脂蛋白(LDL),高水平Lp(a)是一种公认的心血管疾病危险因子。体内存在氧化型Lp(a)更易于促进动脉粥样硬化的发生发展。Lp(a)中的载脂蛋白(a)[apo(a)]存在异质性,研究显示其危险性可能是由于apo(a)等位基因水平差异引起的,而且apo(a)的多态性影响到Lp(a)水平的临床测定,如何降低apo(a)对结果的影响还需要更多深入研究。目前针对高Lp(a)水平的人群尚无统一的治疗标准,但降脂治疗有益于预防心脑血管疾病的发生。     

Lipoxygenase products of arachidonic acid stimulate LHRH release from rat median eminence

Exogenous arachidonic acid (AA) incubated in presence of male rat hypothalamus, shows a low rate of conversion (less than 1%) of the substrate with a major product, identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by reverse phase-high performance liquid chromatography (rpHPLC) and gas chromatography-mass spectrometry (GC-MS). Furthermore, immunoreactive 12-HETE estimated after purification on rpHPLC is produced by hypothalamus slices or median eminences (MEs) incubated in absence of any exogenous precursor. The effect of 12-HETE was tested on the release of LHRH from rat MEs after a 30-min incubation and was compared to the effect of another lipoxygenase product, 5-HETE, and to the well-known stimulatory effect of prostaglandin E2 (PGE2). The three AA metabolites stimulate LHRH release. A significant stimulatory effect on LHRH release is obtained with 10(-9) M of 12-HETE and only with 10(-8) M of 5-HETE or PGE2. Furthermore, the effect of higher concentrations is different according to the eicosanoid tested. The maximal response (176% of the control) is reached with 12-HETE at 10(-8) M. No significant change is observed at 10(-7) and 10(-6) M. The response with 5-HETE is also maximal (162% of the control) at 10(-8) M but decreases significantly (only 117% of the control) at 10(-6) M. The amplitude of the response to PGE2 is larger and higher, reaching a plateau (300% of the control) at 10(-6) M. 12-HETE has no effect on somatostatin (SRIF), release, as already known for PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

At neutral pH, oxidation of CH(3)OH --> CH(2)O by an o-quinone requires general-base catalysis and the reaction is endothermic. The active-site -CO(2)(-) groups of Glu-171 and Asp-297 (Glu-171-CO(2)(-) and Asp-297-CO(2)(-)) have been considered as the required general base catalysts in the bacterial o-quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o-PQQ when MeOH is hydrogen bonded to Glu-171-CO(2)(-) and the crystal water (Wat1) is hydrogen bonded to Asp-297-CO(2)(-) is DeltaG++ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated DeltaG++ when MeOH is hydrogen bonded to Asp-297-CO(2)(-) is >50 kcal/mol. The Asp-297-CO(2)(-)...Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDH.PQQ.Wat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297-CO(2)(-) bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297-CO(2)(-), we find that MeOH returns to be hydrogen bonded to Glu-171-CO(2)(-) and Asp-297-CO(2)(-) coordinates to Ca(2+) during 3 ns simulation. The Asp-297-CO(2)(-)...Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation DeltaG++ = 1.1 kcal/mol for Asp-297-CO(2)(-) general-base catalysis of Wat1 hydration of the immediate CH(2)==O product --> CH(2)(OH)(2). By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH(2)(OH)(2) is exothermic.     

The presence of high-affinity, low-capacity estradiol-17beta binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

High-affinity, low-capacity estradiol-17beta (E(2)) binding is present in rainbow trout scale. The K(d) and B(max) of the scale E(2) binding are similar to those of the liver E(2) receptor (K(d) is 1.6 +/- 0.1 and 1.4 +/- 0.1 nM, and B(max) is 9.1 +/- 1.2 and 23. 1 +/- 2.2 fmol x mg protein(-1), for scale and liver, respectively), but different from those of the high-affinity, low-capacity E(2) binding in plasma (K(d) is 4.0 +/- 0.4 nM and B(max) is 625.4 +/- 63. 1 fmol x mg protein(-1)). The E(2) binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E(2) receptor, where E(2) is displaced by diethylstilbestrol, but not by testosterone. The putative scale E(2) receptor thus appears to bind both E(2) and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E(2) binding could be detected in rainbow trout gill or skin.     

Preferential transfer of the complete glycan is determined by the oligosaccharyltransferase complex and not by the catalytic subunit

Most eukaryotic cells show a strong preference for the transfer in vivo and in vitro of the largest dolichol-P-P-linked glycan (Glc(3)Man(9)GlcNAc(2)) to protein chains over that of biosynthetic intermediates that lack the full complement of glucose units. The oligosaccharyltransferase (OST) is a multimeric complex containing eight different proteins, one of which (Stt3p) is the catalytic subunit. Trypanosomatid protozoa lack an OST complex and express only this last protein. Contrary to the OST complex from most eukaryotic cells, the Stt3p subunit of these parasites transfers in cell-free assays glycans with Man(7-9)GlcNAc(2) and Glc(1-3)Man(9)GlcNAc(2) compositions at the same rate. We have replaced Saccharomyces cerevisiae Stt3p by the Trypanosoma cruzi homologue and found that the complex that is formed preferentially transfers the complete glycan both in vivo and in vitro. Thus, preference for Glc(3)Man(9)GlcNAc(2) is a feature that is determined by the complex and not by the catalytic subunit.     

Preparation and Spectroscopic Studies of Cobalt(II)-Stellacyanin

The cobalt(II) derivative of the "blue" copper protein stellacyanin has been prepared, and its visible-ultraviolet spectrum is reported. Tryptophan fluorescence quenching and p-mercuribenzoate titration results strongly suggest that Co(II) and Cu(II) compete for the same stellacyanin binding site and that a cysteine sulfur atom is coordinated in both cases. This interpretation is supported by the finding of an intense band at 355 nm in Co(II)-stellacyanin attributable to a charge transfer transition of the RS(-) --> Co(II) type. The visible absorption spectrum of Co(II)-stellacyanin exhibits band maxima at 540, 625, and 655 nm. These bands are attributable to d-d transitions originating in a high-spin Co(II) center. It is suggested that a correspondence exists between charge transfer bands observed at 355 and 300 nm in the Co(II) derivative to those found at 604 and 450 nm in the native protein. It is concluded that the intense 604-nm peak in Cu(II)-stellacyanin is attributable to a cys-S --> Cu(II) charge transfer transition.     

Kinetic Studies on Klebsiella pneumoniae Nitrogenase

Purified cell-free extracts of Klebsiella pneumoniae reduce N(2), N(3) (-), CN(-), or C(2)H(2) in the absence of an ATP-generating system when substrate concentrations of ATP are used. The optimum Mg(++)/ATP ratio is 0.5. Michaelis constants for the reduction of substrates calculated from kinetic studies of K. pneumoniae nitrogenase were similar to those that have been reported for Azotobacter vinelandii and Clostridium pasteurianum. Hill plots of the kinetic data are consistent with the view that there is a single binding site for each of the substrates N(2), C(2)H(2), CN(-), N(3) (-), and ATP. Inhibition studies of K. pneumoniae nitrogenase indicate that ADP competitively inhibits C(2)H(2) reduction. Also, the reducible substrates, N(3) (-) and CN(-), inhibit C(2)H(2) reduction. The inhibition by azide is noncompetitive, that by cyanide is mixed.     

Luminescence and Binding Studies on tRNAPhe

The phenylalanine transfer RNA of baker's yeast (tRNA(Phe)) contains a base Y of unknown molecular structure next to the anticodon triplet. Since the base Y fluoresces at room temperature (lambda(max) = 431 nm), its emission properties offer a unique tool for studying conformational and binding properties of tRNA(Phe). The results obtained by these experiments include the following: (1) The quantum yield of fluorescence of Y in tRNA(Phe) (phiF) is 0.07 +/- 0.01 at high Mg(2+) concentrations (>10(-2)M) and about half that at 10(-3)M or less, indicating a [Mg(2+)]-dependent conformational change of the anticodon loop. (2) The fluorescence of Y isolated from tRNAPhe (Y(+)) is red-shifted by 15 nm compared to Y in tRNA(Phe) which suggests a stacked (more hydrophobic) environment for Y in the intact anticodon loop. phiF of Y(+) is 0.035. (3) The solvent isotope effect phiF(D(2)O)/phiF(H(2)O) is 1.5 for tRNA(Phe) and 1.9 for Y(+) i.e., Y in tRNA is still hydrated. (4) The temperature dependence of phiF in a polar glass shows that quenching occurs only at temperatures at which the glass has sufficiently low viscosity to permit solvent shell relaxation in the excited state. The low-temperature (80 degrees K) fluorescence is blue shifted (lambda(max) = 409 nm) and the phosphorescence has a decay time of 1.5 seconds, a threshold at 392 nm and a spectral shape like that of guanine. (5) In the presence of 10(-2)M Mg(2+) penta-uridylate, which contains the codon triplet, a small blue shift and a decrease in phiF are observed. This shift can be used to establish the formation of a binary complex between the codon and the anticodon with an association constant of 4 x 10(2)M(-1), approximately. A similar complex is formed with poly-uridylate but not with poly-cytidylate. In the absence of Mg(2+) the binary complex is not formed.     

Lipoprotein(a) plasma concentrations after renal transplantation: a prospective evaluation after 4 years of follow-up.

The highly atherogenic lipoprotein(a) [Lp(a)] is significantly elevated in patients with renal disease. It is discussed controversially whether Lp(a) concentrations decrease after renal transplantation and whether the mode of immunosuppressive therapy influences the Lp(a) concentrations. In a prospective study the Lp(a) concentrations before and on average 48 months after renal transplantation were measured in 145 patients. The determinants of the relative changes of Lp(a) concentrations were investigated in a multivariate analysis. Patients treated by CAPD showed a larger decrease of Lp(a) than hemodialysis patients, reflecting their markedly higher Lp(a) levels before transplantation. The relative decrease of Lp(a) was higher with increasing Lp(a) concentrations before transplantation in combination with an increasing molecular weight of apolipoprotein(a) [apo(a)]. That means that the relative decrease of Lp(a) is related to the Lp(a) concentration and the apo(a) size polymorphism. With increasing proteinuria and decreasing glomerular filtration rate, the relative decrease of Lp(a) became less pronounced. Neither prednisolone nor cyclosporine (CsA) had a significant impact on the Lp(a) concentration changes. Azathioprine (Aza) was the only immunosuppressive drug which had a dose-dependent influence on the relative decrease of Lp(a) levels. These data clearly demonstrate a decrease of Lp(a) following renal transplantation which is caused by the restoration of kidney function. The relative decrease is influenced by Aza but not by CsA or prednisolone.