Stimulation by iodide of H(2)O(2) generation in thyroid slices from several species

The regulation of thyroid metabolism by iodide involves numerous inhibitory effects. However, in unstimulated dog thyroid slices, a small inconstant stimulatory effect of iodide on H(2)O(2) generation is observed. The only other stimulatory effect reported with iodide is on [1-(14)C]glucose oxidation, i.e., on the pentose phosphate pathway. Because we have recently demonstrated that the pentose phosphate pathway is controlled by H(2)O(2) generation, we study here the effect of iodide on basal H(2)O(2) generation in thyroid slices from several species. Our data show that in sheep, pig, bovine, and to a lesser extent dog thyroid, iodide had a stimulatory effect on H(2)O(2) generation. In horse and human thyroid, an inconstant effect was observed. We demonstrate in dogs that the stimulatory effect of iodide is greater in thyroids deprived of iodide, raising the possibility that differences in thyroid iodide pool may account, at least in part, for the differences between the different species studied. This represents the first demonstration of an activation by iodide of a specialized thyroid function. In comparison with conditions in which an inhibitory effect of iodide on H(2)O(2) generation is observed, the stimulating effect was observed for lower concentrations and for a shorter incubation time with iodide. Such a dual control of H(2)O(2) generation by iodide has the physiological interest of promoting an efficient oxidation of iodide when the substrate is provided to a deficient gland and of avoiding excessive oxidation of iodide and thus synthesis of thyroid hormones when it is in excess. The activation of H(2)O(2) generation may also explain the well described toxic effect of acute administration of iodide on iodine-depleted thyroids.     

Differential tyrosine phosphorylation of plasma membrane Ca2+-ATPase and regulation of calcium pump activity by carbachol and bradykinin

We investigated the effects of thapsigargin (TG), bradykinin (BK), and carbachol (CCh) on Ca(2+) entry via endogenous channels in human embryonic kidney BKR21 cells. After depletion of Ca(2+) stores by either TG, BK, or CCh, the addition of Ca(2+) gave a much larger rise in Ca(2+) levels in CCh-treated and TG-treated cells than in cells treated with BK. However, in experiments performed with Ba(2+), a cation not pumped by Ca(2+)-ATPases, only a modest difference between CCh- and BK-stimulated Ba(2+) entry levels was observed, suggesting that the large difference in the Ca(2+) response is mediated by a differential regulation of Ca(2+) pump activity by CCh and BK. This hypothesis is supported by the finding that when Ca(2+) is removed during the stable, CCh-induced Ca(2+) plateau phase, the decline of cytosolic Ca(2+) is much faster in the absence of CCh than in its presence. In addition, if Ca(2+) is released from a caged Ca(2+) compound after a UV pulse, the resulting Ca(2+) peak is much larger in the presence of CCh than in its absence. Thus, the large increase in Ca(2+) levels observed with CCh results from both the activation of Ca(2+) entry pathways and the inhibition of Ca(2+) pump activity. In contrast, BK has the opposite effect on Ca(2+) pump activity. If Ca(2+) is released from a caged Ca(2+) compound, the resulting Ca(2+) peak is much smaller in the presence of BK than in its absence. An investigation of tyrosine phosphorylation levels of the plasma membrane Ca(2+)-ATPase (PMCA) demonstrated that CCh stimulates an increase in tyrosine phosphorylation levels, which has been reported to inhibit Ca(2+) pump activity, whereas in contrast, BK stimulates a reduction of PMCA tyrosine phosphorylation levels. Thus, BK and CCh have a differential effect both on Ca(2+) pump activity and on tyrosine phosphorylation levels of the PMCA.     

Characterization of Mg2+ inhibition of mitochondrial Ca2+ uptake by a mechanistic model of mitochondrial Ca2+ uniporter

Ca(2+) is an important regulatory ion and alteration of mitochondrial Ca(2+) homeostasis can lead to cellular dysfunction and apoptosis. Ca(2+) is transported into respiring mitochondria via the Ca(2+) uniporter, which is known to be inhibited by Mg(2+). This uniporter-mediated mitochondrial Ca(2+) transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg(2+) inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg(2+) and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg(2+) inhibition and Pi regulation mechanisms. The model is extended from our previous model of the uniporter that is based on a multistate catalytic binding and interconversion mechanism and Eyring's free energy barrier theory for interconversion. The model satisfactorily describes a wide variety of experimental data sets on the kinetics of mitochondrial Ca(2+) uptake. The model also appropriately depicts the inhibitory effect of Mg(2+) on the uniporter function, in which Ca(2+) uptake is hyperbolic in the absence of Mg(2+) and sigmoid in the presence of Mg(2+). The model suggests a mixed-type inhibition mechanism for Mg(2+) inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca(2+) handling to understand the mechanisms by which Ca(2+) mediates signaling pathways and modulates energy metabolism.     

Is N2O3 the main nitrosating intermediate in aerated nitric oxide (NO) solutions in vivo? If so,where, when,and which one?

The widespread opinion that N(2)O(3) as a product of NO oxidation is the only nitros(yl)ating agent under aerobic conditions is based on experiments in homogeneous buffered water solutions. In vivo NO is oxidized in heterogeneous media and this opinion is not correct. The equilibrium in the system being dependent on temperature and DeltaG((sol)) for NO, NO(2), isomers of both N(2)O(3), and N(2)O(4). For polar solvents including water, DeltaG((sol)) for N(2)O(3) is high enough, and a stationary concentration of N(2)O(3) in the mixture with other oxides is sufficient to guarantee the hydrolysis of N(2)O(3) to nitrite. In heterogeneous media, the mixture contains solvates NO(2(sol)), N(2)O(3(sol)), and N(2)O(4(sol)) at stationary nonequilibrium concentrations. As far as DeltaG((sol)) is decreased in heterogeneous mixtures with low polar solvents and/or at increased temperatures, the equilibrium in such a system shifts to NO(2). Although NO(2) is a reactive free radical, it almost does not react with water. In contrast, the reaction with most functional protein groups efficiently proceeds by a radical type with the formation of nitrite and new radicals (X) further stabilized in various forms. Therefore, the ratio of the nitrosylated and nitrated products yields depends on actual concentrations of all NO(x).     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Curcumin, a molecule that inhibits the Ca2+-ATPase of sarcoplasmic reticulum but increases the rate of accumulation of Ca2+

Curcumin, an important inhibitor of carcinogenesis, is an inhibitor of the ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR). Inhibition by curcumin is structurally specific, requiring the presence of a pair of -OH groups at the 4-position of the rings. Inhibition is not competitive with ATP. Unexpectedly, addition of curcumin to SR vesicles leads to an increase in the rate of accumulation of Ca(2+), unlike other inhibitors of the Ca(2+)-ATPase that result in a reduced rate of accumulation. An increase in the rate of accumulation of Ca(2+) is seen in the presence of phosphate ion, which lowers the concentration of free Ca(2+) within the lumen of the SR, showing that the effect is not passive leak across the SR membrane. Rather, simulations suggest that the effect is to reduce the rate of slippage on the ATPase, a process in which a Ca(2+)-bound, phosphorylated intermediate releases its bound Ca(2+) on the cytoplasmic rather than on the lumenal side of the membrane. The structural specificity of the effects of curcumin on ATPase activity and on Ca(2+) accumulation is the same, and the apparent dissociation constants for the two effects are similar, suggesting that the two effects of curcumin could follow from binding to a single site on the ATPase.     

15-Deoxy-delta 12,14-prostaglandins D2 and J2 are potent activators of human eosinophils

15-Deoxy-Delta(12,14)-PDJ(2) (15d-PGJ(2)) is a degradation product of PGD(2) that has been proposed as an anti-inflammatory compound because of its various inhibitory effects, some of which are mediated by peroxisome proliferator-activated receptor-gamma. In contrast to its reported inhibitory effects on macrophages and other cells, we found that this compound is a potent activator of eosinophils, inducing calcium mobilization, actin polymerization, and CD11b expression. It is selective for eosinophils, having little or no effect on neutrophils or monocytes. 15d-PGJ(2) has an EC(50) of approximately 10 nM, similar to that of its precursor, PGD(2). The concentrations of 15d-PGJ(2) required to activate eosinophils are thus much lower than those required for its anti-inflammatory effects (usually micromolar). 15-Deoxy-Delta(12,14)-prostaglandin D(2) (15d-PGD(2)) is also a potent activator of eosinophils, with an EC(50) about the same as that of PGD(2), whereas Delta(12)-PGJ(2) is slightly less potent. Eosinophils pretreated with PGD(2) no longer respond to 15d-PGJ(2), and vice versa, but in both cases the cells still respond to another eicosanoid proinflammatory mediator, 5-oxo-6,8,11,14-eicosatetraenoic acid. This indicates that the effects of 15d-PGJ(2) are mediated by the DP(2)/chemoattractant receptor-homologous molecule expressed on Th2 cells that has recently been identified in eosinophils. 15d-PGJ(2) is selective for the DP(2) receptor, in that it has no effect on DP(1) receptor-mediated adenylyl cyclase activity in platelets. We conclude that 15d-PGJ(2) and 15d-PGD(2) are selective DP(2) receptor agonists that activate human eosinophils with potencies at least 100 times greater than those for the proposed anti-inflammatory effects of 15d-PGJ(2) on other cells.     

Differential dependence of hypoxia-inducible factors 1 alpha and 2 alpha on mTORC1 and mTORC2

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1 alpha and HIF2 alpha has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2 alpha expression is the more important contributor. Expression of HIF1 alpha is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2 alpha expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1 alpha and HIF2 alpha to rapamycin, both HIF1 alpha and HIF2 alpha expression is dependent on mTOR. HIF1 alpha expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2 alpha was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1 alpha is dependent on both mTORC1 and mTORC2, HIF2 alpha is dependent only on mTORC2. We also examined the dependence of HIF alpha expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2 alpha was dependent on Akt2, whereas that of HIF1 alpha was dependent on Akt3. Because HIF2 alpha is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2 alpha has been implicated.     

Effect of ammonia on the synthesis and function of the N 2 -fixing enzyme system in Clostridium pasteurianum

The N(2)-fixing system of Clostridium pasteurianum operates under regulatory controls; no activity is found in cultures growing on excess NH(3). The conditions which are necessary for the synthesis and function of this system were studied in whole cells by using acetylene reduction as a sensitive assay for the presence of the N(2)-fixing system. Nitrogenase of N(2)-fixing cultures normally can fix twice as much N(2) as is needed to maintain the growth rate. When cultures that have grown for four or more generations on NH(3) exhaust NH(3) from the medium, a diauxic lag of about 90 min ensues before growth is resumed on N(2). Neither N(2)-fixing nor acetylene reduction activity can be detected before growth is resumed on N(2). N(2) is not a necessary requirement for this synthesis since under argon that contains less than 10(-8)m N(2), the N(2)-fixing system is made. If NH(3) is added to N(2)-dependent cultures, synthesis of the enzyme system is abruptly stopped, but the enzyme already present remains stable and functional for at least 6 hr (over three generations). Cultures grown under argon in a chemostat controlled by limiting ammonia have derepressed nitrogenase synthesis. If the argon is removed and replaced by N(2), partial repression of nitrogenase occurs.     

Can trans-polyacetylene be formed on single-walled carbon-doped boron nitride nanotubes?

Recently, the grafting of polymer chains onto nanotubes has attracted increasing attention as it can potentially be used to enhance the solubility of nanotubes and in the development of novel nanotube-based devices. In this article, based on density functional theory (DFT) calculations, we report the formation of trans-polyacetylene on single-walled carbon-doped boron nitride nanotubes (BNNTs) through their adsorption of a series of C(2)H(2) molecules. The results show that, rather than through [2 + 2] cycloaddition, an individualmolecule would preferentially attach to a carbon-doped BNNT via "carbon attack" (i.e., a carbon in the C(2)H(2) attacks a site on the BNNT). The adsorption energy gradually decreases with increasing tube diameter. The free radical of the carbon-doped BNNT is almost completely transferred to the carbon atom at the end of the adsorbed C(2)H(2) molecule. When another C(2)H(2) molecule approaches the carbon-doped BNNT, it is most energetically favorable for this C(2)H(2) molecule to be adsorbed at the end of the previously adsorbed C(2)H(2) molecule, and so on with extra C(2)H(2) molecules, leading to the formation of polyacetylene on the nanotube. The spin of the whole system is always localized at the tip of the polyacetylene formed, which initiates the adsorption of the incoming species. The present results imply that carbon-doped BNNT is an effective "metal-free" initiator for the formation of polyacetylene.     

Phosphorylation of Ime2 regulates meiotic progression in Saccharomyces cerevisiae

Ime2p is a meiosis-specific protein kinase in Saccharomyces cerevisiae that controls multiple steps in meiosis. Although Ime2p is functionally related to the Cdc28p cyclin-dependent kinase (CDK), no cyclin binding partners that regulate its activities have been identified. The sequence of the Ime2p catalytic domain is similar to CDKs and mitogen-activated protein kinases (MAPKs). Ime2p is activated by phosphorylation of its activation loop in a Cak1p-dependent fashion and is subsequently phosphorylated on multiple residues as cells progress through meiosis. In this study, we show that Ime2p purified from meiotic cells is phosphorylated on Thr(242) and Tyr(244) in its activation loop and on Ser(520) and Ser(625) in its C terminus. Ime2p autophosphorylates on threonine in its activation loop in vitro consistent with autophosphorylation of Thr(242) playing a role in its activation. Moreover, autophosphorylation in cis is required for Ime2p to become hyperphosphorylated. Phosphorylation of the C-terminal serines is not essential to sporulation. However, Ime2p C-terminal phosphorylation site mutants genetically interact with components of the FEAR network that controls exit from meiosis I. These data suggest that Ime2p plays a role in controlling the exit from meiosis I and demonstrate that a phospho-modification pathway regulates Ime2p during the different phases of meiotic development.     

大麦根细胞质膜Ca~(2+)-ATP酶和Ca~(2+)转运系统的特性

用大麦质膜微囊研究细胞质膜 Ca~(2+)转运过程,发现质膜 Ca~(2+)—ATP酶在反应系统中不存在Mg~(2+)时可正常表现活性。跨膜Ca~(2+)转运按其对Mg~(2+)的需求可分为两个过程,一个是不需Mg~(2+)的、具高Ca~(2+)亲和力和较低的转运能力;另一个则是需Mg~(2+)的、具低Ca~(2+)亲和力和较高的转运能力。前者的动力学特征与Ca~(2+)—ATP酶相近,而后者则相差很大。据此推测,大麦根细胞质膜上除Ca~(2+)—ATP酶外,还存在另一个不同的Ca~(2+)转运系统。由两者分别承担的Ca~(2+)转运过程在细胞钙信使系统中可能起着不同的作用。     

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ～75% and single RyR2 opening frequency ～150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels

Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Effects on phenotypic variability of directional selection arising through genetic differences in residual variability

In standard models of quantitative traits, genotypes are assumed to differ in mean but not variance of the trait. Here we consider directional selection for a quantitative trait for which genotypes also confer differences in variability, viewed either as differences in residual phenotypic variance when individual loci are concerned or as differences in environmental variability when the whole genome is considered. At an individual locus with additive effects, the selective value of the increasing allele is given by ia/sigma + 1/2 ixb/sigma2, where i is the selection intensity, x is the standardized truncation point, sigma2 is the phenotypic variance, and a/sigma and b/sigma2 are the standardized differences in mean and variance respectively between genotypes at the locus. Assuming additive effects on mean and variance across loci, the response to selection on phenotype in mean is isigma2(Am)/sigma + 1/2 ixcov(Amv)/sigma2 and in variance is icov(Amv)/sigma + 1/2 ixsigma2(Av)/sigma2, where sigma2(Am) is the (usual) additive genetic variance of effects of genes on the mean, sigma2(Av) is the corresponding additive genetic variance of their effects on the variance, and cov(Amv) is the additive genetic covariance of their effects. Changes in variance also have to be corrected for any changes due to gene frequency change and for the Bulmer effect, and relevant formulae are given. It is shown that effects on variance are likely to be greatest when selection is intense and when selection is on individual phenotype or within family deviation rather than on family mean performance. The evidence for and implications of such variability in variance are discussed.     

Prostaglandin D2 modulates human neutrophil intracellular calcium flux and inhibits superoxide release via its ring carbonyl

We compared the effects of prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF2) and various ketones on superoxide (OX) release by human neutrophils, which had been stimulated by N-formyl methionyl leucyl phenylalanine (FMLP). Our data suggested that the ring carbonyl of PGD2 is essential to its inhibitory effect on OX release, but the carbonyl group as a ketone, alone is not sufficient. Using the fluorescent Ca2+ probe, Fura-2AM, we found that PGD2 increased the rate of decline of FMLP stimulated intracellular free Ca2+ (Ca)i, but that PGF2 had no effect. cAMP altered FMLP stimulated (Ca)i, in a pattern similar to PGD2. Furthermore, the ring carbonyl of PGD2 is crucial to its effect on OX as well as on (Ca)i.     

Conformational analysis of the tetranucleotides m6(2)A-m6(2)A-U-m6(2)A(m6(2)A = N6-dimethyladenosine) and U-m6(2)A-U-m6(2)A and of the hybrid dA-r(U-A). A one- and two-dimensional NMR study

A 1H-NMR investigation was carried out on the tetranucleotides U-m6(2)A-U-m6(2)A and m6(2)A-m6(2)A-U-m6(2)A (m6(2) = N6-dimethyladenosine) as well as on the hybrid trinucleotide dA-r(U-A). An extensive comparison with m6(2)A-U-m6(2)A and other relevant compounds is made. Previous proton NMR studies on trinucleotides have shown that purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3' side of the bulge is known to have no measurable effect on the bulge population. Chemical-shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)-m6(2)A(2) stack in U-m6(2)A-U-m6(2)A diminishes the population of bulged-out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5'----3' direction is fully confirmed by the fact that the strong m6(2)A(1)-m6(2)A(2) stack in the tetranucleotide m6(2)A-m6(2)A-U-m6(2)A virtually precludes the formation of bulged-out structures. The conformational characteristics of dA-r(U-A) appear comparable with those of m6(2)A-U-m6(2)A, which indicates that the presence of a 2'-hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.     

Characterization of zinc-alpha(2)-glycoprotein as a cell adhesion molecule that inhibits the proliferation of an oral tumor cell line.

Zn-alpha(2)-glycoprotein (Znalpha(2)gp) is a soluble protein widely distributed in body fluids and glandular epithelia. We have found it to be expressed in stratified epithelia as well. Znalpha(2)gp is clinically correlated with differentiation in various epithelial tumors, including oral and epidermal tumors. We have cloned epidermal Znalpha(2)gp and report the preparation of the recombinant protein in a Baculovirus expression system. Like the native molecule, recombinant Znalpha(2)gp has RNase activity. Znalpha(2)gp functions as a matrix protein for the Tu-138 oral squamous cell carcinoma cell line. Cell attachment to Znalpha(2)gp is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. Attachment is also inhibited by the antibody to integrin alpha(5)beta(1) (the fibronectin receptor), but not by antibodies to integrins alpha(v)beta(3), alpha(3)beta(1), and alpha(2)beta(1). We find that the proliferation of Tu-138 cells is inhibited on a Znalpha(2)gp matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. We believe that the role of Znalpha(2)gp in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.