Hydrogen peroxide in the Burkitt's lymphoma cell line Raji provides protection against arsenic trioxide-induced apoptosis via the phosphoinositide-3 kinase signalling pathway

Many anticarcinogenic drugs kill tumour cells by inducing apoptosis. We examined the effects of hydrogen peroxide (H(2)O(2)) on arsenic trioxide (As(2)O(3))-induced cell killing. Low concentrations of H(2)O(2) (200 micromol/l) inhibited the ability of As(2)O(3) to induce apoptosis in the Burkitt's lymphoma cell line Raji. H(2)O(2) altered the form of cell death from apoptosis to pyknosis/necrosis and also lowered the degree of cell killing by As(2)O(3). H(2)O(2) was capable of preventing caspase-3 activation induced by As(2)O(3) in Raji cells. Incubation of cells with a phosphoinositide-3 kinase (PI-3K) inhibitor, wortmannin (100 nmol/l), blocked the effects of H(2)O(2) on As(2)O(3)-induced caspase-3 activation. In addition, the PI-3K inhibitor partially blocked the effects of H(2)O(2) on up-regulation of Bcl-2 and Bcl-X(L) protein expression, down-regulation of Bax protein expression, and phosphorylation of Bcl-2 and IkappaBalpha. This investigation demonstrated for the first time that low concentrations of H(2)O(2) provide protection against the in vivo of As(2)O(3)-induced apoptosis. PI-3K plays a crucial role in enhancing cell survival during H(2)O(2), inhibiting As(2)O(3)-induced apoptosis in the Burkitt's lymphoma cells. As(2)O(3)-induced cancer cell apoptosis may be enhanced by certain antioxidants in the treatment protocol.     

Nitroxyl and its anion in aqueous solutions: spin states,protic equilibria,and reactivities toward oxygen and nitric oxide

The thermodynamic properties of aqueous nitroxyl (HNO) and its anion (NO(-)) have been revised to show that the ground state of NO(-) is triplet and that HNO in its singlet ground state has much lower acidity, pKa((1)HNO/(3)NO(-)) approximately 11.4, than previously believed. These conclusions are in accord with the observed large differences between (1)HNO and (3)NO(-) in their reactivities toward O(2) and NO. Laser flash photolysis was used to generate (1)HNO and (3)NO(-) by photochemical cleavage of trioxodinitrate (Angeli's anion). The spin-allowed addition of (3)O(2) to (3)NO(-) produced peroxynitrite with nearly diffusion-controlled rate (k = 2.7 x 10(9) M(-1) x s(-1)). In contrast, the spin-forbidden addition of (3)O(2) to (1)HNO was not detected (k < 3 x 10(5) M(-1) x s(-1)). Both (1)HNO and (3)NO(-) reacted sequentially with two NO to generate N(3)O as a long-lived intermediate; the rate laws of N(3)O formation were linear in concentrations of NO and (1)HNO (k = 5.8 x 10(6) M(-1) x s(-1)) or NO and (3)NO(-) (k = 2.3 x 10(9) M(-1) x s(-1)). Catalysis by the hydroxide ion was observed for the reactions of (1)HNO with both O(2) and NO. This effect is explicable by a spin-forbidden deprotonation by OH(-) (k = 4.9 x 10(4) M(-1) x s(-1)) of the relatively unreactive (1)HNO into the extremely reactive (3)NO(-). Dimerization of (1)HNO to produce N(2)O occurred much more slowly (k = 8 x 10(6) M(-1) x s(-1)) than previously suggested. The implications of these results for evaluating the biological roles of nitroxyl are discussed.     

Silicon-Carbon bond cleavage reactions of Ansa tungstenocene compounds: the [Me2Si] bridge as a site for metallocene functionalization

[Me(2)Si(Cp(Me(2)))(2)]W(H)Cl is obtained via reaction of WCl(6) with a mixture of [Me(2)Si(Cp(Me(2)))(2)]Li(2) and NaBH(4), from which the dichloride [Me(2)Si(Cp(Me(2)))(2)]WCl(2) is obtained via treatment with CHCl(3). [Me(2)Si(Cp(Me(2)))(2)]WCl(2) provides a means to access other ansa tungstenocene compounds, such as [Me(2)Si(Cp(Me(2)))(2)]WH(2), [Me(2)Si(Cp(Me(2)))(2)]WMe(2), and [Me(2)Si(Cp(Me(2)))(2)]WCO. Of most interest, the reactions of [Me(2)Si(Cp(Me(2)))(2)]W(H)Cl with organolithium reagents do not yield simple ansa tungstenocene derivatives. Specifically, the reactions of [Me(2)Si(Cp(Me(2)))(2)]W(H)Cl with MeLi, Bu(n)Li, or PhLi result in the formation of mixed-ring tungstenocene compounds resulting from C-Si cleavage and functionalization of the ansa bridge, namely (Cp(Me(2)))(eta(5),kappa(1)-C(5)H(2)Me(2)SiMe(2)CH(2))WH, (Cp(Me(2)))[eta(5),kappa(1)-C(5)H(2)Me(2)Si(Me)(Bu(n))CH(2)]WH, and (Cp(Me(2)))[eta(5),kappa(1)-C(5)H(2)Me(2)SiMe(2)(C(6)H(4))]WH, respectively. In contrast to the C-Si cleavage achieved by MeLi, Bu(n)Li, and PhLi, the ansa bridge of [Me(2)Si(Cp(Me(2)))(2)]W(H)Cl is inert to Bu(t)Li and the product obtained is the fulvene ("tuck-in") complex [Me(2)Si(Cp(Me(2)))(eta(6)-C(5)MeH(2)CH(2))]WH derived from dehydrohalogenation.     

Antagonistic effects of 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 on L-type Ca2+ channels and Na+/Ca2+ exchange in enterocytes from Atlantic cod (Gadus morhua)

There is mounting evidence that vitamin D and its metabolites play important roles in regulating plasma calcium concentrations in teleost fish as in other vertebrates. The aims of the present study were to elucidate the possible cellular target mechanisms for the rapid actions of 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in Atlantic cod enterocytes at physiological doses, and to establish the concentration and thus the physiological range of circulating 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in the Atlantic cod. The plasma concentrations of 25(OH)D(3), 1,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) were 15.3 +/- 2.7nM, 125.1 +/- 12.3pM and 10.1 +/- 23.5nM respectively. Exposure of enterocytes to 10mM calcium (Ca(2+)) evoked an increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)). This increase was suppressed by 24R,25(OH)(2)D(3) dose-dependently, with an EC(50) of 4.9nM and a maximal inhibition of 60%. 24R,25(OH)(2)D(3) (20nM) abolished an increase in [Ca(2+)](i) (approximately 252%) in the control enterocytes exposed to 10microM S(-)-BAYK-8644, suggesting that the hormone acts by inhibiting Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Administration of 20nM 24R,25(OH)(2)D(3) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 20%, indicating a release of Ca(2+) from intracellular stores. Administration of 25(OH)D(3) (20nM) resulted in a biphasic change in the enterocyte [Ca(2+)](i): within 1--5s, it decreased to 87 +/- 12nM below its mean basal [Ca(2+)](i) (334 +/- 13nM), followed by a rapid recovery of [Ca(2+)](i) to a new level, 10% lower than the initial [Ca(2+)](i). The rapid decrease, the recovery rate and the final [Ca(2+)](i) were all affected dose-dependently by 25(OH)D(3), with EC(50) values of 8.5, 17.0 and 18.9nM respectively. Furthermore, the effects of 25(OH)D(3) were sensitive to sodium (Na(+)), bepridil (10microM) and nifedipine (5 microM), suggesting that 25(OH)D(3) regulates the activity of both basolateral membrane-associated Na(+)/Ca(2+) exchangers and brush border membrane-associated L-type Ca(2+) channels. Administration of 25(OH)D(3) (10nM) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 18%, indicating a release of Ca(2+) from intracellular stores. 1,25(OH)(2)D(3) also affected enterocyte [Ca(2+)](i) in a biphasic manner: the rapid decrease, the recovery rate, and the mean final [Ca(2+)](i) were all affected dose-dependently, with EC(50) values of 8.3, 24.5 and 7.7nM respectively. The high EC(50) values for 1,25(OH)(2)D(3) compared with circulating concentrations of 1,25(OH)(2)D(3) (130pM) suggest that this effect is pharmacological, rather than of physiological relevance in enterocyte Ca(2+) homeostasis of the Atlantic cod. It is concluded that 24R,25(OH)(2)D(3) has a physiological role in decreasing intestinal Ca(2+) uptake via inactivation of L-type Ca(2+) channels, whereas the physiological role of 25(OH)D(3) is to increase enterocyte Ca(2+) transport via activation of Na(+)/Ca(2+) exchangers, concurrent with activation of L-type Ca(2+) channels.     

Differential D1 and D5 receptor regulation and degradation of the angiotensin type 1 receptor

Renal sodium transport is increased by the angiotensin type 1 receptor (AT(1)R), which is counterregulated by dopamine via unknown mechanisms involving either the dopamine type 1 (D(1)R) or dopamine type 5 receptor (D(5)R) that belong to the D(1)-like receptor family of dopamine receptors. We hypothesize that the D(1)R and D(5)R differentially regulate AT(1)R protein expression and signaling, which may have important implications in the pathogenesis of essential hypertension. D(1)R and D(5)R share the same agonists and antagonists; therefore, the selective effects of either D(1)R or D(5)R stimulation on AT(1)R expression in human renal proximal tubule cells were determined using antisense oligonucleotides selective to either D(1)R or D(5)R. We also determined the role of receptor tyrosine kinase and the proteosome on the D(1)R/D(5)R-mediated effects on AT(1)R expression and internalization. In renal proximal tubule cells, D(5)R (not D(1)R) decreased AT(1)R expression (half-life: 0.47+/-0.18 hours) and AT(1)R-mediated extracellular signal-regulated kinase 1/2 phosphorylation (232+/-18.9 U with angiotensin II [10(-7) mol/L] versus 81+/-8.9 U with angiotensin II [10(-7) mol/L] and fenoldopam [D(1)R/D(5)R agonist; 10(-6) mol/L; P<0.05; n=6). The fenoldopam-induced decrease in AT(1)R expression was reversed by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3,4-d) pyrimidine (c-Src tyrosine-kinase inhibitor) and clasto-lactacystin beta-lactone (proteasome inhibitor), demonstrating that the fenoldopam-mediated decrease in total cell AT(1)R expression is a result of a c-Src- and proteasome-dependent process. D(5)R stimulation decreases AT(1)R expression and is c-Src and proteasome dependent. The discovery of differential regulation by D(1)R and D(5)R opens new avenues for the development of agonists selective to either receptor subtype as targeted antihypertensive agents that can decrease AT(1)R-mediated antinatriuresis.     

Significance of valine/leucine247 polymorphism of beta2-glycoprotein I in antiphospholipid syndrome: increased reactivity of anti-beta2-glycoprotein I autoantibodies to the valine247 beta2-glycoprotein I variant

OBJECTIVE: To clarify the consequences of the valine/leucine polymorphism at position 247 of the beta(2)-glycoprotein I (beta(2)GPI) gene in patients with antiphospholipid syndrome (APS), by investigating the correlation between genotypes and the presence of anti-beta(2)GPI antibody. The reactivity of anti-beta(2)GPI antibodies was characterized using recombinant Val(247) and Leu(247) beta(2)GPI. METHODS: Sixty-five Japanese patients with APS and/or systemic lupus erythematosus who were positive for antiphospholipid antibodies and 61 controls were analyzed for the presence of the Val/Leu(247) polymorphism of beta(2)GPI. Polymorphism assignment was determined by polymerase chain reaction followed by restriction enzyme digestion. Recombinant Val(247) and Leu(247) beta(2)GPI were established to compare the reactivity of anti-beta(2)GPI antibodies to beta(2)GPI between these variants. The variants were prepared on polyoxygenated plates or cardiolipin-coated plates, and the reactivity of a series of anti-beta(2)GPI antibodies (immunized anti-human beta(2)GPI monoclonal antibodies [Cof-19-21] and autoimmune anti-beta(2)GPI monoclonal antibodies [EY1C8, EY2C9, and TM1G2]) and IgGs purified from patient sera was investigated. RESULTS: A positive correlation between the Val(247) allele and the presence of anti-beta(2)GPI antibodies was observed in the patient group. Human monoclonal/polyclonal anti-beta(2)GPI autoantibodies showed higher binding to recombinant Val(247) beta(2)GPI than to Leu(247) beta(2)GPI, although no difference in the reactivity of the immunized anti-beta(2)GPI between these variants was observed. Conformational optimization showed that the replacement of Leu(247) by Val(247) led to a significant alteration in the tertiary structure of domain V and/or the domain IV-V interaction. CONCLUSION: The Val(247) beta(2)GPI allele was associated with both a high frequency of anti-beta(2)GPI antibodies and stronger reactivity with anti-beta(2)GPI antibodies compared with the Leu(247) beta(2)GPI allele, suggesting that the Val(247) beta(2)GPI allele may be one of the genetic risk factors for development of APS.     

Pharmacokinetics of traditional Chinese syndrome and recipe: a hypothesis and its verification (I)

AIM:To propose a hypothesis defining the absorption, distribution, metabolism and elimination of traditional Chinese recipe (TCR)component in blood of healthy subjects and patients, and estimate its correctness.METHODS:The pharmacokinetics (PK) of same dose of drug was studied in the animal model of traditional Chinese syndrome (S)and healthy animals. The classification, termi-nology, concept and significance of the hypothesis were set forth with evidence provided in the present study. The hypotheses consisted of traditional Chinese syndrome PK (S-PK) and traditional Chinese recipe PK (R-PK). Firstly, the observed tetramethylpyrazine (TMP) PK in healthy, chronically reserpinized rats (rat model of spleen deficiency syndrome, RMSDS) and RMSDS treated with Sijunzi decoction (SJZD) for confirmation were used to verify S-PK; secondly, the ferulic acid (FA) PK in healthy and high molecular weight dextran (HMWD)-induced rabbit model with blood stasis syndrome (RDBSS) was also used to verify S-PK; and lastly, TMP PK parameters in serum of healthy rats after orally taken Ligusticum wallichii (LW), LW and Salvia miltiorrhiza (LW&SM) decoctions were compared to verify R-PK.RESULTS:The apparent first-order absorption Ka,(13.61 plus minus 2.56)h(-1) ,area under the blood drug concentration-time curve AUC, (24.88 plus minus 9.76)&mgr;gcenter doth(-1)mL(-1) , maximum drug concentration C(max), (4.82 plus minus 1.23)&mgr;gcenter dotmL(-1) of serum TMP in RMSDS were increased markedly(P< 0.05) compared with those Ka = (5.41 plus minus1.91)h(-1), AUC = (5.20 plus minus 2.57)&mgr;gcenter doth(-1)center dotmL(-1), C(max) = (2.33 plus minus 1.77)&mgr;gcenter dotmL(-1) of healthy rats (HR). The apparent first-order rate constant for alpha and beta distribution phase alpha = (0.38 plus minus 0.09)h(-1), beta = (0.06 plus minus 0.03)h(-1) , the apparent first-order intercompartmental transfer rate constants K10 = (0.24 plus minus 0.07)h(-1), K(12) = (0.11 plus minus 0.02)h(-1), K(21) = (0.11 plus minus 0.02)h(-1) of serum TMP in RMSDS were decreased significantly (P <0.01) compared with those K(10) = (0.88 plus minus 0.20)h(-1), K(12) = (1.45 plus minus 0.47)h(-1), K(21) = (0.72 plus minus 0.22)h(-1) of HR. However, no apparent differences occurred between HR and RMSDS treated with SJZD. The serum FA concentration and its AUC (5.6690 plus minus 2.3541)&mgr;gcenter doth(-1)center dotmL(-1) in RMBSS were also higher than those AUC =(2.7566 plus minus0.8232)&mgr;gcenter doth(-1)center dotmL(-1) of healthy rabbits (P <0.05). The Ka (11.51 plus minus 2.82)h(-1), AUC (0.84 plus minus0.17)&mgr;gcenter doth(-1)center dotmL(-1) of LW & SM-derived TMP in serum were much lower (P <0.05) than those Ka = (19.58 plus minus 4.14)h(-1),AUC = (1.27 plus minus 0.26)&mgr;gcenter doth(-1)center dotmL(-1) of LW-derived TMP in serum after oral decoctions.CONCLUSION:The SDS and blood stasis syndrome state could affect significantly the pharmacokinetic parameters of drugs and the abnormal SDS pharmacokinetic parameters could be normalized by SJZD. The combination of Chinese medicine in TCR could reciprocally affect the pharmacokinetic parameters of other components absorbed into the systemic circulation. These results support the S and R-PK hypothesis.     

Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes

The possible molecular basis for the previously described antagonistic interactions between adenosine A(1) receptors (A(1)R) and dopamine D(1) receptors (D(1)R) in the brain have been studied in mouse fibroblast Ltk(-) cells cotransfected with human A(1)R and D(1)R cDNAs or with human A(1)R and dopamine D(2) receptor (long-form) (D(2)R) cDNAs and in cortical neurons in culture. A(1)R and D(1)R, but not A(1)R and D(2)R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A(1)R/D(1)R heteromerization disappeared after pretreatment with the D(1)R agonist, but not after combined pretreatment with D(1)R and A(1)R agonists. A high degree of A(1)R and D(1)R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A(1)R and D(2)R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A(1)R agonist caused coclustering (coaggregation) of A(1)R and D(1)R, which was blocked by combined pretreatment with the D(1)R and A(1)R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D(1)R and A(1)R agonists, but not with either one alone, substantially reduced the D(1)R agonist-induced accumulation of cAMP. The A(1)R/D(1)R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A(1)R of D(1)R receptor signaling in the brain. The persistence of A(1)R/D(1)R heteromerization seems to be essential for the blockade of A(1)R agonist-induced A(1)R/D(1)R coclustering and for the desensitization of the D(1)R agonist-induced cAMP accumulation seen on combined pretreatment with D(1)R and A(1)R agonists, which indicates a potential role of A(1)R/D(1)R heteromers also in desensitization mechanisms and receptor trafficking.     

Release of endothelium-derived relaxing factor from human umbilical vessels

The ability of human umbilical endothelial cells to release relaxing substance(s) in response to different agonists was investigated. Endothelium-denuded aortic rings of rats were used for the bioassay and tension recording. After precontraction, this preparation showed no response to histamine, acetylcholine, A 23187, or adenosine triphosphate while serotonin elicited further contraction. Superfusion of the precontracted preparations with the perfusate from umbilical veins and arteries stimulated with histamine (10(-7)-10(-5) M), A23187 (10(-7)-10(-6) M), or adenosine triphosphate (10(-5)-10(-4) M) elicited a relaxation. No relaxation was obtained with acetylcholine (10(-8)-10(-6) M) or serotonin (10(-8)-10(-6) M). The relaxation of bioassay aortic rings under the influence of the perfusate from histamine-stimulated umbilical vessels was inhibited by mepyramine (10(-5) M) but not by cimetidine (10(-4) M) suggesting the involvement of H1-receptors. The relaxation was also inhibited by increasing the transit time between the donor and the detector preparation, by methylene blue (5 X 10(-5) M), and by nordihydroguaiaretic acid (5 X 10(-5) M) but not by indomethacin (5 X 10(-5) M), and which have been reported for endothelium-derived relaxing factor. The involvement of umbilical endothelial cells in the relaxation response was further confirmed by studying precontracted, rubbed rat aortic rings seeded with cultured endothelial cells from human umbilical veins. Such preparations relaxed in response to histamine (10(-7)-10(-4) M) in contrast with the control preparations. No relaxations of these preparations were observed in response to acetylcholine (10(-9)-10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia and reoxygenation increase H2O2 production in rats

To test the effect of transition from sustained hypoxia to normoxia on production of reactive oxygen species (ROS) in lungs, the authors measured hydrogen peroxide (H(2)O(2)) output in the expired air of rats breathing hypoxic, normoxic, and hyperoxic gas mixtures at the end of exposure to 72 hours of hypoxia. Twenty-one male Wistar rats (200 to 280 g) were randomly assigned to 1 of 3 groups. First two groups (experimental) were kept for 3 days in normobaric hypoxic chamber (F(1)O(2) 0.1), rats of the third group (controls) breathed air. The rats were then anesthetized, intubated, placed in the plethysmograph, and their ventilation measured. Two periods of exhaled breath condensate (EBC) collection, each lasting 1 hour, were then performed to assay H(2)O(2) output. The controls breathed during both samplings air, the first experimental group breathed during first sampling period hypoxic mixture (F(1)O(2) 0.1; SH-H measurement) and then, during second period, air (SH-H-A measurement), the second experimental group breathed first air (SH-A measurement) and then hyperoxic mixture (F(1)O(2) 1.0; SH-A-O(2) measurement). Concentration of H(2)O(2) in the EBC was assayed by chemiluminescence. H(2)O(2) production in the control group was low and similar in both measurements (20+/-10 and 13+/-5 pmol/h, mean+/-SEM). Exposure to 72 hours of hypoxia increased the H(2)O(2) production to 105+/-18 pmol/h (SH-H). Transition from hypoxia to normoxia resulted in an increase in the H(2)O(2) production (SH-A 421+/-24 pmol/h, and SH-H-A 366+/-19 pmol/h). Following transition from air breathing to hyperoxia did not affect the H(2)O(2) production (SH-A-O(2) 373+/-25 pmol/h). The results showed that sustained hypoxia and transition from sustained hypoxia to normoxia increased H(2)O(2) formation in the lungs.     

Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR

(13)C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing (13)C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-(13)C(1)]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved (13)CO(2), the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of approximately 1 s along with H(13)CO(3)(-), the hydrated form of (13)CO(2) generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-(13)C(1)]pyruvate, production of (13)CO(2) and H(13)CO(3)(-) was suppressed by approximately 90%, whereas the signal from [1-(13)C(1)]lactate was enhanced. In separate experiments, it was shown that O(2) consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of (13)CO(2) and H(13)CO(3)(-) from [1-(13)C(1)]pyruvate does not reflect production of CO(2) in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.     

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).     

Accumulation of CO(2) in reservoir devices during simulated neonatal mechanical ventilation

Aerosolized albuterol is frequently administered to mechanically ventilated neonates by metered dose inhaler (MDI) and a reservoir device. These reservoirs are often placed between the Y-piece and endotracheal tube, thereby creating mechanical dead space and increasing the risk of rebreathing carbon dioxide (CO(2)). The objectives of this study were: 1) to quantify CO(2) accumulation in two commonly used reservoirs (ACE(R), Aerochamber(R)-MV) and a bidirectional nonreservoir actuator (Airlife(R) Minispacer) during mechanical ventilation of a neonatal lung model; and 2) to determine the effect of tidal volume (V(T)) on CO(2) accumulation. We hypothesized that the accumulation of CO(2) in these devices is clinically insignificant at the small tidal volumes used in mechanically ventilated premature neonates. The model was constructed to simulate CO(2) exhalation by a ventilated neonate and consisted of a neonatal ventilator circuit (rate = 40/min; peak inspiratory pressure (PIP) = 20 cm H(2)0) attached to a reservoir/actuator and neonatal test lung. The ventilator delivered inspiratory gas (room air) to the test lung, which was vented into the atmosphere by a small adjustable leak. Expiration was simulated by manually ventilating 7.1% CO(2) (partial pressure of CO(2) (PCO(2)) = 48 mm Hg) back through the model. Accumulation of CO(2) within the reservoir/actuator was measured using an end-tidal CO(2) monitor. Each 4-min experiment was conducted at three V(T) (7.5 mL, 15 mL, and 25 mL), and the median PCO(2) was calculated in 0.5-min increments. For V(T) = 7.5 mL, CO(2) accumulated slowly in the ACE(R) and Minispacer(R) and reached a maximum at 4.0 min (PCO(2) = 2.3 mm Hg and 7.3 mm Hg, respectively). In contrast, the Aerochamber(R)-MV rapidly reached a PCO(2) of 9.5-10.0 mm Hg by 1-1. 5 min. A similar trend occurred with V(T) = 15 mL; however, higher partial pressures (approximately 10-12 mm Hg) were achieved with all devices. At V(T) = 25 mL, PCO(2) rose rapidly with the ACE(R), Aerochamber(R)-MV, and Minispacer(R), reaching peaks of 17.2, 12.3, and 20.3 mm Hg, respectively (P < 0.05). In conclusion, accumulation of CO(2) in reservoir/actuator depends on V(T) as well as the chamber design and internal volume. Due to the short duration of use when administering drugs via MDI, accumulation of CO(2) in these devices is not likely to be clinically relevant for the majority of ventilated newborns.     

Analysis of the three dimensional structure of the CXGXC motif in the CMGCC and CAGYC regions of alpha-and beta-subunits of human chorionic gonadotropin: importance of glycine residue (G) in the motif

The three dimensional structures of the C(1)X(2)G (3)(X(3))X(4)C(5) motif of hCG, which is considered to be important for noncovalent assembly of the alpha- and beta-subunits of glycoprotein hormones were analyzed to assess the importance of glycine (Gly) (G) at site X(3) in the motif by the conformational energy calculation using computational procedures. In the C(1)M(2)G (3)(X(3))C(4)C(5) motif of the alpha-subunit, Ramachandran plot analysis showing the allowed area of the dihedral angles demonstrated that only a Gly residue was allowed at site X(3). In calculating collision with surrounding atoms as a monomer the possible main chain models of the C(1)A(2)G(3)(X(3))Y(4)C(5) motif in the beta-subunit showed that only alanine (Ala) (A) or Gly at site X(3) is allowed to alleviate the collision with the cysteine (Cys) (C) residues which form a disulfide bridge. A mutant of the beta-subunit with the C(1)A(2)A(3)(X(3))Y(4)C(5) motif (Ala at site X(3)) may not compose a heterodimer with the alpha-subunit because of interference of intermolecular hydrogen bond formation. These findings indicate that the Gly residue at site X(3) (G(3)) in the motif is essential for heterodimer formation of glycoprotein hormones. The significance of similar motifs found in various human proteins other than glycoprotein hormones was suggested.     

Upregulation of 25-hydroxyvitamin D3-1α-hydroxylase by butyrate in Caco-2 cells

AIM: To investigate the possible involvement of 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase [1alpha-25(OH) (2) D(3)] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells. METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 micromol/L 25(OH) (2) D(3) or with 1 micromol/L 1alpha-25(OH) (2) D(3) for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH) (2) D(3) or 1alpha-25(OH) (2) D(3). 1alpha-25(OH) (2) D(3) mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1alpha-25(OH) (2) D(3) protein. Finally, enzymatic activity was measured by ELISA. RESULTS: Both butyrate and 1alpha-25(OH) (2) D(3) stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH) (2) D(3) had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1alpha-25(OH) (2) D(3) mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites. CONCLUSION: Our experimental data provide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1alpha-25(OH) (2) D(3) followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH) (2) D(3) in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.     

Autoimmune and lymphoproliferative disease in (B6-GIX+ × 129) F1 mice: Relation to naturally occurring antibodies against murine leukemia virus-related cell surface antigens

G(IX) congeneic mouse strains, C57BL/6-G(IX) (+)(B6-G(IX) (+)) and 129-G(IX) (-), have been derived from the prototype strains, B6(G(IX) (-)) and 129(G(IX) (+)). The hybrids, (B6-G(IX) (+) x 129)F(1) (G(IX) (+)F(1)) and (B6 x 129-G(IX) (-))F(1) (G(IX) (-)F(1)), differ only in regard to genetic loci controlling G(IX) antigen expression. G(IX) (+)F(1) mice spontaneously produce G(IX) antibody and often show signs of autoimmune disease and lymphoproliferative disease. G(IX) (-)F(1) mice and mice of the two parental strains (B6-G(IX) (+) and 129) of G(IX) (+)F(1) do not produce G(IX) antibody and seldom show signs of these diseases. G((ERLD)), and G((RADA1)), antibodies, natural thymocytotoxic autoantibody, and antinuclear antibodies were produced by G(IX) (+)F(1) mice. However, these four antibodies were also found in the other strains. G(IX) (+)F(1) mice develop pronounced diffuse glomerulonephritis similar to that found in systemic lupus erythematosus in man. Incidence studies in which mice were examined according to age rather than state of health showed that the lesions occurred in 38% of G(IX) (+)F(1) mice but not in G(IX) (-)F(1), B6-G(IX) (+), or 129 mice. Lymphoproliferative lesions were either reticulum cell sarcoma (RCS) type A or reactive lymphoid hyperplasia (RLH). RCS occurred more often in G(IX) (+)F(1) (38%) than in G(IX) (-)F(1) (12%) or B6-G(IX) (+) (8%). No RCS occurred in mice of the 129 strain. RLH occurred in G(IX) (+)F(1) mice (10%) but not in the other strains. From these results, the following conclusions are drawn: (i) Severe glomerulonephritis and the increased occurrence of lymphoproliferative lesions in these animals depend on the presence of G(IX) antigen; (ii) besides genes controlling G(IX) antigen expression, other genes from both parental strains are required to create the basis in the progeny F(1) mice for the development of these diseases; and (iii) the chronic production of G(IX) antibody may be necessary for the development of the severe glomerulonephritis and for the increased occurrence of lymphoproliferative diseases in G(IX) (+)F(1) mice.     

The nature of analogue-based free thyroxine estimates.

Clinical laboratories often use analogue-based immunoassays to estimate serum free thyroxine (FT(4)) concentrations. These assays yield FT(4) estimates that correlate closely with thyroxine (T(4)) binding protein concentrations. This correlation implies that either T(4) binding proteins or protein bound T(4) contribute to analogue-based FT(4) values. To study the contributions made by T(4) binding proteins to these FT(4) estimates further, four analogue-based FT(4) assays were applied to: (1) FT(4) solutions without T(4) binding proteins, (2) to T(4) binding protein solutions without T(4), and (3) to total T(4) solutions containing T(4) binding protein, FT(4), and protein-bound T(4). The FT(4) estimates obtained with these solutions ranged from 0.2-8.6 ng/dL, when FT(4) concentrations ranged from less than 0.2-12,000 ng/dL. In the FT(4) solutions, gravimetrically determined FT(4) concentrations were 500-12,000 ng/dL (0.5-12.0 microg/dL) without protein-bound T(4), and the FT(4) estimates obtained were 0.3-6.9 ng/dL. In the total T(4) solutions, dialyzable FT(4) concentrations were less than 0.2-59 ng/dL, retained T(4) concentrations were 499.8-11,441 ng/dL, and the analogue-based FT(4) estimates obtained were 0.2-8.6 ng/dL. Similar FT(4) estimates (0.2-8.6 ng/dL and 0.3-6.9 ng/dL) were obtained with similar concentrations of either protein-bound T(4) or FT(4). Similar test results were associated with similar total T(4) concentrations, not similar FT(4) concentrations. Protein-bound T(4) and T(4) binding protein contributed variably to test results. T(4) quantifications included large analytical losses that are unaccounted for. These assays passed tests of correlation with FT(4) concentrations, but they failed tests of specificity for FT(4) and accuracy in T(4) quantification.     

Thermophilic ATP synthase has a decamer c-ring: indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling

In a rotary motor F(o)F(1)-ATP synthase that couples H(+) transport with ATP synthesis/hydrolysis, it is thought that an F(o)c subunit oligomer ring (c-ring) in the membrane rotates as protons pass through F(o) and a 120 degrees rotation produces one ATP at F(1). Despite several structural studies, the copy number of F(o)c subunits in the c-ring has not been determined for any functional F(o)F(1). Here, we have generated and isolated thermophilic Bacillus F(o)F(1), each containing genetically fused 2-mer-14-mer c (c(2)-c(14)). Among them, F(o)F(1) containing c(2), c(5), or c(10) showed ATP-synthesis and other activities. When F(1) was removed, F(o) containing c(10) worked as an H(+) channel but F(o)s containing c(9), c(11) or c(12) did not. Thus, the c-ring of functional F(o)F(1) of this organism is a decamer. The inevitable consequence of this finding is noninteger ratios of rotation step sizes of F(1)/F(o) (120 degrees /36 degrees ) and of H(+)/ATP (10:3). This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by H(+) transport at F(o) and elementary events in catalysis at F(1).     

Configurationally homogeneous diastereomers of a linear hexa(tertiary phosphine): enantioselective self-assembly of a double-stranded parallel helicate of the type (P)-[Cu(3)(hexaphos)(2)](PF(6))(3)

Three configurationally homogeneous diastereomers of the linear hexa(tertiary phosphine) Ph(2)PCH(2)CH(2)P(Ph)CH(2)CH(2)P(Ph)CH(2)CH(2)P(Ph)CH(2)CH(2)P(Ph)CH(2)CH(2)PPh(2) (hexaphos) have been isolated in enantiomerically pure form, namely (R,S,S,R)-, (R,S,S,S)-, and (S,S,S,S)-hexaphos. The strongly helicating (R,S,S,R)-(-) form of the ligand combines with copper(I) ions to generate by stereoselective self-assembly the P enantiomer of a parallel helicate of the type [Cu(3)(hexaphos)(2)](PF(6))(3), which has been characterized by x-ray crystallography. Theoretical modeling of the cation indicates that it is the relationship between the helicities of the two 10-membered rings containing the three copper ions, each of which has the twist-boat-chair-boat conformation, and the configurations of the three chiral, tetrahedral copper stereocenters of P configuration that determines the stereochemistry of the parallel and double alpha-helix conformers of the double-stranded trinuclear metal helicate.