Insulin-like growth factor I increases alpha Vbeta 3 affinity by increasing the amount of integrin-associated protein that is associated with non-raft domains of the cellular membrane.

Insulin-like growth factor I (IGF-I) stimulates an increase in alpha(V)beta(3) ligand binding. Stimulation of smooth muscle cells by IGF-I requires alpha(V)beta(3) ligand occupancy, and enhanced alpha(V)beta(3) ligand occupancy augments IGF-I actions. Therefore, IGF-I-induced changes in alpha(V)beta(3) ligand binding may act to further enhance IGF-I actions. Integrin-associated protein (IAP) has been shown to be associated with alpha(V)beta(3) and is required for the binding of alpha(V)beta(3) to vitronectin-coated beads. We therefore investigated whether IGF-I could stimulate IAP-alpha(V)beta(3) association resulting in enhanced ligand binding. IGF-I stimulated an increase in IAP-alpha(V)beta(3) association. This was due, at least in part, to an IGF-I-stimulated redistribution of IAP from the Triton-insoluble fraction of the cell to the Triton-soluble fraction of the cell, where most of the alpha(V)beta(3) was located. Inhibition of the phosphatidylinositol 3-kinase pathway blocked both the redistribution of IAP and the increase in IAP-alpha(V)beta(3) association, providing further evidence that the redistribution of IAP is essential for the increase in association. An anti-IAP monoclonal antibody, blocked both the IGF-I-stimulated increase in IAP-alpha(V)beta(3) complex formation and cell migration. IGF-I-stimulated translocation of IAP and increase in IAP-alpha(V)beta(3) association represent an important process by which IGF-I modulates alpha(V)beta(3) ligand binding and cellular responses.     

Photosynthetic pathway influences xylem structure and function in Flaveria (Asteraceae)

Higher water use efficiency (WUE) in C(4) plants may allow for greater xylem safety because transpiration rates are reduced. To evaluate this hypothesis, stem hydraulics and anatomy were compared in 16 C(3), C(3)-C(4) intermediate, C(4)-like and C(4) species in the genus Flaveria. The C(3) species had the highest leaf-specific conductivity (K(L)) compared with intermediate and C(4) species, with the perennial C(4) and C(4)-like species having the lowest K(L) values. Xylem-specific conductivity (K(S)) was generally highest in the C(3) species and lower in intermediate and C(4) species. Xylem vessels were shorter, narrower and more frequent in C(3)-C(4) intermediate, C(4)-like and C(4) species compared with C(3) species. WUE values were approximately double in the C(4)-like and C(4) species relative to the C(3)-C(4) and C(3) species. C(4)-like photosynthesis arose independently at least twice in Flaveria, and the trends in WUE and K(L) were consistent in both lineages. These correlated changes in WUE and K(L) indicate WUE increase promoted K(L) decline during C(4) evolution; however, any involvement of WUE comes late in the evolutionary sequence. C(3)-C(4) species exhibited reduced K(L) but little change in WUE compared to C(3) species, indicating that some reduction in hydraulic efficiency preceded increases in WUE.     

Highly efficient technetium-99m labeling procedure based on the conjugation of N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine ligand with poly(ethylene glycol)

The PN(2)S N-(N-(3-diphenylphosphinopropionyl)glycyl)cysteine ligand was conjugated to methoxy-poly(ethylene glycol)-amino (mPEG-NH(2)) 5 and 20 kDa to yield PN(2)S(Trt)-PEG(5000) 1 and PN(2)S(Trt)-PEG(20000) 2, and then detritylated to PN(2)S-PEG(5000) 4 and PN(2)S-PEG(20000) 5. When an acidic solution of (99m)TcO(4)(-) is added to 4 or 5 in solid form, a quantitative yield in a single labeled species, (99m)Tc-labeled PN(2)S-PEG(5000) 9 and (99m)Tc-labeled PN(2)S-PEG(20000) 10, respectively, is obtained. The reaction occurs in less than 15 min at room temperature for 4 and 35 degrees C for 5. This labeling procedure avoids the use of an external reducing agent, and it is based on the amphiphilic properties of PN(2)S-PEGs. Once in water, 4 and 5 self-assemble in micelles, which catalyze the metal reduction by means of an electron pair transfer from the phosphorus to technetium. The [(99m)TcO](3+) species is then coordinated, and at micelle level, both the (P)ON(2)S and the PN(2)S coordinations are possible, as demonstrated by reacting (99m)Tc-gluconate and ReOCl(3)(PPh(3))(2) with 4 and 5 and with the oxidized analogous (P)ON(2)S-PEG(5000) 6. Compounds 9 and 10 exhibited a high stability both in vitro and in vivo. Biodistribution studies in mice also indicated that PN(2)S linking and (99m)Tc labeling do not modify PEG behavior in water and in vivo since the polymer dictates the fate of the conjugate.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

Energy gain and energy gap in normal-weight children: longitudinal data of the KOPS

Population-based prevention of overweight needs evidence-based goals consistent with our present knowledge about energy gap (i.e., daily imbalance between energy intake and energy expenditure resulting in overweight). Longitudinal data of normal-weight children (1,029 girls and 1,028 boys; Kiel Obesity Prevention Study, KOPS) were used to calculate energy gain (i.e., increase in fat mass (FM) and fat-free mass (FFM)) in normal-weight children staying normal weight (persistent children) or becoming overweight (incident children). Taking into account weight gain in proportion to height gain (normal development) energy gap was calculated from increases in FM and FFM exceeding normal development. Children were divided into two groups and were followed from age 6 to 10 (group A) and 10 to 14 years (group B). FM and FFM were measured. Medians of 4-year BMI- (kg/m(2))/weight changes (kg) were +1.8/+13.2 (A) and +3.0/+18.7 (B) in girls, and +1.6/+12.8 (A) and +2.6/21.7 (B) in boys. Corresponding data for FM/FFM (kg) were +3.1/+10.2 (A) and +5.1/12.7 (B) in girls, and +2.3/10.8 (A) and +3.0/18.6 (B) in boys. The 4-year-incidence of overweight (%) were 9.4 (A) and 5.4 (B) in girls, and 11.0 (A) and 3.8 (B) in boys, respectively. Mean energy gains (kcal/day) were 26.8 (A) and 46.4 (B) in girls, and 22.1 (A) and 32.5 (B) in boys. The 90th percentile of energy gap (kcal/day) in incident children were 58.1 (A) and 72.0 (B) in girls and 46.0 (A) and 53.2 (B) in boys. To prevent overweight in children energy gap should not exceed 46-72 kcal/day.     

Studies of the receptor-bound conformation of alphaIIbbeta3 antagonists by 15N-edited NMR spectroscopy

We report the results of NMR studies and computer simulations of potent antagonists reflective of the alpha(IIb)beta(3) receptor-bound conformations. The peptides c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Phe(4)-(15)N-Arg(5)-Cys]-NH(2) (Phe-Arg analog) (Mpa: 3-mercaptopropionic acid) and c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Asp(4)-(15)N-Val(5)-Cys]-NH(2) (Asp-Val analog) were subjected to (15)N-edited NMR experiments to study the conformations of these peptides in the absence and in the presence of alpha(IIb)beta(3) receptor. The NMR studies of the Phe-Arg analog, a selective alpha(IIb)beta(3) antagonist, resulted in distinctly different experimental data in the presence and absence of the receptor. The computer simulations for this peptide resulted in one large family of structures consistent with the experimental data. This conformation suggests a type I beta-turn spanning residues Arg(1) and Gly(2) when bound to the receptor and we were able to establish a model for the three dimensional arrangement of the pharmacophores. The studies on the Asp-Val analog, an alpha(v)beta(3) antagonist that binds to the alpha(IIb)beta(3) with moderate affinity, resulted in conformations that are not as well defined as those for the Phe-Arg analog but are consistent with the model established for this analog. These results are important for the design of novel alpha(IIb)beta(3) antagonists.     

Up-regulation of alphavbeta3 integrin expression is a novel molecular response to chemotherapy-induced cell damage in a heregulin-dependent manner

alpha(v)beta(3) integrin has a dual role in apoptosis. Whereas ligated alpha(v)beta(3) activates cell survival pathways and suppresses pro-apoptotic signals, unligated alpha(v)beta(3) or integrins bound to soluble ligands promote apoptosis. In this study, we assessed the role of alpha(v)beta(3) in chemosensitivity of breast cancer cells expressing different levels of heregulin (HRG). Expression levels of the RGD-binding integrins alpha(v)beta(3) were measured in MDA-MB-231 human breast cancer cells and its low HRG-expressing derivative (MDA-MB-231/AS31) treated with the microtubule-interfering agents (MIAs) paclitaxel and vincristine. Following treatment, only alpha(v)beta(3) levels were significantly increased in MDA-MB-231 cells. Interestingly, alpha(v)beta(3) expression was more significantly up-regulated in the MDA-MB-231/AS31 cells than in the parental cells. This MIA-induced increase of alpha(v)beta(3) expression was correlated with a decrease in cell viability and an increase in apoptosis in MDA-MB-231/AS31 cells, indicating that overexpression of alpha(v)beta(3) is linked to chemotherapy-induced cell death in low HRG-expressing breast cancer models. Moreover, a paclitaxel-induced increase of alpha(v)beta(3) was also observed in MCF-7 cells but not in an doxorubicin-resistant derivative that shows cross-resistance to paclitaxel, further providing evidence that the extent of alpha(v)beta(3) up-regulation is related to cell damage. These results indicate that alpha(v)beta(3) integrin is dramatically up-regulated in low HRG-expressing breast cancer models that are highly responsive to MIAs, thus providing a novel molecular marker of chemosensitivity influenced by HRG levels in breast cancer cells.     

Selective inhibition of ammonium oxidation and nitrification-linked n(2)o formation by methyl fluoride and dimethyl ether

Methyl fluoride (CH(3)F) and dimethyl ether (DME) inhibited nitrification in washed-cell suspensions of Nitrosomonas europaea and in a variety of oxygenated soils and sediments. Headspace additions of CH(3)F (10% [vol/vol]) and DME (25% [vol/vol]) fully inhibited NO(2) and N(2)O production from NH(4) in incubations of N. europaea, while lower concentrations of these gases resulted in partial inhibition. Oxidation of hydroxylamine (NH(2)OH) by N. europaea and oxidation of NO(2) by a Nitrobacter sp. were unaffected by CH(3)F or DME. In nitrifying soils, CH(3)F and DME inhibited N(2)O production. In field experiments with surface flux chambers and intact cores, CH(3)F reduced the release of N(2)O from soils to the atmosphere by 20- to 30-fold. Inhibition by CH(3)F also resulted in decreased NO(3) + NO(2) levels and increased NH(4) levels in soils. CH(3)F did not affect patterns of dissimilatory nitrate reduction to ammonia in cell suspensions of a nitrate-respiring bacterium, nor did it affect N(2)O metabolism in denitrifying soils. CH(3)F and DME will be useful in discriminating N(2)O production via nitrification and denitrification when both processes occur and in decoupling these processes by blocking NO(2) and NO(3) production.     

Effect of hydrogen peroxide on neuron sensitivity to acetylcholine

The dose-dependent effect of hydrogen peroxide on snail neuromembrane chemosensitivity was studied by means of standard voltage-clamp method. Short-term exposure (7 min) of neurons to H(2)O(2) (10(-11)-10(-4) M) caused dose-dependent depression of Acetylcholine (Ach)-induced ionic currents in the membrane. The H(2)O(2)-induced depression of Ach-sensitivity of membrane was more pronounced in K(+)-free solution than in normal physiological solution and it disappeared in cold medium (5 degrees C). The H(2)O(2) (10(-11)-10(-4) M) decreased membrane electrical conductivity and cell volume. The dose-dependent decrease in Ach-sensitivity of the snail neuromembrane by H(2)O(2) may be due to a decrease in the number of functionally active membrane receptors caused by a decrease in membrane active surface. H(2)O(2)-induced decrease in Ach-sensitivity has a metabolic but Na(+)-K(+) pump independent character, the nature of which is the subject for current investigation.     

Secondary stabilization reactions and proton-coupled electron transport in photosystem II investigated by electroluminescence and fluorescence spectroscopy

The oxidized primary electron donor in photosystem II, P(680)(+), is reduced in several phases, extending over 4 orders of magnitude in time. Especially the slower phases may reflect the back-pressure exerted by water oxidation and provide information on the reactions involved. The kinetics of secondary electron-transfer reactions in the microseconds time range after charge separation were investigated in oxygen-evolving thylakoids suspended in H2O or D2O. Flash-induced changes of chlorophyll fluorescence yield and electric field-induced recombination luminescence were decomposed into contributions from oxidation states S(0), S(1), S(2), and S(3) of the oxygen-evolving complex and interpreted in terms of stabilization kinetics of the initial charge-separated state S(j)Y(Z)P(680)(+)Q(A)(-)Q(B). In approximately 10% of the centers, only charge recombination took place. Otherwise, no static heterogeneity was involved in the microsecond reduction of P(680)(+) by Y(Z) (stabilization) or Q(A)(-) (recombination). The recombination component in active centers occurs mainly upon charge separation in S(3), and, in the presence of D2O, in S(2) as well and is tentatively attributed to the presence of Y(Z)(ox)S(j-1) in equilibrium with Y(Z)S(j). A 20-30 micros stabilization occurs in all S-states, but to different extents. Possible mechanisms for this component are discussed. D2O was found to decrease: (i) the rate of the reaction Y(Z)(ox)S(1) to Y(Z)S(2), (ii) the equilibrium constant between P680(+)Y(Z)S(2) and P(680)Y(Z)(ox)S(2), (iii) the rate of the slow phase of P(680)(+) reduction for the S(3) --> S(0) transition, and (iv) the rate of electron transfer from Q(A)(-) to Q(B) /Q(B)(-). The increased 'miss probability' in D2O is due to (iii).     

Reactions of thiocyanate in the mixture of nitrite and hydrogen peroxide under acidic conditions: investigation of the reactions simulating the mixture of saliva and gastric juice

Nitrite and SCN(-) in saliva can mixes with H(2)O(2) in the stomach. The mixing can result in the formation of ONOOH. It is not yet known how salivary SCN(-) reacts with ONOOH. An objective of the present study was to elucidate the reaction between ONOOH and SCN(-). In nitrite/H(2)O(2) systems at pH 2, SCN(-) inhibited the consumption of nitrite and the formation of O(3)(-). SCN(-) enhanced the decomposition of ONOOH and H(2)O(2) in HNO(2)/H(2)O(2) systems. Accompanying the reactions, sulfate was formed, suggesting that ONOOH oxidized SCN(-). SCN(-) inhibited the nitration of phenolics induced by HNO(2)/H(2)O(2). The inhibition is discussed taking SCN(-)-dependent reduction of ONOOH to HNO(2) into consideration. SCN(-) also inhibited H(2)O(2)-induced consumption of nitrite and nitration of phenolics in acidified saliva. The result obtained in this study suggests that salivary SCN(-) can reduce ONOOH to O(2)(-)/HNO(2) inhibiting nitrating reactions in the stomach.     

Conformational preferences of anticodon 3'-adjacent hypermodified nucleic acid base cis-or trans-zeatin and its 2-methylthio derivative, cis-or trans- ms(2)zeatin

Conformational preferences of the hypermodified nucleic acid bases N6-(Delta(2)-cis-hydroxyisopentenyl)adenine, cis-io(6)Ade also known as cis-zeatin, and N(6)-(Delta(2)-trans-hydroxyisopentenyl)adenine, trans-io(6)ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms(2)io(6)Ade or cis-ms(2)zeatin, and trans-ms(2)io6Ade or trans-ms(2)zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AM1 and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io(6)Ade, trans-io(6)Ade, cis-ms(2)io(6)Ade and trans-ms(2)io(6)Ade are such that in each of these molecules the isopentenyl substituent spreads away (has "dista" conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io(6)Ade as well as cis-ms(2)io(6)Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io(6)Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms(2)io(6)Ade, possible O-H...S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms(2)io(6)Ade as well as trans-ms(2)io(6)Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this non-obstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i(6)Ade and ms(2)i(6)Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms(2)-zeatin (ms(2)io(6)Ade) modification.     

Antioxidant properties of cystic fibrosis sputum

Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H(2)O(2)), a physiological oxidant, is not elevated in CF exhalates. H(2)O(2) may be neutralized by antioxidants in CF airway secretions. The H(2)O(2)-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H(2)O(2) cytotoxicity model. 16HBE14o- cells were exposed to H(2)O(2) in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H(2)O(2) neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 h and cell viability (propidium iodide) after 24 h of H(2)O(2) exposure. Furthermore, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H(2)O(2) in both control media (i.e., 0 or 10% FBS). Furthermore, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H(2)O(2). In contrast, there was 100% cytotoxicity in cells exposed to 600 microM H(2)O(2) in both control media. The H(2)O(2)-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H(2)O(2) and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H(2)O(2).     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice

Adenosine plays a role in physiological and pathological conditions, and A(2) adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A(2B)AR and its relationship to the A(2A)AR in coronary flow (CF) changes using A(2B) single-knockout (KO) and A(2A/2B) double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A(2A)KO and A(2B)KO and A(2A/2B)DKO mice. BAY 60-6583 (a selective A(2B) agonist) had no effect on CF in A(2B)KO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min(-1)·g(-1)). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A(2B)KO mice (maximum of 34.6 ± 4.7 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Also, CGS-21680 (a selective A(2A) agonist) increased CF in A(2B)KO mice (maximum of 29 ± 1.9 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min(-1)·g(-1)). SCH-58261 (an A(2A)-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A(2B)KO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min(-1)·g(-1)) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min(-1)·g(-1)). NECA did not induce any increase in CF in A(2A/2B)DKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Furthermore, the mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker 5-hydroxydecanoate had no effect on the NECA-induced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min(-1)·g(-1)), A(2A)KO (12.5 ± 2.3 ml·min(-1)·g(-1)), and A(2B)KO (16.2 ± 0.8 ml·min(-1)·g(-1)) mice was significantly blunted by the K(ATP) channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min(-1)·g(-1), respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min(-1)·g(-1)) and BAY 60-6583-induced (16.4 ± 1.60 ml·min(-1)·g(-1)) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min(-1)·g(-1), respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A(2A)ARs in A(2B)KO mice and demonstrates that both A(2A)ARs and A(2B)ARs induce CF changes through K(ATP) channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders.     

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.     

Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.     

Up-regulation of dopamine D(2)L mRNA levels in the ventral tegmental area and dorsal striatum of amphetamine-sensitized C57BL/6 mice: role of Ca(v)1.3 L-type Ca(2+) channels

Dopamine D(2) long (D(2)L) and D(2) short (D(2)S) isoforms of the D(2) receptor play an important role in psychostimulant-induced neuronal adaptations. In this study, we used quantitative real-time PCR to specifically amplify these two splice variants to examine their mRNA expression in the dorsal striatum (dStr), nucleus accumbens (NAc) and the ventral tegmental area (VTA) of amphetamine-sensitized C57BL/6 mice. We found a significant increase in D(2)L mRNA in the VTA and dStr of amphetamine-treated mice that positively correlated with the sensitized locomotor response. We also found a significant increase in D(2)S mRNA in the VTA. We further examined the role of the Ca(v)1.3 subtype of L-type Ca(2+) channels in up-regulation of D(2)L and D(2)S mRNA in the VTA. Amphetamine-pretreated Ca(v)1.3 wild-type (Ca(v)1.3(+/+)) mice exhibited sensitized behavior and a significant increase in D(2)L and D(2)S mRNA compared with saline-pretreated mice Amphetamine-pretreated homozygous Ca(v)1.3 knockout (Ca(v)1.3(-/-)) mice did not exhibit sensitized behavior. There was a significant increase in D(2)S mRNA, but not D(2)L mRNA. In conclusion, our results find that amphetamine increases D(2)L mRNA expression in the dStr and the VTA, an adaptation that correlates with expression of sensitized behavior and dependence on Ca(v)1.3 Ca(2+) channels.     

Lipoolygosaccharide indirectly enhances inflammatory lesions in lungs as a primary infection site by non-encapsulated and type B Haemophilus influenzae through production of cytokines

We investigated the role of cytokines in differences in histopathologic changes in the lung between bronchopneumonia caused by non-encapsulated Haemophilus influenzae strain 770235f(0)b(0)and systemic disease caused by type b H. influenzae strain 770235f(0)b(+). Tumour necrosis factor-alpha (TNF-alpha), interleukin-(IL)-6 and IL-1 beta levels in bronchoalveolar lavage fluid (BALF) samples of mice infected with strain 770235f(0)b(0)were higher than in those infected with strain 770235f(0)b(+)until 24 h post-infection. Serum IL-6 rapidly increased in strain 770235f(0)b(0)infection after 72 h post-infection. Serum TNF-alpha level in strain 770235f(0)b(0)infection appeared earlier than in strain 770235f(0)b(+)infection. IL-1 beta production in strain 770235f(0)b(+)infection was later than in strain 770235f(0)b(0)infection. Moreover, a biphasic concentration pattern of TNF-alpha and IL-6 was noted in BALF of mice with strain 770235f(0)b(0)infection.