Bifunctional combined Au-Fe(2)O(3) nanoparticles for induction of cancer cell-specific apoptosis and real-time imaging

We demonstrate bifunctional combined Au-Fe(2)O(3) nanoparticles (NPs) for selectively induction of apoptosis in cancer cells and real-time imaging. The as-prepared Au-Fe(2)O(3) NPs combine the merits of both Au and γ-Fe(2)O(3) NPs, maintaining excellent fluorescence quenching property and catalytic activity. Conjugated with α(Ⅴ)β(3) integrin-targeting peptide (RGD) and fluorescein isothiocyanate (FITC)-labeled capsase-3 recognition sequence (DEVD) on the Au surface, the resulting RGD/FITC-DEVD-Au-Fe(2)O(3) NPs bind preferentially to integrin α(Ⅴ)β(3)-rich human liver cancer cells (HepG2), sequentially initiate catalytic formation of hydroxyl radicals (·OH) and enable the real-time monitoring of·OH-induced caspase-3-dependent apoptosis in these cancer cells. Furthermore, the catalytic activity of RGD/FITC-DEVD-Au-Fe(2)O(3) NPs is much higher than that of individual γ-Fe(2)O(3) NPs due to the polarization effect at the Au-Fe(2)O(3) interface. Such bifunctional Au-Fe(2)O(3) NPs exhibit simultaneous targeting, therapeutic and imaging functions and are therefore promising for future therapeutic applications in cancer.     

Combinatorial roles for histamine H1-H2 and H3-H4 receptors in autoimmune inflammatory disease of the central nervous system

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (HA) and its receptors have been implicated in disease pathogenesis. HA exerts its effects through four different G protein-coupled receptors designated H(1)-H(4). We previously examined the effects of traditional single HA receptor (HR) knockouts (KOs) in experimental allergic encephalomyelitis (EAE), the autoimmune model of MS. Our results revealed that H(1) R and H(2) R are propathogenic, while H(3) R and H(4) R are antipathogenic. This suggests that combinatorial targeting of HRs may be an effective disease-modifying therapy (DMT) in MS. To test this hypothesis, we generated H(1) H(2) RKO and H(3) H(4) RKO mice and studied them for susceptibility to EAE. Compared with wild-type (WT) mice, H(1) H(2) RKO mice developed a less severe clinical disease course, whereas the disease course of H(3) H(4) RKO mice was more severe. H(1) H(2) RKO mice also developed less neuropathology and disrupted blood brain barrier permeability compared with WT and H(3) H(4) RKO mice. Additionally, splenocytes from immunized H(1) H(2) RKO mice produced less interferon(IFN)-γ and interleukin(IL)-17. These findings support the concept that combined pharmacological targeting of HRs may be an appropriate ancillary DMT in MS and other immunopathologic diseases.     

The α1β1 and α2β1 Integrins Provide Critical Support for Vascular Endothelial Growth Factor Signaling, Endothelial Cell Migration, and Tumor Angiogenesis

Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the alpha(1)beta(1) and alpha(2)beta(1) integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either alpha(1)beta(1) or alpha(2)beta(1) supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on alpha(1)beta(1) and alpha(2)beta(1) as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of alpha(1)beta(1) and alpha(2)beta(1) in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of alpha(1)beta(1) and alpha(2)beta(1) substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the alpha(1)beta(1) and alpha(2)beta(1) integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.     

Molecular identification of pathogenetic IdLNF+1 autoantibody idiotypes derived from the NZBxSWR F1 model for systemic lupus erythematosus

The acceleration of nephritis in SNF(1) mice by CD4(+) T-cell clones reactive with a nephritogenic idiotype, Id(LN)F(1) [1], as well as the ability of anti-Id(LN)F(1) antisera to down-regulate the production of Id(LN)F(+)(1) immunoglobulin (Ig) in vivo and delay nephritis [2], suggests that dysregulation of this idiotype may contribute to the development of SNF(1) nephritis. Herein, we show that a monoclonal Id(LN)F(1)-expressing antibody, 540, significantly (P< or = 0.01) stimulated Id(LN)F(1)-reactive T-cell clones B6 and D2 to proliferate, while other Id(LN)F+1 antibodies did not. Further, injection of 540-producing hybridoma cells into nonautoimmune (SWRxBalb/c)F(1) mice resulted in the deposition of Id(LN)F(+)(1) Ig in the kidneys, in a pattern indicative of early nephritis. To identify the pathogenetic Id(LN)F(1) epitope(s) at the molecular level, we compared the deduced amino acid sequences of the heavy and light chain variable regions of pathogenetic and non-pathogenetic Id(LN)F(1)-expressing Igs 540, 317, and 533. Two overlapping peptides derived from the V(H) sequence of 540 (aa 54-66 and 62-73), which both contain the triple basic amino acid motif K(X)K(X)K, stimulated SNF(1) T cells and T-cell clones B6 and D2. These results further support the involvement of a subset of Id(LN)F(1)-expressing Ig in SNF(1) nephritis.     

End-tidal pressure of CO<Subscript>2</Subscript> and exercise performance in healthy subjects

High arterial CO(2) pressure (P(a)CO(2)) measured in athletes during exercise suggests inadequate hyperventilation. End-tidal CO(2) pressure (P (ET)CO(2)) is used to estimate P(a)CO(2.) However, P(ET)CO(2) also depends on exercise intensity (CO(2) production, .VCO2) and ventilation efficiency (being P(ET)CO(2) function of respiratory rate). We evaluated P(ET)CO(2) as a marker, which combines efficiency of ventilation and performance. A total of 45 well-trained volunteers underwent cardiopulmonary tests and were grouped according to P(ET)CO(2) at respiratory compensation (RC): Group 1 (P(ET)CO(2) 35.1-41.5 mmHg), Group 2 (41.6-45.7) and Group 3 (45.8-62.6). At anaerobic threshold, RC and peak exercise, ventilation (.VE) was similar, but in Group 3, a greater tidal volume (Vt) and lower respiratory rate (RR) were observed. Peak exercise workload and .VO2 were lowest in Group 1 and similar between Group 2 and 3. Group 3 subjects also showed high peak .VCO2 suggesting a greater glycolytic metabolism. In conclusion, a high P(ET)CO(2) during exercise is useful in identifying a specific respiratory pattern characterized by high tidal volume and low respiratory rate. This respiratory pattern may belong to subjects with potential high performance.     

Adult mesenchymal stem cells support cisplatin-treated dorsal root ganglion survival

Human cannabinoid receptors 1 (hCB(1)R) and 2 (hCB(2)R) are expressed in the CNS and couple to G(i)/G(o)-proteins. The aim of this study was to compare coupling of hCB(1)R and hCB(2)R to G(alpha)(i2)beta(1)gamma(2) in Sf9 insect cells. High-affinity agonist binding at hCB(1)R, but not at hCB(2)R, was resistant to guanine nucleotides. hCB(1)R activated G(alpha)(i2)beta(1)gamma(2) much more rapidly than hCB(2)R in the [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTPgammaS) binding assay. Moreover, hCB(1)R exhibited a higher constitutive activity than hCB(2)R as assessed by the relative inhibitory effects of inverse agonists on [(35)S]GTPgammaS binding and steady-state high-affinity GTPase activity compared to the stimulatory effects of the hCB(1/2)R agonist CP 55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol]. G(alpha)(i2)beta(1)gamma(2) coupled to hCB(2)R exhibited higher GDP- and GTPgammaS-affinities than G(alpha)(i2)beta(1)gamma(2) coupled to hCB(1)R. NaCl effectively reduced constitutive activity of hCB(1)R but not of hCB(2)R. Collectively, hCB(1)R and hCB(2)R couple differentially to G(alpha)(i2)beta(1)gamma(2). Moreover, hCB(1)R exhibits higher constitutive activity than hCB(2)R. These differences point to distinct functions of hCB(1)R and hCB(2)R in the CNS.     

Differential antiherpes activity of the (E)-and (Z)-isomers of 5-(2-fluorovinyl)-2'-deoxyuridine (FVUdR)

Four novel analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine [(E)-BrVUdR]--the (E)- and (Z)-isomer of 5-(2-fluorovinyl)-UdR (FVUdR), (Z)-5-(2-carboxy-2-fluorovinyl)-UdR [(Z)-COOH-FVUdR], and (E)-5-(2-ethoxyvinyl)-UdR [(E)-EOVUdR] were compared with the reference compounds (E)-BrVUdR and 5-vinyl-UdR (VUdR) for their inhibitory effects on plaque formation of herpes simplex virus type 1 (HSV-1 strain 77) and type 2 (HSV-2 strain 82) in human embryonic lung fibroblast (HELF) cell cultures. (Z)-FVUdR and (Z)-COOH-FVUdR were completely inactive against HSV-1 and HSV-2 (ID50 greater than 500 microM). For the other analogues the following order of decreasing potency was found: (E)-BrVUdR greater than VUdR greater than (E)-FVUdR much greater than (E)-EOVUdR (against HSV-1) and VUdR much greater than (E)-BrVUdR greater than (E)-FVUdR much greater than (E)-EOVUdR (against HSV-2).     

Engagement of the EP2 prostanoid receptor closes the K+ channel KCa3.1 in human lung mast cells and attenuates their migration

Human lung mast cells (HLMC) express the Ca(2+)-activated K(+) channel K(Ca)3.1, which plays a crucial role in their migration to a variety of diverse chemotactic stimuli. K(Ca)3.1 activation is attenuated by the beta(2)-adrenoceptor and the adenosine A(2A) receptor through a G(s)-coupled mechanism independent of cyclic AMP. Prostaglandin E(2) promotes degranulation and migration of mouse bone marrow-derived mast cells through the G(i)-coupled EP(3) prostanoid receptor, and induces LTC(4) and cytokine secretion from human cord blood-derived mast cells. However, PGE(2) binding to the G(s)-coupled EP(2) receptor on HLMC inhibits their degranulation. We show that EP(2) receptor engagement closes K(Ca)3.1 in HLMC. The EP(2) receptor-specific agonist butaprost was more potent than PGE(2) in this respect, and the effects of both agonists were reversed by the EP(2) receptor antagonist AH6809. Butaprost markedly inhibited HLMC migration induced by chemokine-rich airway smooth muscle-conditioned media. Interestingly, PGE(2) alone was chemotactic for HLMC at high concentrations (1 microM), but was a more potent chemoattractant for HLMC following EP(2) receptor blockade. Therefore, the G(s)-coupled EP(2) receptor closes K(Ca)3.1 in HLMC and attenuates both chemokine- and PGE(2)-dependent HLMC migration. EP(2) receptor agonists with K(Ca)3.1 modulating function may be useful for the treatment of mast cell-mediated disease.     

Behavior of junction channels between rat glomus cells during normoxia and hypoxia

The activity of gap junction channels between cultured and clustered carotid body glomus cells of the rat was studied with dual voltage clamping during normoxia (PO(2) 300 Torr) and hypoxia induced by sodium dithionite (Na(2)S(2)O(4)) or 100% N(2). Na(2)S(2)O(4) reduced the saline PO(2) to approximately 10 Torr, whereas 100% N(2) reduced ambient O(2) to approximately 60 Torr. The following observations were made. 1) In normoxia, the intercellular macroconductance (G(j) = 3.0 +/- 1.01 ns, mean +/- SE) was changed unevenly (increased and decreased) under hypoxic conditions by either agent, although N(2) produced the largest changes. 2) The intercellular microconductances of the channels (g(j) = 104.44 +/- 10.16 pS under normoxic conditions) significantly decreased in 100% N(2) but showed depressions and enhancements in Na(2)S(2)O(4). 3) The conductance of single-junction channels (SChs), calculated as g(j) variance/mean g(j), yielded a mean of approximately 17.6 pS. Larger values were obtained with manual measurements of the data (approximately 34 pS). Hypoxic hypoxia (induced by 100% N(2)) significantly depressed the conductance of SChs when calculated from digitized records or from manual measurements. Hypoxia induced by Na(2)S(2)O(4) did not significantly change junctional conductance. 4) The number of intercellular channels, calculated as g(j)/SCh g(j), had a mean of approximately 452 (range 1 to 2,471). During N(2)-induced hypoxia, this number significantly decreased to approximately 84 but remained unchanged during Na(2)S(2)O(4) hypoxia. 5) The mean open time of junction channels varied from 4 to 30 ms in different experiments, having an overall mean of mu = 11.33 +/- 0.33 ms. This value was significantly reduced by 100% N(2) but was not changed by Na(2)S(2)O(4). 6) Intracellular calcium ([Ca(2+)](i)), 46.2 +/- 4.84 nM under normoxia, significantly increased to 77.32 +/- 11.27 nM with Na(2)S(2)O(4) and to 66.39 +/- 11.64 nM with 100% N(2). It is concluded that 100% N(2) uncouples glomus cells by significantly reducing intercellular macro- and microconductances. Hypoxia induced by Na(2)S(2)O(4) had variable effects. The coupling effects of hypoxia may depend on, or be aided by, increases in [Ca(2+)](i) and/or intracellular pH changes. However, secreted transmitters and ATP plus the effects of hypoxia on second messengers and other cytoplasmic components may also play an important role in this phenomenon.     

Prostaglandin E2 release in gastric antral mucosa of guinea-pigs: basal PGE2 release by cyclo-oxygenase 2 and ACh-stimulated PGE2 release by cyclo-oxygenase 1

Prostaglandin E(2) (PGE(2)), which is generated by two isoforms of cyclo-oxygenase (COX(1) and COX(2)), is a key mediator in gastric mucosal defense. In the present study, antral mucosa of guinea-pigs was incubated with various agonists or antagonists in a medium, the PGE(2) concentration of which was measured using a PGE(2) EIA kit. Prostaglandin E(2) was released from the antral mucosa spontaneously (basal PGE(2) release) and acetylcholine (ACh, 10 microM) enhanced the PGE(2) release (ACh-stimulated PGE(2) release) was mediated via intracellular Ca(2+) concentration ([Ca(2+)](i)). Arachidonic acid enhanced both forms of PGE(2) release, and a phospholipase A(2) inhibitor (amylcinnamoyl anthranilic acid) and COX inhibitors (acetylsalicylic acid and indomethacin) decreased them. 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol (SC560, 100 nm, a COX(1)-selective inhibitor) inhibited ACh-stimulated PGE(2) release without any decrease in basal PGE(2) release. N-(2-Cyclohexyloxy-4-nitrophenyl) methanesulphonamide (NS398, 20 microM, a COX(2)-selective inhibitor) decreased basal PGE(2) release without any reduction of ACh-stimulated PGE(2) release. However, ionomycin (a Ca(2+) ionophore) increased PGE(2) release from antral mucosa in the presence of SC560 or NS398, suggesting that COX(1) and COX(2) are regulated by [Ca(2+)](i). These findings indicate that COX(1)-containing cells have ACh receptors but COX(2)-containing cells do not. Moreover, in isolated antral epithelial cells, SC560 decreased basal and ACh-stimulated PGE(2) release, but NS398 did not. In conclusion, in antral mucosa, basal PGE(2) release is mainly maintained by COX(2) of non-epithelial cells, and ACh-stimulated PGE(2) release is maintained by COX(1) of epithelial cells.     

Effect of sodium carbonate solution on self-setting properties of tricalcium silicate bone cement

In this study, the effects of sodium carbonate (Na(2)CO(3) ) solution with different concentrations (10, 15, 20, and 25 wt%) as liquid phase on the setting time and compressive strength of tricalcium silicate bone cements are investigated. The in vitro bioactivity and degradability of the resultant Ca(3)SiO(5)-Na(2)CO(3) solution paste was also studied. The results indicate that as the concentration of Na(2)CO(3) solution varies from 0 to 25 wt%, the initial and final setting time of the cement decrease significantly from 90 to 20 min and from 180 to 45 min, respectively. After setting for 24 h, the compressive strength of Ca(3)SiO(5)-Na(2)CO(3) solution paste reaches 5.1 MPa, which is significantly higher than that of Ca( 3)SiO(5)-water cement system. The in vitro bioactivity of the cements is investigated by soaking in simulated body fluid (SBF) for 7 days. The results show that the Ca(3)SiO(5)-Na(2)CO( 3) solution bone cement has a good bioactivity and can degrade in Ringer's solution. The results indicate that Na(2)CO(3) solution as a liquid phase significantly improves the self-setting properties of Ca( 3)SiO(5) cement as compared to water. The Ca(3)SiO( 5) cement paste prepared using Na(2)CO(3) solution shows good bioactivity and moderate degradability, and the Ca(3)SiO( 5)-Na(2)CO(3) solution system may be used as degradable and bioactive bone defect filling materials.     

Targeted dual-contrast T1- and T2-weighted magnetic resonance imaging of tumors using multifunctional gadolinium-labeled superparamagnetic iron oxide nanoparticles

Development of a multifunctional nanoparticle (NP) system allowing for dual-contrast T(1)- and T(2)-weighted targeted magnetic resonance (MR) imaging of tumors could significantly improve the diagnosis accuracy. In this study, superparamagnetic silica-coated iron oxide core-shell nanoparticles (Fe(3)O(4)@SiO(2) NPs) with a diameter of approximately 21 nm were synthesized via a thermal decomposition approach and were aminated through silanization. The amine-functionalized Fe(3)O(4)@SiO(2) NPs enabled the covalent conjugation of a paramagnetic gadolinium complex (Gd-DTPA, DTPA: diethylenetriamine pentaacetic acid) and an arginine-glycine-aspartic acid (RGD) peptide as a targeting ligand onto their surface. The formed Fe(3)O(4)@SiO(2)(Gd-DTPA)-RGD NPs are water-dispersible, stable, and biocompatible as confirmed by MTT cell viability assay. Relaxivity measurements show that they have a T(1) relaxivity (r(1)) of 4.2 mM(-1) s(-1) and T(2) relaxivity (r(2)) of 17.4 mM(-1) s(-1) at the Gd/Fe molar ratio of 0.3:1, suggesting a possibility to use them as both T(1) positive and T(2) negative contrast agents. In vitro and in vivo MR imaging experiments show that the developed multifunctional Fe(3)O(4)@SiO(2)(Gd-DTPA)-RGD NPs enable targeted dual-contrast T(1)- and T(2)-weighted MR imaging of tumor cells over-expressing high-affinity α(v)β(3) integrin in vitro and in vivo. Our results clearly indicate that the approach to forming multifunctional Fe(3)O(4)@SiO(2)(Gd-DTPA)-RGD NPs could be extended for fabricating other biologically active NPs for T(1)- and T(2)-weighted MR imaging of other biological systems with high accuracy.     

Betamethasone modulates the biological function of human polymorphonuclear leukocytes

We have examined the influence of betamethasone (BT) on cyclic AMP (cAMP) metabolism and lysosomal enzyme release from highly purified (approximately equal to 99%) human polymorphonuclear leukocytes (PMNs). Preincubation (1-24 h) of human PMNs with BT (10(-9)-10(-5) M) had no effect on either cAMP content or on beta-glucuronidase release induced by formyl-containing tripeptide (f-met peptide). Preincubation (16-24 h) of PMNs with BT (10(-8)-10(-7) M) dose-dependently potentiated the cAMP accumulation caused by beta-agonists (isoproterenol), adenosine A2/Ra agonist (NECA), prostaglandin E1 (PGE1) and histamine in PMNs. Similarly, BT potentiated the inhibition of f-met peptide-induced beta-glucuronidase release from human PMNs caused by PGE1 (10(-6) M), histamine (2 X 10(-5) M), NECA (10(-4) M) and isoproterenol (10(-6) M).     

Construction of novel Brassica napus genotypes through chromosomal substitution and elimination using interploid species hybridization

A synthetic Brassica napus rapeseed with genome composition of A(r)A(r)C(c)C(c), made by combining A(r) from B. rapa (A(r)A(r)) and C(c) from B. carinata (B(c)B(c)C(c)C(c)), is valuable for making new genes available to breeders and gaining heterosis in crosses. An intergenomic hybrid A(n)A(r)C(n)C(c) was made from a hybrid between natural Brassica napus (A(n)A(n)C(n)C(n)) and a synthetic rapeseed. To construct the synthetic Brassica napus, hexaploid plants (2n=54, A(r)A(r)B(c)B(c)C(c)C(c)) were first obtained through chromosome doubling of trigenomic hybrids (2n=27, A(r)B(c)C(c)) between Brassica carinata (2n=34) and B. rapa (2n=20). Pentaploid hybrids (2n=46, A(r)A(n)B(c)C(c)C(n)) were then produced by crossing the hexaploid with the pollen of natural B. napus (2n=38). Chromosomes with dual and single B(c) genomes were observed in somatic cells of hexaploid and pentaploid plants. About 80% of pollen mother cells of pentaploid hybrids had 19 or more bivalents, indicating that the bivalents from A(r)/A(n) and C(c)/C(n) chromosomes were normally formed. The occurrence of trivalents and quadrivalents at diakinesis suggested that B(c), A(n) and A(r) or B(c), C(n) and C(c) homologous pairing and exchange might happen. The variable number of laggards, 3 and 4 in most cases, were observed in the majority of PMCs at anaphase. Results from genomic in situ hybridization showed that the laggards belonged mainly to the B(c) genome, suggesting that the B(c) genome could be eliminated in the gametes of pentaploid hybrids. 16.15% of seeds derived from self-pollinated pentaploids have 38 chromosomes, and 90% of 38-chromosome seeds were completely excluded B(c) genome. The cytological results of this experiment suggested that it is possible to obtain new materials with genome composition of A(r)A(r)C(c)C(c) for rapeseed breeding.     

Fluorine-18-labeled Gd3+/Yb3+/Er3+ co-doped NaYF4 nanophosphors for multimodality PET/MR/UCL imaging

Molecular imaging modalities provide a wealth of information that is highly complementary and rarely redundant. To combine the advantages of molecular imaging techniques, (18)F-labeled Gd(3+)/Yb(3+)/Er(3+) co-doped NaYF(4) nanophosphors (NPs) simultaneously possessing with radioactivity, magnetic, and upconversion luminescent properties have been fabricated for multimodality positron emission tomography (PET), magnetic resonance imaging (MRI), and laser scanning upconversion luminescence (UCL) imaging. Hydrophilic citrate-capped NaY(0.2)Gd(0.6)Yb(0.18)Er(0.02)F(4) nanophosphors (cit-NPs) were obtained from hydrophobic oleic acid (OA)-coated nanoparticles (OA-NPs) through a process of ligand exchange of OA with citrate, and were found to be monodisperse with an average size of 22 × 19 nm. The obtained hexagonal cit-NPs show intense UCL emission in the visible region and paramagnetic longitudinal relaxivity (r(1) = 0.405 s(-1)·(mM)(-1)). Through a facile inorganic reaction based on the strong binding between Y(3+) and F(-), (18)F-labeled NPs have been fabricated in high yield. The use of cit-NPs as a multimodal probe has been further explored for T(1)-weighted MR and PET imaging in vivo and UCL imaging of living cells and tissue slides. The results indicate that (18)F-labeled NaY(0.2)Gd(0.6)Yb(0.18)Er(0.02) is a potential candidate as a multimodal nanoprobe for ultra-sensitive molecular imaging from the cellular scale to whole-body evaluation.     

Interactions among adenosine deaminase,adenosine A(1) receptors and dopamine D(1) receptors in stably cotransfected fibroblast cells and neurons

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding.The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.     

Restoration by VIP of the carbachol-stimulated Cl- secretion in TTX-treated guinea pig distal colon

To determine if vasoactive intestinal peptide (VIP) restores neural activity from tetrodotoxin (TTX) blockade, we studied the effects of VIP and related agents on carbachol (Cch)-induced Cl(-) secretion in control-isolated guinea pig distal colon and in that treated with TTX. The short circuit current (I(sc)) increased dose-dependently after serosal applications of Cch (10(-6) - 2 x 10(-5) M) and VIP (5 x 10(-9) - 10(-7) M). But no additive or synergistic increase in I(sc) was observed. Cch- and VIP-induced I(sc) was completely abolished by a serosal application of TTX (10(-6) M). However, a serosal application, not mucosal, of VIP (10(-7) M) and 8-bromo-cAMP (10(-3) M) restored the Cch-stimulated, TTX-inhibited I(sc) by 113% and 75.8%, respectively. Furthermore, mucosal and serosal applications of forskolin (aden late cyclase activator) restored the I(sc) by 43.9% and 65.3%, respectively. The restored I(sc) was completely abolished by atropine (muscarinic receptor antagonist). These results suggest that VIP may restore the cholinergic activity by increasing the level of intracellular cAMP, and that cholinergic neuron is very likely to be responsible for the regulation of Cl(-) secretion at neuroepithelial junctions. The exact mechanism of VIP's effect on the TTX-inhibited epithelial Cl(-) secretion, and its possible usefulness in the treatment of TTX-induced pathophysiological conditions, remain to be determined.     

Possible release of an ArgGlyArgProGln pentapeptide with innate immunity properties from acidic proline-rich proteins by proteolytic activity in commensal streptococcus and actinomyces species

This study suggests degradation of salivary acidic proline-rich proteins (PRPs) into potential innate-immunity-like peptides by oral Streptococcus and Actinomyces species. PRP degradation paralleled cleavage of Pro-containing substrates. PRP degradation by S. gordonii strain SK12 instantly released a Pyr(1)-Pro(104)Pro(105) and a Gly(111)-Pro(149)Gln(150) peptide together with a presumed Arg(106)Gly(107)Arg(108)Pro(109)Gln(110) pentapeptide. The synthetic Arg(106)Gly(107)Arg(108)Pro(109)Gln(110) peptide desorbed bound bacteria and counteracted sucrose-induced decrease of dental plaque pH in vitro.     

Molecular components of transient outward potassium current in cultured neonatal rat ventricular myocytes

We have previously reported that K(v1.4), K(v4.2), and K(v4.3) mRNAs are present in adult and neonatal rat ventricular myocytes, and that transient outward potassium current (I(to)) recovers from inactivation with a slow (I(to,s)) and a fast (I(to,f)) time course. This study was designed to determine the molecular correlates of I(to,s) and I(to,f) in cultured neonatal rat ventricular myocytes (NRVM) employing dominant-negative adenoviral infections to manipulate the function of endogenous I(to)-encoding K+ channels. Western blot data from cultured NRVM showed that K(v1.4), K(v4.2), and K(v4.3) channel proteins are present in these myocytes. The biphasic recovery from inactivation of I(to) in control GFP-infected myocytes demonstrated equal contribution of I(to,s) and I(to,f) in NRVM. Infection of cultured NRVM with adenoviruses expressing full-length K(v1.4) or K(v4.2) genes generated currents with recovery from inactivation kinetics similar to native I(to,s) and I(to,f) in GFP-infected myocytes, respectively. Overexpression of dominant-negative truncated K(v1.4) transgene (K(v1.4)N) caused a 51% reduction in I(to), selectively removing the slowly recovering I(to,s). Overexpression of dominant-negative K(v4.2)N reduced I(to) by 53% and eliminated the fast-recovering I(to,f). Our results establish that, in neonatal rat ventricular myocytes, the shaker K(v1) family (probably K(v1.4) and/or K(v1.7)) underlies I(to,s), and that the shal K(v4) family (probably K(v4.2) and K(v4.3)) is responsible for I(to,f).