Phenols, a major group of antioxidant phytochemicals, have profound importance due to their biological and free radical scavenging activities. To identify their potential sources, extracts of some plants were studied for their total phenolic content (TPC), antioxidant (AOA) and free radical scavenging activities (FRSA) by different methods at multiple concentrations followed by specific phenolic composition. The amount of TPC varied from 2.8 mg/g (Withania somnifera, roots) to 107.8 mg/g (Cassia fistula, fruits) and the AOA from 24.2% (Curcuma zeoderia, leaves) to 96.9% (Trewia nudiflora, leaves). Bark of Azadirachta indica, fruits of C. fistula, and leaves and fruits of T. nudiflora were found to have high TPC (89.8- 107.8 mg/g) and high AOA (84.8-96.9%). Promising plant parts were studied for their FRSA and reducing power (RP), where the bark of A. indica, Casuarina equisetifolia and Cinnamomum zeylanicum, flowers of Indigofera tinctoria, fruits of Lawsonia inermis, and fruits and leaves of T. nudiflora showed a very low inhibitory concentration value ranging from 0.14 to 0.26 mg/ml, efficiency concentration value from 6.1 to 11.6 mg/mg DPPH and reducing power value from 0.6 to 2.8 ascorbic acid equivalents (ASE/ml), and reasonably high values (8.5 -16.2) of anti-radical power (ARP), indicating their strong FRSA. They also showed better inhibition of hydroxyl radical induced deoxyribose degradation than that of reference standard. Fruits of C. fistula with high phenols (107.8 mg/g) showed poor reducing power (5.9 ASE/ml) and ARP (4.7); in contrast, the bark of C. equisetifolia and fruits of L. inermis were with comparatively lower phenols (72.1 and 75.8 mg/g) but exhibited good ARP (16.2 and 14.4) and reducing power (0.7 and 0.6 ASE/ ml, respectively). Some of the plants were also found effective in protecting plasmid DNA nicking induced by hydroxyl radicals generated by Fenton's reaction. They were further assayed for their specific phenolic composition through high-performance liquid chromatography and MS/MS, where the amount of caffeic acid varied from 0.312 to 0.797 mg/g, chlorogenic acid from 0.018 to 2.109 mg/g, ellagic acid from 0.009 to 0.902 mg/g, ferulic acid from 0.036 to 0.078 mg/g, gallic acid from 0.192 to 3.597 mg/g, kaempferol from 0.011 to 0.910 mg/g, quercetin from 0.047 to 1.106 mg/g and rutin from 0.059 to 2.029 mg/g.

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA alpha-isoforms RIalpha, RIIalpha, and Calpha and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.

OBJECTIVE

To identify the genetic basis of a large consanguineous Spanish pedigree affected with autosomal recessive retinitis pigmentosa (arRP) with premature macular atrophy and myopia.

METHODS

After a high-throughput cosegregation gene chip was used to exclude all known RP and Leber congenital amaurosis (LCA) candidates, genome-wide screening and linkage analysis were performed. Direct mutational screening identified the pathogenic mutation, and primers were designed to obtain the RT-PCR products for isoform characterization.

RESULTS

Mutational analysis detected a novel homozygous PROM1 mutation, c.869delG in exon 8 cosegregating with the disease. This variant causes a frameshift that introduces a premature stop codon, producing truncation of approximately two-thirds of the protein. Analysis of PROM1 expression in the lymphocytes of patients, carriers, and control subjects revealed an aberrant transcript that is degraded by the nonsense-mediated decay pathway, suggesting that the disease is caused by the absence of the PROM1 protein. Three (s2, s11 and s12) of the seven alternatively spliced isoforms reported in humans, accounted for 98% of the transcripts in the retina. Given that these three contained exon 8, no PROM1 isoform is expected in the affected retinas.

CONCLUSIONS

A remarkable clinical finding in the affected family is early macular atrophy with concentric spared areas. The authors propose that the hallmark of PROM1 truncating mutations is early and severe progressive degeneration of both rods and cones and highlight this gene as a candidate of choice to prioritize in the molecular genetic study of patients with noncanonical clinical peripheral and macular affectation.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, CCCCCRp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.

The inhibition of voltage-activated Ca2+ channel currents by cortisol (hydrocortisone), the principal glucocorticoid in man and guinea pig, was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region using whole-cell voltage-clamp recordings. Steady-state inhibition by cortisol of the peak Ca2+ channel current evoked by depolarization from -80 to -10 mV increased in a concentration-dependent fashion, with a maximal inhibition of 63 +/- 4% of the total current at 100 microM. Cortisone had a maximal 17 +/- 2% inhibition at 10 microM. Corticosterone and the metabolite allotetrahydrodeoxycorticosterone exhibited a plateau of inhibition of around 15% and 25%, respectively, between 10 pM and 100 nM; both compounds continued to inhibit at concentrations>> 10(-7) M. Analysis of tail currents at -80 mV showed that cortisol and corticosterone had no effect on the voltage-dependent activation or deactivation of the Ca2+ channel current. However, cortisol slowed the activation of the current. Cortisol inhibited both the N-type or omega-conotoxin (CgTX)-sensitive, and the L-type or nifedipine (NIF)-sensitive Ca2+ channel current but had no effect on the CgTX/NIF-insensitive Ca2+ channel current. In neurons isolated from pertussis toxin (PTX)-treated animals, the cortisol inhibition was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) or with the specific inhibitors of protein kinase C (PKC), the pseudosubstrate PKC inhibitor (PKCI 19-31) (2 microM) and bisindolylmaleimide (BIS) (1 microM) significantly diminished the cortisol inhibition of the Ca2+ channel current. The specific inhibitor of cAMP-dependent protein kinase (PKA) inhibitor, Rp-cAMPS (100 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide-targeted alpha-therapy with 7.4 MBq of (212)Pb-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ReO-[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) ((212)Pb-DOTA-Re(Arg(11))CCMSH) cured 45% of B16/F1 murine melanoma-bearing CCCMSH therapy. The purpose of this study was to evaluate the potential of (203)Pb-DOTA-Re(Arg(11))CCMSH as a matched-pair SPECT agent for (212)Pb-DOTA-Re(Arg(11))CCMSH.

METHODS

DOTA-Re(Arg(11))<em>C</em><em>C</em>MSH was labeled with (203)Pb in 0.5 M NH(4)OAc buffer at pH 5.4. The internalization and efflux of (203)Pb-DOTA-Re(Arg(11))<em>C</em><em>C</em>MSH were determined in B16/F1 melanoma cells. The pharmacokinetics of (203)Pb-DOTA-Re(Arg(11))<em>C</em><em>C</em>MSH was examined in B16/F1 melanoma-bearing <em>C</em>57 mice. A micro-SPE<em>C</em>T/<em>C</em>T study was performed with (203)Pb-DOTA-Re(Arg(11))<em>C</em><em>C</em>MSH in a B16/F1 melanoma-bearing <em>C</em>57 mouse at 2 h after injection.

RESULTS

(203)Pb-DOTA-Re(Arg(11))CCMSH was easily prepared in NH(4)OAc buffer and completely separated from the excess nonradiolabeled peptide by reversed-phase high-performance liquid chromatography (RP-HPLC). (203)Pb-DOTA-Re(Arg(11))CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of (203)Pb-DOTA-Re(Arg(11))CCMSH activity internalized after a 20-min incubation at 25 degrees C. After incubation of the cells in culture medium for 20 min, 78% of internalized activity remained in the cells. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a biodistribution pattern similar to that of (212)Pb-DOTA-Re(Arg(11))CCMSH in B16/F1 melanoma-bearing mice. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a peak tumor uptake of 12.00+/-3.20 percentage injected dose per gram (%ID/g) at 1 h after injection. The tumor uptake gradually decreased to 3.43+/-1.12 %ID/g at 48 h after injection. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a peak tumor-to-kidney uptake ratio of 1.53 at 2 h after injection. The absorbed doses to the tumor and kidneys were 4.32 and 4.35 Gy, respectively, per 37 MBq. Whole-body clearance of (203)Pb-DOTA-Re(Arg(11))CCMSH was fast, with approximately 89% of the injected activity cleared through the urinary system by 2 h after injection. (203)Pb showed 1.6-mm SPECT resolution, which was comparable to (99m)Tc. Melanoma lesions were visualized through SPECT/CT images of (203)Pb-DOTA-Re(Arg(11))CCMSH at 2 h after injection.

CONCLUSIONS

(203)Pb-DOTA-Re(Arg(11))CCMSH exhibited favorable pharmacokinetic and tumor imaging properties, highlighting its potential as a matched-pair SPECT agent for (212)Pb-DOTA-Re(Arg(11))CCMSH melanoma treatment.

The three-dimensional supramolecular structures and the states of order of the acyl chains of lipid A from different Gram-negative species were investigated at 40 degrees C, high water content (80-90%), and different [lipid A]/[Mg2+] molar ratios using synchrotron radiation X-ray diffraction and Fourier-transform infrared spectroscopy. Measurements were made on free lipid A from Salmonella minnesota R595, mono- and bi-phosphoryl, as well as those from the non-enterobacterial strains Rhodobacter capsulatus 37b4, Rhodopseudomonas viridis F, and Rhodocyclus gelatinosus 29/1. Parallel to differences in their chemical primary structure, the structural polymorphisms and states of order at 37 degrees C of the non-enterobacterial lipid A were found to be different from those of enterobacterial lipid A. A clear correlation between the supramolecular structure and previously determined biological activities was found. Lipid A with a strong preference for lamellar structures (Rb. capsulatus and Rp. viridis) are endotoxically inactive and lack cytokine-inducing capacity; the compounds assuming a mixed lamellar/nonlamellar structure (monophosphoryl lipid A from S. minnesota) are of lower toxicity in vivo, but may induce cytokines in vitro; those lipid A with a strong tendency to form non-lamellar inverted structures (lipid A from S. minnesota and Rc. gelatinosus) exhibit full endotoxicity in vitro and in vivo. In contrast, anti-complementary activity is most pronounced for compounds with lamellar and least expressed for those with inverted structures. The states of order at 37 degrees C vary non-systematically, exhibiting the highest values for lipid A of S. minnesota and the lowest for that of Rc. gelatinosus. We propose to extend the term 'endotoxic conformation', which is used to describe the conformation of a single lipid A molecule required for optimal triggering of biological effects, to 'endotoxic supramolecular conformation' which denotes the particular organization of lipid A aggregates in physiological fluids causing biological activity.

1. Inward unitary currents through inwardly rectifying K+ channels of myotubes derived from newborn rats or from a murine, clonal myoblast cell line were studied in the cell-attached configuration. Open-closed transitions of the channel were observed in the absence of blocking ions. 2. The single-channel conductance was 26.3 +/- 2.9 pS (mean + S.D., n = 14) with 150 mM-K+ pipette solution at room temperature (19-22 degrees C). The channel showed substates of conductance in addition to the main conductance state. A channel with a smaller conductance (8.9 +/- 2.6 pS, n = 4) was also but less frequently observed. 3. The probability of the channel being open is weakly voltage dependent: it decreased from 0.94 to 0.84 as the membrane was hyperpolarized from the resting potential (RP) + 20 mV to RP - 50 mV. 4. The lifetimes of the openings were distributed according to a single exponential. At least three exponentials were required to fit the frequency histogram of the lifetimes of all closed states. The mean open time showed a weak voltage dependence, while the mean closed times had little voltage dependence. 5. In the presence of external Na+, the open probability decreased from 0.89 to 0.43 and the mean open time decreased from 203 to 28 ms (40 mM-K+, 200 mM-Na+ pipette solution) when the patch membrane was hyperpolarized from RP - 40 mV to RP - 110 mV. The mean closed times were not different from those with 150 mM-K+, Na+-free pipette solution and showed little voltage dependence. 6. It is suggested that inactivation of the macroscopic inward currents during hyperpolarization results mainly from a voltage-dependent block by Na+ with relatively slow kinetics.

The involvement of cyclic AMP-dependent protein kinase A (PKA) in the exocytotic release of insulin from rat pancreatic islets was investigated using the Rp isomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS). Preincubation of electrically permeabilised islets with Rp-cAMPS (1 mM, 1 h, 4 degrees C) inhibited cAMP-induced phosphorylation of islet proteins of apparent molecular weights in the range 20-90 kDa, but did not affect basal (50 nM Ca2+) nor Ca2(+)-stimulated (10 microM) protein phosphorylation. Similarly, Rp-cAMPS (500 microM) inhibited both cAMP- (100 microM) and 8BrcAMP-induced (100 microM) insulin secretion from electrically permeabilised islets without affecting Ca2(+)-stimulated (10 microM) insulin release. In intact islets, Rp-cAMPS (500 microM) inhibited forskolin (1 microM, 10 microM) potentiation of insulin secretion, but did not significantly impair the insulin secretory response to a range of glucose concentrations (2-20 mM). These results suggest that cAMP-induced activation of PKA is not essential for either basal or glucose-stimulated insulin secretion from rat islets.

BACKGROUND

Parathyroid hormone (PTH), the major systemic calcium regulating hormone has been implicated in the development of hypertension and the occurrence of uraemic vascular changes. As nitric oxide synthase (NOS) is involved in the production of nitric oxide, and acute PTH effect is characterized by vasodilation, the effect of PTH on the endothelial NOS (eNOS) system was measured in cultured human umbilical cord vein endothelial cells (HUVEC) and the pathways possibly involved were studied.

METHODS

The presence of the PTH receptor-1 (PTHR1) on the HUVEC membrane was examined by RT-PCR, immunocytochemistry and western blot. HUVEC were stimulated with 10(-12) to 10(-10) mol/l PTH. The eNOS mRNA expression was established by RT-PCR and the eNOS protein levels were determined by western blot. The eNOS activity was measured by the conversion of [(14)C]arginine to [(14)C]citrulline.

RESULTS

PTHR1 has been found to be expressed in HUVEC and its expression is depressed by increasing concentrations of PTH. PTH induced a significant increase in eNOS mRNA (10(-11) mol/l: 1.87 +/- 0.16, P = 0.012; 10(-10) mol/l: 1.96 +/- 0.28, P = 0.007, fold of control), and protein expression. The eNOS activity was also significantly stimulated (10(-11) mol/l: 1139 +/- 203; 10(-10) mol/l: 1323 +/- 216 vs control: 621 +/- 154 cpm/150 mug protein, P < 0.01). The addition of calphostin C (PKC inhibitor) or Rp-cAMP (PKA inhibitor) reduced the eNOS mRNA, protein expression and activity of PTH-stimulated HUVEC. The combined treatment of calphostin C and Rp-cAMP abolished the eNOS protein expression and activity.

CONCLUSIONS

PTH induces an increased activity of the eNOS system; probably both PKA and PKC pathways are involved in this activation. Such data may explain the vasodilation observed after acute treatment with PTH.

Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, Mr approximately 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes. The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior. Both the adsorption of DNA, directly measured with [32P]dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations [Dt] and [Cat] of DNA and of calcium, respectively. At [Cat] = 1 mM, the half-saturation or equilibrium constant is KD = 15 +/- 1 nM at 293 K (20 degrees C). The optimal transformation frequency is TFopt = 4.1 +/- 0.4 X 10(-5) if a single exponential pulse of initial field strength E0 = 4 kV cm-1 and decay time constant tauE = 45 ms is applied at [Dt] = 2.7 nM and 10(8) cells in 0.1 ml. The dependence of TF on [Cat] yields the equilibrium constants KCazero = 1.8 +/- 0.2 mM (in the absence of DNA) and K'Ca (at 2.7 nM DNA), comparable with and derived from electrophoresis data. In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At Eo = 4.0 kV cm-1 and T = 293 K, the flow coefficient of DNA through the porous membrane patches is Kto = 7.0 +/- 0.7 x 10(3)S-1 and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D0 approximately 10.3. The mean radius of these pores is rp = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size ø approximately 5.5 microns) is Np = 2.2 +/- 0.2 x 10(4). The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface. The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive translocation of the inserted DNA into the cell interior. For practical purposes of optimal transformation efficiency, 1 mM calcium is necessary for sufficient DNA binding and the relatively long pulse duration of 20-40 ms is required to achieve efficient electrodiffusive transport across the cell wall and into the outer surface of electroporated cell membrane patches.

The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.

We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20 healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1) removal of six high-abundant proteins; (2) tryptic digestion of low- and high-abundant proteins of serum; (3) separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification. A total of 944 nonredundant proteins were identified under a stringent filter condition (X(corr)>> or = 1.9,>> or = 2.2, and>> or = 3.75, < or = C(n)>> or = 0.1, and R(sp)>> or = 4.0) in both pooled male and female samples, in which 594 and 622 entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206 proteins were identified with at least two distinct peptides per protein and 185 proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement CCA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery.

A central step in promoter activation by RNA polymerase (RNAP) is the localized separation of the DNA strands to form the transcription bubble. We have used potassium permanganate footprinting to monitor DNA strand-separation by the Bacillus subtilis sigmaD RNAP at the strong promoter, Phag, directing transcription of flagellin. The susceptibility of individual thymine bases to permanganate oxidation is influenced by temperature, Mg2+, nucleotides, and the RNAP delta subunit. In the absence of delta, sigmaD RNAP establishes a partially opened complex even at 0 degrees C with permanganate reactivity localized between -11 and -4 (RP(-4)). The region of strand separation expands to near -1 at 20 degrees C (RP(-1)) and to +3 at 40 degrees C (RP(+3)). The delta subunit inhibits the downstream propagation of the transcription bubble and thereby increases the concentration of early intermediates in the melting pathway. Indeed, E delta sigmaD forms a distinct nucleated complex (RPn) at 0 degrees C with a structural distortion localized to an AT base step within the -10 element. We propose a model for promoter melting in which strand separation nucleates within the conserved -10 consensus and subsequently propagates downstream. Mg2+ and nucleoside triphosphates (NTPs) favor the downstream propagation of the transcription bubble and strongly stimulate the RP(-1) to RP(+3) conversion. The NTP effects are apparently mediated by binding of substrate to the initiating NTP site: purines are more effective than pyrimidines and GMP alone can greatly increase the level of DNA-melting. The binding of substrates, but not Mg2+ alone, can effectively overcome the anti-melting effect of delta.

The signaling pathways by which mechanical forces modulate fetal lung development remain largely unknown. In the present study, we tested the hypothesis that strain-induced fetal type II cell differentiation is mediated via the cAMP signaling pathway. Freshly isolated E19 fetal type II epithelial cells were cultured on collagen-coated silastic membranes and exposed to mechanical strain for varying intervals, to simulate mechanical forces during lung development. Unstretched samples were used as controls. Mechanical strain activated heterotrimeric G-protein alpha(s) subunit, cAMP, and the transcription factor cAMP response element binding protein (CREB). Incubation of E19 cells with the PKA inhibitor H-89 significantly decreased strain-induced CREB phosphorylation. Moreover, adenylate cyclase 5 and CREB genes were also mechanically induced. In contrast, components of the PKA-independent (Epac) pathway, including Rap-1 or B-Raf, were not phosphorylated by strain. The addition of forskolin or dibutyryl cAMP to unstretched E19 monolayers markedly upregulated expression of the type II cell differentiation marker surfactant protein C, whereas the Epac agonist 8-pCPT-2'-O-Me-cAMP had no effect. Furthermore, incubation of E19 cells with the PKA inhibitor Rp-2'-O-monobutyryladenosine 3',5'-cyclic monophosphorothioate or transient transfection with plasmid DNA containing a PKA inhibitor expression vector significantly decreased strain-induced surfactant protein C mRNA expression. In conclusion, these studies indicate that the cAMP-PKA-dependent signaling pathway is activated by force in fetal type II cells and participates in strain-induced fetal type II cell differentiation.

1. The aim of this study was to investigate the mechanism of control of Na+,K+-ATPase activity by the cAMP-protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10-3 M dibutyryl-cAMP (db-cAMP) or 8-bromo-cAMP) and endogenous cAMP (direct activation of adenylyl cyclases by 10-5 M forskolin) on Na+,K+-ATPase activity and membrane trafficking. 2. PKA activation stimulated both the cation transport and hydrolytic activity of Na+,K+-ATPase by about 40%. Transport activity stimulation was specific to the PKA signalling pathway since (1) db-cAMP stimulated the ouabain-sensitive 86Rb+ uptake in a time- and dose-dependent fashion; (2) this effect was abolished by addition of H-89 or Rp-cAMPS, two structurally different PKA inhibitors; and (3) this stimulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db-cAMP on the hydrolytic activity of Na+,K+-ATPase was accounted for by an increased maximal ATPase rate (Vmax) without alteration of the efficiency of the pump, suggesting that cAMP-PKA pathway was implicated in membrane redistribution control. 3. To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP-PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+-ATPase activity by db-cAMP was associated with an increase (+40%) in Na+, K+-ATPase units expressed at the cell surface which was assessed by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (-30%) in pump units located in the subcellular fraction corresponding to early endosomes. 4. In conclusion, PKA stimulates Na+,K+-ATPase activity, at least in part, by increasing the number of Na+-K+ pumps in the plasma membrane in proximal convoluted tubule cells.

Membrane parameters at the respective resting potentials in low, normal, and high extracellular potassium solutions were determined in intercostal muscle fibers from 15 patients with no known neuromuscular disease. In synthetic interstitial fluid (normal potassium concentration 3.5 mmol/liter), we found the following mean values: resting membrane potential RP = -83.3 mV, space constant lambda = 2364 micron, fiber diameter d = 49.3 micron, fiber input resistance Rin = 795 k omega, specific membrane capacitance Cm = 4.7 muF/cm2, and specific membrane resistance Rm = 5970 omega X cm2. The specific membrane conductance was gm = 168 muS/cm2, 76% of it being chloride conductance, 24% being potassium conductance. The dependence of the membrane parameters on the extracellular potassium concentration followed the predictions by the constant field theory. There was no indication of active chloride transport. The resting membrane conductance decreased with temperature with a Q10 of 1.3. Excitability parameters were nearly independent of temperature between 37 and 27 degrees C.

Hereditary cataract is a phenotypically and genetically heterogeneous lens disease that is responsible for a significant proportion of the visual impairment and blindness that occurs in children. In a five-generation Chinese family with autosomal dominant inherited congenital cataract, clinical examination showed three cataract phenotypes: punctuate, nuclear, and total cataracts. Linkage analysis was performed and positive two-point LOD scores (with maximum of 4.43 and 4.27 at theta=0) were obtained for markers D21S1411 and D21S1890 on chromosome 21q22.3, flanking the CRYAA (alphaA-crystallin-encoding gene) locus. Sequencing of CRYAA revealed a novel heterozygous G>A transition (c.346G>A) in exon 3 that cosegregated with the disease phenotype and results in a conservative substitution of Arg to His at codon 116 (p.R116H). To understand the molecular basis of cataract formation, mutant and wild-type alphaA-crystallins were expressed in E. coli. RP-HPLC (reverse phase-high-performance liquid chromatography) suggested an increased hydrophobicity of the mutant recombinant protein, compared to that of wild-type alphaA-crystallins. Furthermore, loss of chaperone activity of the mutant was seen in DTT (DL-dithiothreitol)-induced insulin aggregation assay. FPLC (fast protein liquid chromatography) purification showed that the His-116 mutant protein had increased binding affinity to lysozyme. Gain of activated lysozyme binding, elevation of hydrophobicity and loss of chaperone activity of the mutant protein may be some of the molecular mechanisms underlying cataract in this large family.

The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPCC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of beta-arrestin-2 (betaarr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, Cftr(-/-) MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls.

The inhibition of Ca2+ channel currents by endogenous brain steroids was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarizing steps from -80 to -10 mV occurred in a concentration-dependent manner with the following ICCC), 298 nM. THCC, PE, and PES depressed a fraction of the Ca2+ channel current with a maximal inhibition of 60% of the total current. However, substitution of an acetate group for the sulfate group on PES resulted in a complete loss of activity. Progesterone had no effect (4% inhibition at 100 microM). Intracellular dialysis of PES had no effect on the Ca2+ current; concomitant extracellular perfusion of PES showed normal inhibitory activity, suggesting that the steroid binding site can only be accessed extracellularly. Analysis of tail currents at -80 mV demonstrated that THCC and PES slowed the rate of Ca2+ current activation and deactivation with no change in the voltage dependence of activation. Inhibition of the Ca2+ channel current by THCC and PES was voltage dependent. THCC primarily inhibits the omega-conotoxin (CgTX)-sensitive or N-type Ca2+ channel current. PE was nonselective in inhibiting both the CgTX- and the nifedipine (NIF)-sensitive Ca2+ channel current. These neurosteroids had no effect on the CgTX/NIF-insensitive current. In neurons isolated from pertussis toxin (PTX)-treated animals by chronic intracerebroventricular infusion (1000 ng/24 hr for 48 hr), the Ca2+ channel current inhibition by PES, PE, and THCC was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) also significantly diminished the neurosteroid inhibition of the Ca2+ channel current. Intracellular dialysis with the general kinase inhibitors H-7 (100 microM), staurosporine (400 nM), and a 20 amino acid protein kinase inhibitor (1 microM) also significantly prevented the THCC and PES inhibition of the Ca2+ channel current. Intracellular dialysis with the more specific inhibitors of protein kinase C (PKC), the pseudosubstrate inhibitor (PKCI 19-36) (1-2 microM) and bisindolylmaleimide (1 microM) significantly diminished the THCC and PE inhibition of the Ca2+ channel current. Rp- cAMP (100 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the THCC and PE inhibition of the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

Mobile elements (MEs) constitute greater than 50% of the human genome as a result of repeated insertion events during human genome evolution. Although most of these elements are now fixed in the population, some MEs, including ALU, L1, SVA and HERV-K elements, are still actively duplicating. Mobile element insertions (MEIs) have been associated with human genetic disorders, including Crohn's disease, hemophilia, and various types of cancer, motivating the need for accurate MEI detection methods. To comprehensively identify and accurately characterize these variants in whole genome next-generation sequencing (NGS) data, a computationally efficient detection and genotyping method is required. Current computational tools are unable to call MEI polymorphisms with sufficiently high sensitivity and specificity, or call individual genotypes with sufficiently high accuracy.

RESULTS

Here we report Tangram, a computationally efficient MEI detection program that integrates read-pair (RP) and split-read (SR) mapping signals to detect MEI events. By utilizing SR mapping in its primary detection module, a feature unique to this software, Tangram is able to pinpoint MEI breakpoints with single-nucleotide precision. To understand the role of MEI events in disease, it is essential to produce accurate individual genotypes in clinical samples. Tangram is able to determine sample genotypes with very high accuracy. Using simulations and experimental datasets, we demonstrate that Tangram has superior sensitivity, specificity, breakpoint resolution and genotyping accuracy, when compared to other, recently developed MEI detection methods.

CONCLUSIONS

Tangram serves as the primary MEI detection tool in the 1000 Genomes Project, and is implemented as a highly portable, memory-efficient, easy-to-use C++ computer program, built under an open-source development model.

BACKGROUND

We and others have reported that transient accumulation of cyclic AMP (cAMP) in the myocardium during ischemic preconditioning (IP) limits infarct size independent of protein kinase C (PKC). Accumulation of cAMP activates protein kinase A (PKA), which has been demonstrated to cause reversible inhibition of RhoA and Rho-kinase. We investigated the involvement of PKA and Rho-kinase in the infarct limitation by IP.

RESULTS

Dogs were subjected to 90-minute ischemia and 6-hour reperfusion. We examined the effect on Rho-kinase activity during sustained ischemia and infarct size of (1) preischemic transient coronary occlusion (IP), (2) preischemic activation of PKA/PKC, (3) inhibition of PKA/PKC during IP, and (4) inhibition of Rho-kinase or actin cytoskeletal deactivation during myocardial ischemia. Either IP or dibutyryl-cAMP treatment activated PKA, which was dose-dependently inhibited by 2 PKA inhibitors (H89 and Rp-cAMP). IP and preischemic PKA activation substantially reduced infarct size, which was blunted by preischemic PKA inhibition. IP and preischemic PKA activation, but not PKC activation, caused a substantial decrease of Rho-kinase activation during sustained ischemia. These changes were cancelled by preischemic inhibition of PKA but not PKC. Furthermore, either Rho-kinase inhibition (hydroxyfasudil or Y27632) or actin cytoskeletal deactivation (cytochalasin-D) during sustained ischemia achieved the same infarct limitation as preischemic PKA activation without affecting systemic hemodynamic parameters, the area at risk, or collateral blood flow.

CONCLUSIONS

Transient preischemic activation of PKA reduces infarct size through Rho-kinase inhibition and actin cytoskeletal deactivation during sustained ischemia, implicating a novel mechanism for cardioprotection by ischemic preconditioning independent of PKC and a potential new therapeutic target.

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal degeneration characterized by photoreceptor death, which results in visual failure. Here, we used a combination of homozygosity mapping and exome sequencing to identify mutations in ARL2BP, which encodes an effector protein of the small GTPases ARL2 and ARL3, as causative for autosomal-recessive RP (RPcted by RP and situs inversus, a homozygous, splice-acceptor mutation, c.101-1G>C, which alters pre-mRNA splicing of ARLBP2 in blood RNA, was identified. In another family, a homozygous c.134T>G (p.Met45Arg) mutation was identified. In the mouse retina, ARL2BP localized to the basal body and cilium-associated centriole of photoreceptors and the periciliary extension of the inner segment. Depletion of ARL2BP caused cilia shortening. Moreover, depletion of ARL2, but not ARL3, caused displacement of ARL2BP from the basal body, suggesting that ARL2 is vital for recruiting or anchoring ARL2BP at the base of the cilium. This hypothesis is supported by the finding that the p.Met45Arg amino acid substitution reduced binding to ARL2 and caused the loss of ARL2BP localization at the basal body in ciliated nasal epithelial cells. These data demonstrate a role for ARL2BP and ARL2 in primary cilia function and that this role is essential for normal photoreceptor maintenance and function.

Patients with head and neck squamous cell carcinoma harbor T cell-inflamed and non-T cell-inflamed tumors. Despite this, only 20% of patients respond to checkpoint inhibitor immunotherapy. Lack of induction of innate immunity through pattern-recognition receptors, such as the stimulator of interferon (IFN) genes (STING) receptor, may represent a significant barrier to the development of effective antitumor immunity. Here, we demonstrate robust control of a T cell-inflamed (MOC1), but not non-T cell-inflamed (MOC2), model of head and neck cancer by activation of the STING pathway with the synthetic cyclic dinucleotide RP,RP dithio-c-di-GMP. Rejection or durable tumor control of MOC1 tumors was dependent upon a functional STING receptor and CD8 T lymphocytes. STING activation resulted in increased tumor microenvironment type 1 and type 2 IFN and greater expression of PD-1 pathway components in vivo Established MOC1 tumors were rejected and distant tumors abscopally controlled, after adaptive immune resistance had been reversed by the addition of PD-L1 mAb. These findings suggest that PD-1 pathway blockade may reverse adaptive immune resistance following cyclic dinucleotide treatment, enhancing both local and systemic antitumor immunity. Cancer Immunol Res; 4(12); 1061-71. ©2016 AACR.