AFDX-116 discriminates between muscarinic M2 receptors of the heart and the iris smooth muscle

Summary Studies with the atypical muscarinic antagonist pirenzepine provide convincing evidence for the classification of muscarinic acetylcholine receptors (mAChRs) into two subtypes, M₁ and M₂. The present study examines the heterogeneity of the M₂ subtype employing the newly developed competitive muscarinic antagonist, AFDX-116. Comparison of the binding affinities of pirenzepine, atropine, and AFDX-116 to mAChRs in microsomes from the rabbit cerebral cortex, heart, and iris smooth muscle shows that iris mAChRs, which are pharmacologically of the M₂ subtype, can be distinguished from M₂ cardiac receptors based on their affinity for AFDX-116. These results are consistent with the hypothesis that the M₂ receptor subtype consists of a heterogeneous population of receptors.Abbreviations mAChRs Muscarinic Acetylcholine Receptors - CCh Carbachol - NMS N-Methylscopolamine - AFDX-116 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepine-6-one     

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin-(1–24) -tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH₄)₂SO₄ precipitate; ASP_30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The ³²P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ-³²P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP_30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg²⁺ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca²⁺. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP_30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca²⁺-independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca²⁺ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.     

Requirement for calcium ions in acetylcholine-stimulated phosphodiesteratic cleavage of phosphatidyl-myo-inositol 4,5-bisphosphate in rabbit iris smooth muscle

1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca(2+) was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50mum) increased the breakdown of phosphatidylinositol bisphosphate in [(3)H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of (3)H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the (3)H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20mum), but not tubocurarine (100mum); and it was abolished by depletion of extracellular Ca(2+) with EGTA, but restored on addition of low concentrations of Ca(2+) (20mum). 5. Calcium-antagonistic agents, such as verapamil (20mum), dibenamine (20mum) or La(3+) (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (;microsomes') was stimulated by 43% in the presence of 50mum-Ca(2+). 7. The results indicate that increased Ca(2+) influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca(2+) activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the (3)H radioactivity of this phospholipid.     

Identification of miRNA signatures and their therapeutic potentials in prostate cancer

Molecular Biology Reports - Herein, we identified miRNA signatures that were able to differentiate malignant prostate cancer from benign prostate hyperplasia and revealed the therapeutic potential...     

Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity

Cell capacity for cytosolic NADPH regeneration by NADP‐dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O₃) treatment (120 ppb for 17 days). Two genotypes with differential O₃ sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O₃ flux (POD₀), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O₃ flux, CO₂ assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP‐dehydrogenases above their constitutive level, i.e. NADP‐G6PDH (EC 1.1.1.49), NADP‐ME (malic enzyme) (EC 1.1.1.40) and NADP‐ICDH (NADP‐isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non‐phosphorylating NADP‐GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP‐G6PDH and NADP‐ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O₃‐treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.     

Upregulation of SATB1 Is Associated with Prostate Cancer Aggressiveness and Disease Progression

Disease aggressiveness remains a critical factor to the progression of prostate cancer. Transformation of epithelial cells to mesenchymal lineage, associated with the loss of E-cadherin, offers significant invasive potential and migration capability. Recently, Special AT-rich binding protein (SATB1) has been linked to tumor progression. SATB1 is a cell-type restricted nuclear protein, which functions as a tissue-specific organizer of DNA sequences during cellular differentiation. Our results demonstrate that SATB1 plays significant role in prostate tumor invasion and migration and its nuclear localization correlates with disease aggressiveness. Clinical specimen analysis showed that SATB1 was predominantly expressed in the nucleus of high-grade tumors compared to low-grade tumor and benign tissue. A progressive increase in the nuclear levels of SATB1 was observed in cancer tissues compared to benign specimens. Similarly, SATB1 protein levels were higher in a number of prostate cancer cells viz. HPV-CA-10, DU145, DUPro, PC-3, PC-3M, LNCaP and C4-2B, compared to non-tumorigenic PZ-HPV-7 cells. Nuclear expression of SATB1 was higher in biologically aggressive subclones of prostate cancer cells with their respective parental cell lines. Furthermore, ectopic SATB1 transfection conferred increased cell motility and invasiveness in immortalized human prostate epithelial PZ-HPV-7 cells which correlated with the loss of E-cadherin expression. Consequently, knockdown of SATB1 in highly aggressive human prostate cancer PC-3M cells inhibited invasiveness and tumor growth in vivo along with increase in E-cadherin protein expression. Our findings demonstrate that SATB1 has ability to promote prostate cancer aggressiveness through epithelial-mesenchymal transition.     

Alkaline protease production by an actinomycete MA1-1 isolated from marine sediments

Alkaliphilic actinomycetes isolated from sediment samples of the Izmir Gulf, Turkey were studied for the production of protease activity. Strain MA1-1 was selected as a good alkaline protease producer as measured by the clear zone diameter by the hydrolysis of skim-milk and casein. The alkaline protease production from the marine alkaliphilic actinomycete MA1-1 was studied by using different carbon and nitrogen sources in medium containing glycerol, peptone, KCl, MgSO₄, K₂HPO₄, and trace elements at 30°C for 72 h. Among the different carbon and nitrogen sources, fructose, starch, maltose, D(+) glucose, yeast extract, malt extract, beef extract and peptone provided higher production of protease. Starch was also found to be effective for growth and enzyme production with highest specific activity at 699 U mg^?1. Purification was achieved by adsorption on Diaion HP 20 which resulted in a recovery rate of 68% with a specific activity of 7618 U mg^?1 protein and 40-fold purification. The optimum pH and temperature of the partially purified protease were determined as pH 9.0 and 50°C, but high activity was also observed at pH 8.0–13.0 and 35–50°C. The inhibition profile exhibited by phenylmethylsulphonyl fluoride (PMSF) showed that this enzyme belongs to the serine-protease group.     

Involvement of Ca2+ channels in endothelin-1-induced MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle

The purpose of the present study was to investigate the role and type of Ca2+ channels involved in the stimulatory effects of endothelin-1 (ET-1) on the Ca2+-dependent functional responses, p42/p44 MAP kinase phosphorylation, 20-kDa myosin light chain (MLC) phosphorylation and contraction, in rabbit iris sphincter, a nonvascular smooth muscle. ET-1 induced inositol phosphates production, MAP kinase phosphorylation, MLC phosphorylation (MLC20-P plus MLC20-2P) and contraction in a concentration-dependent manner with EC50 values of 71, 8, 6 and 25 nM, respectively. ET-1-induced MAP kinase phosphorylation, MLC phosphorylation and contraction were not significantly affected by nifedipine (1-60 microM), an L-type Ca2+ channel blocker, or by LOE 908 (1-100 microM), a blocker of Ca2+-permeable nonselective cation channels. However, SKF96365, a receptor-operated Ca2+ channel (ROCC) blocker, inhibited MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 28, 30 and 42 microM, respectively. 2-APB, a store-operated Ca2+ channel (SOCC) blocker, inhibited ET-1-induced MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 12.7 and 19 microM, respectively, but was without effect on MAP kinase phosphorylation. The combined effects of submaximal concentrations of SKF96365 and 2-APB on ET-1-induced MLC phosphorylation and contraction were not additive, implying that their inhibitory actions could be mediated through a common Ca2+ entry channel. PD98059, a MAP kinase inhibitor, had no effect on ET-1-induced MLC phosphorylation and contraction, suggesting that these ET-1 effects in the rabbit iris muscle are MAP kinase-independent. In conclusion, the present study demonstrated for the first time that in rabbit iris sphincter (a) ET-1, through the ETA receptor, stimulates MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner, (b) that these Ca2+-dependent functional responses are not significantly affected by nifedipine or LOE908, and (c) that ET-1-induced MLC phosphorylation and contraction are inhibited by SKF96365 and 2-APB, suggesting that these effects are mainly due to store- and/or receptor Ca2+ entry.     

A review of 35 cases of asymmetric crying facies

A review of 35 cases of asymmetric crying facies: Congenital asymmetric crying facies (ACF) is caused by congenital hypoplasia or agenesis of the depressor anguli oris muscle (DAOM) on one side of the mouth. It is well known that this anomaly is frequently associated with cardiovascular, head and neck, musculoskeletal, respiratory, gastrointestinal, central nervous system, and genitourinary anomalies. In this article we report 35 ACF patients (28 children and 7 adults) and found additional abnormalities in 16 of them (i.e. 45%). The abnormalities were cerebral and cerebellar atrophy, mega-cisterna magna, mental motor retardation, convulsions, corpus callosum dysgenesis, cranial bone defect, dermoid cyst, spina bifida occulta, hypertelorism, micrognatia, retrognatia, hemangioma on the lower lip, short frenulum, cleft palate, low-set ears, preauricular tag, mild facial hypoplasia, sternal cleft, congenital heart defect, renal hypoplasia, vesicoureteral reflux, hypertrophic osteoarthropathy, congenital joint contractures, congenital hip dislocation, polydactyly, and umbilical and inguinal hernia. Besides these, one infant was born to a diabetic mother, and had atrial septal defect and the four other children had 4p deletion, Klinefelter syndrome, isolated CD4 deficiency and Treacher-Collins like facial appearance, respectively Although many of these abnormalities were reported in association with ACF, cerebellar atrophy, sternal cleft, cranial bone defect, infant of diabetic mother, 4p deletion, Klinefelter syndrome, isolated CD4 deficiency and Treacher-Collins like facial appearance were not previously published.