Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.     

Halide abstraction reactions of Sb(V) chloride: Synthesis and characterisation of hexachloroantimonate salts of M(II) M = Zn,Mg; M(III) M = Cr; and M(IV) M = Ti,Sn and related Cp2M(IV) M = Ti,Zr and Hf

Reactions of SbCl₅ with various covalent metal halides in MeCN have been studied as a convenient and direct route to metal hexachloroantimonate salts via Sb(V) halide abstraction. The isolation and characterization (Ir, Vis-UV, ¹H NMR spectroscopic and microanalytical) of the complexes [Zn(MeCN)₆][SbCl₆]₂, [CrCl₂(MeCN)₄][SbCl₆], [SnCl₃(MeCN)₃][SbCl₆], [TiCl₂(MeCN)₄][SbCl₆]₂, [Cp₂M(Cl)(MeCN)_x][SbCl₆] M = ti, x = 1; M = Zr, Hf, x = 2, and [Cp₂M(MeCN)_y][SbCl₆]₂ M = Ti, y = 2; M = Zr, Hf, y = 3, is described. The reaction of MgCl₂ with SbCl₅ was carried out in EtOAC as solvent and gave [Mg(EtOAc)₆][SbCl₆]₂. ¹²¹Sb NMR, IR and UV spectroscopic measurements provide positive identification of the SbCl_6⁻ anion.     

Vibrational spectra and structure of M(CO2) and M2(CO2) molecules

Atomic Na, K and Cs were codeposited with CO₂ in excess of matrix gas at the temperature of 12 K. The IR spectra revealed the presence of ionic aggregates corresponding to the molecules M(CO)₂ and M₂(CO₂) (M=Na, K, Cs). Both molecular species have C_2v symmetry; M(CO₂) species have a planar ring structure while M₂(CO₂) have a W-shape structure. M₂(CO₂) molecules with C_s symmetry were also identified. The geometrical parameters of all the molecules were determined by ¹²C/¹³C and ¹⁶O/¹⁸O isotopic shifts. Raman spectra were also recorded and the results are reported in this study. The effect of photolysis on the structure of these molecules was examined. It was determined that photolysis promotes the formation of Na(CO₂) and transforms the M₂(CO₂) molecules with C_2v symmetry into C_s symmetry isomers.     

Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures

Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21(CIP1/WAF1) gene in presenescent human fibroblasts. p21 -/- cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 -/- cultures were positive for the senescence-associated beta-galactosidase (SA-beta-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 -/- cells was high (>30%). The observation that p21 -/- and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-beta-gal, cannot distinguish between senescence and crisis. We propose that all SA-beta-gal-positive cultures should be further examined for their BrdU labeling index.     

Cholinergically stimulated gastric acid secretion is mediated by M(3) and M(5) but not M(1) muscarinic acetylcholine receptors in mice

Muscarinic acetylcholine receptors play an important role in the regulation of gastric acid secretion stimulated by acetylcholine; nonetheless, the precise role of each receptor subtype (M(1)-M(5)) remains unclear. This study examined the involvement of M(1), M(3), and M(5) receptors in cholinergic regulation of acid secretion using muscarinic receptor knockout (KO) mice. Gastric acid secretion was measured in both mice subjected to acute gastric fistula production under urethane anesthesia and conscious mice that had previously undergone pylorus ligation. M(3) KO mice exhibited impaired gastric acid secretion in response to carbachol. Unexpectedly, M(1) KO mice exhibited normal intragastric pH, serum gastrin and mucosal histamine levels, and gastric acid secretion stimulated by carbachol, histamine, and gastrin. Pirenzepine, known as an M(1)-receptor antagonist, inhibited carbachol-stimulated gastric acid secretion in a dose-dependent manner in M(1) KO mice as well as in wild-type (WT) mice, suggesting that the inhibitory effect of pirenzepine on gastric acid secretion is independent of M(1)-receptor antagonism. Notably, M(5) KO mice exhibited both significantly lower carbachol-stimulated gastric acid secretion and histamine-secretory responses to carbachol compared with WT mice. RT-PCR analysis revealed M(5)-mRNA expression in the stomach, but not in either the fundic or antral mucosa. Consequently, cholinergic stimulation of gastric acid secretion is clearly mediated by M(3) (on parietal cells) and M(5) receptors (conceivably in the submucosal plexus), but not M(1) receptors.     

Autopoietic and (M,R) systems

From the many attempts to produce a conceptual framework for the organization of living systems, the notions of (M,R) systems and Autopoiesis stand out for their rigor, their presupposition of the circularity of metabolism, and the new epistemologies that they imply. From their inceptions, these two notions have been essentially disconnected because each has defined its own language and tools. Here we demonstrate the existence of a deep conceptual link between (M,R) systems and Autopoietic systems. This relationship permits us to posit that Autopoietic systems, which have been advanced as capturing the central aspects of living systems, are a subset of (M,R) systems. This result, in conjunction with previous theorems proved by Rosen, can be used to outline a demonstration that the operation of Autopoietic systems cannot be simulated by Turing machines. This powerful result shows the potential of linking these two models. Finally, we suggest that the formalism of (M,R) systems could be used to model the circularity of metabolism.     

Enzymatic hydrolysis of n-substituted met-tRNA(M) and met-tRNA(F)

By addition of 1-(14)C-sodium acedate to the growth medium of Nocardia asteroides, it can be shown that the lipid content increases during the exponential phase, but does not vary during the stationary phase of the growth. Nocardic acid biosynthesis from the medium molecular weight fatty acids occurs chiefly during te stationary phase. As these compounds are localised in the cell walls, it becomes evident that the lipid envelope of the walls is still increasing when the cell growth and division have stopped.     

Stereoselectivity of (R)- and (S)-hexahydro-difenidol binding to neuroblastoma M1, cardiac M2, pancreatic M3, and striatum M4 muscarinic receptors

(R)-Hexahydro-difenidol has a higher affinity for M1 receptors in NB-OK 1 cells, pancreas M3 and striatum M4 receptors (pKi 7.9 to 8.3) than for cardiac M2 receptors (pKi 7.0). (S)-Hexahydro-difenidol, by contrast, is nonselective (pKi 5.8 to 6.1). Our goal in the present study was to evaluate the importance of the hydrophobic phenyl, and cyclohexyl rings of hexahydro-difenidol for the stereoselectivity and receptor selectivity of hexahydro-difenidol binding to the four muscarinic receptors. Our results indicated that replacement of the phenyl ring of hexahydro-difenidol by a cyclohexyl group (----dicyclidol) and of the cyclohexyl ring by a phenyl moiety (----difenidol) induced a large (4- to 80-fold) decrease in binding affinity for all muscarinic receptors. Difenidol had a significant preference for M1, M3, and M4 over M2 receptors; dicyclidol, by contrast, had a greater affinity for M1 and M4 than for M2 and M3 receptors. The binding free energy decrease due to replacement of the phenyl and the cyclohexyl groups of (R)-hexahydro-difenidol by, respectively, a cyclohexyl and a phenyl moiety was almost additive in the case of M4 (striatum) binding sites. In the case of the cardiac M2, pancreatic M3, or NB-OK 1 M1 receptors the respective binding free energies were not completely additive. These results suggest that the four (R)-hexahydro-difenidol "binding moieties" (phenyl, cyclohexyl, hydroxy, and protonated amino group) cannot simultaneously form optimal interactions with the M1, M2, and M3 muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human esophageal smooth muscle cells express muscarinic receptor subtypes M(1) through M(5)

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10-14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific alpha-actin. mRNA encoding muscarinic receptor subtypes M(1)-M(5) was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura 2 fluorescence. Basal [Ca(2+)](i), which was 135 +/- 22 nM, increased transiently to 543 +/- 29 nM in response to 10 microM ACh in CM cells (n = 8). This response was decreased <95% by 0.01 microM 4-diphenylacetoxy-N-methylpiperidine, a M(1)/M(3)-selective antagonist, whereas 0.1 microM methoctramine, a M(2)/M(4)-selective antagonist, and 0.1 microM pirenzepine, a M(1)-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca(2+)](i) mediated primarily by the M(3) receptor and involving release of Ca(2+) from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.     

Histochemical identification of non-culturable mycobacteria (M. leprae and M. lepramurium)

Summary The possibility of the histochemical demonstration of the dopa-oxidase reaction in Mycobacterium leprae from the granulomas of untreated cases of human lepromatous leprosy was studied. Fresh frozen sections were taken, fixed for one hour, and incubated with DOPA (3,4-dihydroxyphenylalanine) for two hours at pH 6.8 and at 37° C. The results obtained did not confirm the biochemical phenomena observed in human liver and spleen.Investigation supported by Grant No. DF. S1-153 from Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICIT)     

Theoretical study on the topotactic transformation and memory effect of M (II) M (III)-layered double hydroxides

Abstract

The topotactic transformation mechanism and memory effect of NiAl- and MgFe- layered double hydroxides (LDHs) are investigated by density functional theory (DFT)-based molecular simulation under their two key thermal decomposition temperatures (365, 800 °C for NiAl-LDHs, and 380, 800 °C for MgFe-LDHs). The results show that at the first temperature, the interlayer carbonate in both LDHs decompose to CO₂ and H₂O via a monodentate intermediate. During the dehydroxylation of the layers, for both LDHs the metal cations maintain their original distribution within the LDH (0?0?1) facet, while migrating substantially along the c-axis direction, and the layered structure of MgFe-LDHs is destroyed earlier than those of NiAl-LDH. Meanwhile, MgFe-LDHs can keep the memory effect longer than NiAl-LDHs, and the memory effect will disappear when the four-coordinated metal cations increased. At 800 °C, the layered structure of NiAl-LDHs is slightly destroyed, while a complete collapse of layered structure occurs in MgFe-LDHs. These results agree well with the experimental findings. This work will be helpful for the design and preparation of nanocatalysts derived from LDHs precursors.     

Primary structures of M6 and M7 of mugiline beta (Mugil japonicus)

Mugiline beta isolated from mature sperm nuclei of the Formosan grey mullet, belonging to Perciformes, was fractionated into seven components (M1-M7), by chromatography on CM-Sephadex C-25. The amino acid sequences of the two major components (M6 and M7) were then determined. M6 contained 33 amino acid residues per molecule: Arg, 21; Thr, 1; Ser, 1; Glu, 1; Pro, 3; Ala, 2; Val, 2; Met, 0.3 and Ile, 1.7. The amino acid sequence of M6 is: Pro-Arg-Arg-Arg-Arg-Glu-Thr-Ser-Arg-Pro-Ile-Arg-Arg-Arg-Arg-Arg-Ala-Pro- Ile (Met)-Arg-Arg-Arg-Arg-Arg-Val-Val-Arg-Arg-Arg-Arg. Isoleucine at position 22 is partially replaced by methionine. M7 had an amino acid sequence similar to that of M6 except that glutamic acid at position 6 of M6 was replaced by glutamine. A high degree of homology in the sequences was found between mugiline beta from mullet and thynnine from tuna fish, which also belongs to Perciformes.     

Preparation,structure and properties of dicyano silver complexes of the M(en)3Ag2(CN)4 and M(en)2Ag2(CN)4 type

Complexes of the M(en)₃Ag₂(CN)₄ (M = Ni, Zn, Cd) and M(en)₂Ag₂(CN)₄ (M = Ni, Cu, Zn, Cd) type were prepared and identified by elemental analysis, infrared spectroscopy, measurement of magnetic susceptibility, and X-ray powder diffractometry. The crystal structures of Ni(en)₃Ag₂(CN)₄ (I) and Zn(en)₂Ag₂(CN)₄ (II) were determined by the method of monocrystal structure analysis. Complex I crystallizes in the space group C2/c, a = 1.2639(5), b = 1.3739(4), c = 1.2494(4) nm, β = 113.25(4)°, D_m = 1.86(1), D_c = 1.86 gcm⁻³ Z = 4, R = 0.0429. The crystal structure of I consists of complex cations [Ni(en)₃]²⁺ and complex anions [Ag(CN)₂]⁻. Complex II crystallizes in the space group I2/m, a = 0.9150(3), b = 1.3308(4), c = 0.6442(2) nm, β = 95.80(3)°, D_m = 2.14(1), D_c = 2.15 gcm⁻³, Z = 2, R = 0.0334. Its crystal structure consists of infinite, positively charged chains of the [-NCAgCNZn- (en)₂]_nⁿ⁺ type and isolated [Ag(CN)₂]⁻ anions. The atoms of Ag are positioned parallely to the z axis and the AgAg distance is equal to 0.3221(2) nm.     

Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.