Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis

Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.     

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

Localization ofd-amino acid oxidase mRNA in the mouse kidney and the effect of testosterone treatment

d-Amino acid oxidase (DAO), which catalyzes oxidative deamination ofd-amino acids, is known to be highly expressed in the kidney. This study was designed to examine the localization of DAO mRNA in the mouse kidney using in situ hybridization histochemistry (ISH). For comparison, ISH for mRNA of ornithine decarboxylase (ODC), which is also highly expressed in the mouse kidney, was simultaneously performed. Adult, male mice which received 1 mg of testosterone propionate or vehicle injection, were sacrificed 14 h after injection and their kidneys were removed and processed for ISH. Hybridization signals for both mRNAs were exclusively located over the epithelial cells of the proximal tubule in the vehicle-treated animals. Signals for the DAO mRNA were observed at nearly the same hybridization intensity throughout the proximal tubule, whereas hybridization signals for the ODC mRNA were observed exclusively in the pars convoluta. Following testosterone treatment, ODC mRNA in the pars convoluta was expressed with a stronger intensity than that in the vehicle-injected animals. ODC mRNA was also expressed in the pars recta with a weaker intensity than in the pars convoluta. On the other hand, DAO mRNA expression was little affected by testosterone treatment. These results indicate that, although both genes are possibly expressed in the same cells, the expression of these genes is regulated by different mechanisms.     

Monolignol and dilignol glycosides from Pinus contorta leaves

Four monolignol glucosides have been isolated and identified from the needles of Pinus contorta. Chavicol 4-O-α-L-arabinofuranosyl-(1 →     

Acylated flavonol glucosides of Pinus contorta needles

From fresh Pinus contorta Doug (Coastal) needles four flavonol acylated glucosides and 6-methyl-kaempferol 3-β-D-glucoside were isolated. The three monoacylated glucosides were kaempferol-3-β-D(6-O-p- coumaryl)glucoside, isorhamnetin-3-β-D-(6-O-acetyl)glucoside, quercetin-3-β-D-(p-coumaryl)glucoside and the diacyl compound was kaempferol-3-β-D-(di-p-coumaryl)glucoside.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.