Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

Dark inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase in legumes: A biosystematic study

2-carboxyarabinitol 1-phosphate (CA 1-P) is a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Members of the Fabaceae exhibit a particularly wide range in the extent of CA 1-P accumulation during darkness and include Phaseolus vulgaris, whose dark/light regulation of Rubisco activity is principally achieved by synthesis/degradation of CA 1-P. An extensive survey of the degree of dark inhibition of Rubisco was undertaken for the subfamily Papilionoideae to elucidate evolutionary patterns in the occurrence of this regulatory mechanism. Seventy-five species from 21 tribes were examined. Dark inhibition of Rubisco was found in ancestral tribes such as the Sophoreae, but was substantially reduced or absent in representative species of three more recently evolved tribes, Cicereae, Hedysareae and Vicieae. We conclude that regulation of Rubisco by CA 1-P is neither of recent origin nor of restricted distribution among the Papilionoideae. On the contrary, it becomes lost or less pronounced only in a minority of the more evolutionarily advanced species in this important subfamily.     

Physical reasons for secondary structure stability: alpha-helices in short peptides

It was recently found that some short peptides (including C- and S-peptide fragments of RNase A) can have considerable helicity in solution, ^1–12 which was considered to be surprising. Does the observed helicity require a new explanation, or is it consistent with previous understanding? In this work we show that this helicity is consistent with the physical theory of secondary structure^12–19 based on an extension of the conventional Zimm-Bragg model.²⁰ Without any special modifications, this theory explains reasonably well almost all the experimentally observed dependencies of helicity on pH, temperature, and amino acid replacements. We conclude that the observed “general level” of helicity of C- and S-peptides (5–30% at room temperature and 10–50% near 0°C) is “normal” for short peptides consisting mainly of helix-forming and helix-indifferent residues. The helicity is modified by a multitude of weak specific side chain interactions, many of which are taken into account by the present theory;^13–19 some discrepancies between the theory and experiment can be explained by weak side-chain-side chain interactions that were neglected. A reasonable coincidence of the theory with experiment suggests that it had been used to investigate the role of local interactions in the formation of α-helical “embryos” in unfolded protein chains.     

Chiral resolution of alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene, a novel antiplatelet compound.

alpha,alpha'-Bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, a novel antiplatelet agent, was resolved into three isomers A, B, and C, on a chiral alpha 1-acid glycoprotein analytical column using a mobile phase of 0.025 M phosphate buffer containing 0.025 M tetrabutylammonium hydrogen sulfate, at a pH of 6.5. The effect of molarity, temperature, pH, flow rate, and organic modifiers on the enantioselectivity was examined. Based on circular dichroic spectra at 220 nm, A and C appear to be the (-)- and (+)-enantiomers, respectively, and B the meso diastereomer. Attempts at resolution using Pirkle type columns gave unsatisfactory results. It appears that both hydrophobic and polar interactions between the compound and the stationary phase are important determinants of resolution.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

An approach to the identification of adenosine''s inhibitory site on adenylate cyclase

The separate and combined effects of molybdate and dithiothreitol on the stability of human uterine 9 S estrogen receptor were studied. Maximal, short-term, protection of the 9 S estrogen receptor was achieved by the joint inclusion of both stabilizing agents in cytosol buffers. This molybdate—dithiothreitol-mediated stability was dependent on reducing agent concentration inferring sulphydryl involvement in 9 S receptor protection by molybdate. The study also showed that molybdate—dithiothreitol could not prevent the gradual decay of the 9 S estrogen receptor to the 4 S form in cytosols stored at 4°C over prolonged periods.     

Comparative phytotoxicity of nitrapyrin and ATC to several leguminous species

Summary The relative toxicity of nitrapyrin 2-chloro-6-(trichloromethyl) pyridine and ATC (4-amino-1, 2, 4-triazole) on the growth of chick peas (Cicer arietinum L.) cow peas (Vigna sinensis L.), green beans (Phaseolus vulgaris L.), green peas (Pisum sativum L.) and mung beans (Phaseolus aureus Roxb.) and their effectiveness as nitrification inhibitor were studied under greenhouse conditions. ATC produced no toxicity symptoms in green peas, whereas resulted in leaf chlorosis in cow peas, chick peas and green beans. However, nitrapyrin toxicity appeared as leaf chlorosis in cow peas, and interveinal chlorosis in chick peas. Moreover, nitrapyrin-treated green beans and peas developed leaf curling and cupping. Although ATC had no significant effect on growth, a suppression in plant growth was associated with nitrapyrin application. Furthermore, green beans was the most resistant and chick peas the most sensitive to nitrapyrin. Nitrapyrin was more effective nitrification inhibitor than ACT, especially at the lower rates.     

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.