Application of antimony microelectrodes to intracellular pH monitoring

Some novel studies of the properties of the antimony microelectrode used for intracellular pH measurements are described. First, it is shown that currents in the picoampere range, such as those encountered as leakage in some electrometers, induce important changes in pH sensitivity. The response time of the electrode has also been measured and indicates that the electrode exhibits a rapid time course which would be very useful for dynamic cytoplasmic pH investigations. An example of internal pH recording during cellular acidification in Xenopus laevis oocyte is also presented.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

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.     

A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.     

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.