STUDIES ON THE CDP-ETHANOLAMINE-1,2-DIGLYCERIDE ETHANOLAMINEPHOSPHOTRANSFERASE OF RAT BRAIN

Abstract— The ethanolaminephosphotransferase (EC 2.7.8.1) of rat brain is found largely in the microsomal fraction and is active towards both diacyl glycerol and alkenyl acyl glycerol. Manganese ions were found to be more effective activators of the enzyme than magnesium ions at low concentrations. The K_m for CDP-ethanolamine was found to be about 2.5 × 10⁻⁴ M in the presence of either lipid acceptor and the K _m for the two lipid acceptors about 1.6 × 10⁻³ M. Under the most favourable conditions rates of 270 nmol phosphatidylethanolamine and 70 nmol ethanolamine plasmalogen/mg microsomal protein/h at 39°C were obtained. The effect of temperature on the reaction rate depended on whether diacyl glycerol or alkenyl acyl glycerol was the lipid acceptor. Although diacyl glycerol inhibited the formation of ethanolamine plasmalogen the inhibition was not a simple competitive one. In terms of microsomal protein the activity was maximal during the period of active myelination but at3 days and 150 days of ageitwasat least 50 percent of this maximal activity.     

Physiological studies on the effect of Ca2+ on the duration of the lag phase of Saccharomyces cerevisiae

Abstract Cell multiplication and growth of Saccharomyces cereviseae were followed in 2-ml test tubes containing Wickerham's synthetic medium or very dilute synthetic media supplemented in various ways. The ability of the cell cultures to leave the lag phase and enter the exponential phase of growth was investigated. Multiplication was assessed by microscopical observation. The results showed great differences in times required for the cultures to leave the lag phases and begin multiplication. In Wickerham's medium, all cultures grew well 6 h after inoculation. In the dilute medium, several days elapsed before all the cultures grew. These cultures went into exponential growth with approximately first order kinetics. In the unsupplemented medium, the 'half-lives' in the lag phase were about 28 h. Addition of either Ca²⁺ or Ca²⁺ plus A23187 (calcimycin) reduced the half-lives to 10 and 6 h, respectively. The doubling times in the exponential phases of growth were not shortened by these additions. We suggest that Ca²⁺ plays a crucial role as a signal to switch on the mode of cell proliferation in S. cerevisiae .     

Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis

Abstract: In fetal rat brain neurons, activation of voltage-dependent Na⁺ channels induced their own internalization, probably triggered by an increase in intracellular Na⁺ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na⁺ channel activation. An increase in intracellular Ca²⁺ concentration induced either by thapsigargin, a Ca²⁺-ATPase blocker, or by A23187, a Ca²⁺ ionophore, was unable to provoke Na⁺ channel internalization. However, Ca²⁺ seems to be necessary because both neurotoxin- and amphotericin B-induced Na⁺ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na⁺ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na⁺ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na⁺ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na⁺ channel internalization in neurons.     

Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osm^s) on agar plates containing high concentrations of either non-ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1 ⁺ and gpd2 ⁺, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1 ⁺ was regulated at the mRNA level in response to osmotic upshift, (ii) It was demonstrated that wild-type cells markedly accumulated internal glycerol under high-osmolarity growth conditions. (iii) Δ gpd1 mutants, however, failed to do so even in a high-osmolarity medium, and thus exhibited an Osm^s phenotype. On the other hand, the gpd2 ⁺ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.     

Glucose 6-Phosphate and Fructose 1,6-Bisphosphate Can Be Used as ATP-Regenerating Systems by Cerebellum Ca2+-Transport ATPase

Abstract : In this work, it is shown that the Ca²⁺-transport ATPase found in the microsomal fraction of the cerebellum can use both glucose 6-phosphate/hexokinase and fructose 1,6-bisphosphate/phosphofructokinase as ATP-regenerating systems. The vesicles derived from the cerebellum were able to accumulate Ca²⁺ in a medium containing ADP when either glucose 6-phosphate and hexokinase or fructose 1,6-bisphosphate and phosphofructokinase were added to the medium. There was no Ca²⁺ uptake if one of these components was omitted from the medium. The transport of Ca²⁺ was associated with the cleavage of sugar phosphate. The maximal amount of Ca²⁺ accumulated by the vesicles with the fructose 1,6-bisphosphate system was larger than that measured either with glucose 6-phosphate or with a low ATP concentration and phosphoenolpyruvate/pyruvate kinase. The Ca²⁺ uptake supported by glucose 6-phosphate was inhibited by glucose, but not by fructose 6-phosphate. In contrast, the Ca²⁺ uptake supported by fructose 1,6-bisphosphate was inhibited by fructose 6-phosphate, but not by glucose. Thapsigargin, a specific SERCA inhibitor, impaired the transport of Ca²⁺ sustained by either glucose 6-phosphate or fructose 1,6-bisphosphate. It is proposed that the use of glucose 6-phosphate and fructose 1,6-bisphosphate as an ATP-regenerating system by the cerebellum Ca²⁺-ATPase may represent a salvage route used at early stages of ischemia ; this could be used to energize the Ca²⁺ transport, avoiding the deleterious effects derived from the cellular acidosis promoted by lactic acid.     

Glucose transport throughout fermentation by Saccharomyces cerevisiae NCYC 1108

The rate of fermentation of glucose by a polyploid strain of Saccharomyces cerevisiae growing in a defined salts medium depends on the availability of NH₄+⁺. Its decline after exhaustion of the nitrogen source corresponded with the ability of the cells to accumulate the glucose analogue 2-deoxyglucose. Addition of NH₄+⁺to a nitrogen-depleted culture stimulated both glucose utilization and 2-deoxyglucose uptake. Since stimulation was inhibited by cycloheximide, maintenance of glucose transport during fermentation is dependent on protein synthesis.     

Regulation of the L-Type Ca2+ Channel Current in Rat Pinealocytes

Abstract : In the present study, the role of phosphoprotein phosphatase in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes was investigated using the whole-cell version of the patch-clamp technique. The effects of three phosphatase inhibitors, calyculin A, tautomycin, and okadaic acid, were compared. Although all three inhibitors were effective in inhibiting the L-type Ca²⁺ channel current, calyculin A was more potent than either tautomycin or okadaic acid, suggesting the involvement of phosphoprotein phosphatase-1. To determine the kinase involved in the regulation of these channels, cells were pretreated with H7 (a nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic AMP-dependent kinase), KT5823 (a specific inhibitor of cyclic GMP-dependent kinase), or calphostin C (a specific inhibitor of protein kinase C). Pretreatment with either H7 or calphostin C decreased the inhibitory effect of calyculin A on the L-type Ca²⁺ channel current. In contrast, pretreatment with H89 or KT5823 had no effect on the inhibition caused by calyculin A. Based on these observations, we conclude that basal phosphatase activity, probably phosphoprotein phosphatase-1, plays an important role in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes by counteracting protein kinase C-mediated phosphorylation.     

Effect of Shaking on the Growth of Diluted Cultures of Tetrahymena

ABSTRACT. Cultures of Tetrahymena pyriformis, T. thermophila and T. pigmentosa have been studied with regard to growth rates in shaken and unshaken flasks. In the standard medium, a minimum doubling time of 170 min was obtained for T. pyriformis at 28° C in the unshaken cultures. If the depth of the medium was less than 1 cm, the gyratoric shaking increased the doubling time to 340 min. The effect of shaking could be reduced by the addition of dextrane. Cells subjected to shaking were observed in different media and at different growth temperatures. If cultures were inoculated with 10⁴ cell·ml⁻¹ or more, the effect of shaking was absent. However, with inoculates of 10³ or 10² cell·ml⁻¹, the doubling times for T. pyriformis increased to 240 and 275 min, respectively. Periods of 2 min shaking followed by rest for 60 min could not induce an effect.     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Endothelin Induces a Calcium-Dependent Phosphorylation of PEA-15 in Intact Astrocytes: Identification of Ser104 and Ser116 Phosphorylated, Respectively, by Protein Kinase C and Calcium/Calmodulin Kinase II In Vitro

Abstract: PEA-15 (phosphoprotein enriched in astrocytes, M_r = 15,000) is an acidic serine-phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine-induced apoptosis. PEA-15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA-15 phosphorylation. Endothelin-1 or endothelin-3 (0.1 µ M ) induced a robust phosphorylation of PEA-15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA-15 following endothelin-1 treatment identified two phosphorylated residues: Ser¹⁰⁴, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser¹¹⁶, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin-dependent protein kinase II (CaMKII). Partly purified PEA-15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two-dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser¹¹⁶ and had no effect on Ser¹⁰⁴, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA-15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.     

Author idex volume 36 (1986)

Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO₃), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO⁻₂→ NH₃) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h⁻¹) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min⁻¹ (mg protein)⁻¹. The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M _r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule).     

Effects of oxidative stress on chlorophyll biosynthesis in cucumber (Cucumis sativus) cotyledons

We conducted a series of experiments to assess the effects of oxidative stress on chlorophyll biosynthesis in the vascular plant Cucumis sativus (cucumber). Specifically, cucumber cotyledons were treated with 100 μ M methyl viologen (MV) and subsequently exposed to dark (0 μE m⁻² s⁻¹), low light (40–45 μE m⁻² s⁻¹), or high light (1500–1600 μE m⁻² s⁻¹). Following treatment, extracts of these samples were subjected to high-performance liquid chromatography (HPLC) to quantitate the accumulation of chlorophyll biosynthetic pathway intermediates. The results of these analyses revealed significant accumulation of Mg-protoporphyrin IX monomethyl ester (Mg-proto IX ME) in green (14-h illuminated) as well as in etiolated cotyledons with MV treatment. These data suggest that MV-induced oxidative stress may have inhibited Mg-proto IX ME cyclase activity. Upon exposure to high light, in the presence or absence of MV, both green and etiolated cotyledons predominantly accumulated protoporphyrin IX (Proto IX). These elevated levels of Proto IX might be attributable to attenuated activity of any or all of the following enzymes: Mg-chelatase, Fe-chelatase and protoporphyrinogen IX oxidase. We also observed that MV-induced oxidative stress impacts on chlorophyll biosynthesis to a greater extent than on photosystem II. These results demonstrate that oxidative stress impedes key steps in chlorophyll biosynthesis by either directly or indirectly inhibiting the activity of these enzymes.     

Elevated Levels of Glucose and L-Fucose Reduce 22Na+ Uptake and Whole Cell Na+ Current in Cultured Neuroblastoma Cells

Abstract: Na⁺ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L - fucose concentrations. Chronic exposure of neuroblastoma cells to 30 m M glucose or 30 m M L-fucose caused a decrease in ouabain-sensitive and veratridine-stimulated ²²Na⁺ uptake compared with cells cultured in unsupplemented medium. The Na⁺ current, determined by using whole-cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 μ M ), which blocked whole cell Na⁺ currents, also blocked veratridine-stimulated ²²Na⁺ accumulation. Culturing cells in medium containing 30 m M fructose as an osmotic control had no effect on Na⁺ flux. Specific [³H] saxitoxin binding was not affected by 30 m M glucose or 30 m M L-fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na⁺ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na⁺ transport and Na⁺-channel activity.     

Activation of Protein Kinase C by the Capsaicin Analogue Resiniferatoxin in Sensory Neurones

Abstract: Resiniferatoxin and capsaicin are sensory neurone-specific excitotoxins that operate a common cation channel in nociceptors. Resiniferatoxin is structurally similar to capsaicin and to phorbol esters. Specific [³H]-resiniferatoxin binding, which was detected in the membrane ( K _D value 1.8 ± 0.2 n M ) but not cytosolic fraction of rat dorsal root ganglia, could not be displaced by phorbol 12,13-dibutyrate. Conversely, resiniferatoxin did not displace [³H]phorbol 12,13-dibutyrate binding in either the cytosolic or membrane fraction. Resiniferatoxin and capsaicin both caused translocation of protein kinase C in dorsal root ganglion neurones (EC₅₀ value 18 ± 3 n M ). This translocation was greatly reduced but not abolished, in the absence of external Ca²⁺, suggesting that it was secondary to Ca²⁺ entry. Resiniferatoxin also caused direct activation of a Ca²⁺- and lipid-dependent kinase (or kinases) in the cytosolic fraction of dorsal root ganglia, at concentrations (100 n M to 10 µ M ) higher than required for displacement of [³H]resiniferatoxin binding or translocation of protein kinase C. Capsaicin (up to 10 µ M ) was unable to mimic this effect. These data imply that although resiniferatoxin-induced translocation of protein kinase C in dorsal root ganglion neurones was mainly indirect, it also caused direct activation of a protein kinase C-like kinase in these cells.     

Basic studies on gene therapy of human malignant melanoma by use of the human interferon beta gene entrapped in cationic multilamellar liposomes. 1. Morphology and growth rate of six melanoma cell lines used in transfection experiments with the human interferon beta gene

Six cell lines of human malignant melanoma: A375, A375.2, G361, HMV-1, MM8.1 and WM115 were seeded at densities of 1 × 10⁴ cells/ml, 2 × 10⁴ cells/ml or 3 × 10⁴ cells/ml of RPMI medium supplemented with 10% fetal calf serum and antibiotics in a humidified atmosfere of 5% CO₂ at 37°C. A375 cells were also grown in Dulbecco's minimum Eagle's medium (DMEM medium). The morphology was studied by phase contrast light microscopy. At 4 days after seeding the colonies of A375 cells and HMV-1 cells were oval-shaped, the cells were polyhedrical and were making contact with each other regularly. The remaining cells were scattered, more elongated, and made contact randomly. G361 cells and MM8.1 cells tended to form superposed layers before 100% confluency was achieved. There were great differences in the growth rate and doubling time of melanoma cells. The doubling time in day 1 was short (around 6-12 h) in the case of A375, G361 and HMV-1 cells, longer (around 18h) in the case of MM8.1 cells and very long (ranging between 26 and 89 h) for A375.2 and WM115 cells. There were also differences in the doubling time of cells as a function of the cell density at seeding. On the other hand, except for MM8.1 cells, there were differences between the doubling time in day 2 compared to day 1.     

Ca2+/Calmodulin-Mediated Regulation of Agonist-Induced Sequestration of Gq Protein-Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells

Abstract: We investigated the regulation by intracellular Ca²⁺ of agonist-induced sequestration of G_q protein-coupled histamine H₁ receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H₁ receptors from the cell surface membrane was detected as the loss of [³H]mepyramine binding sites on intact cells accessible to the hydrophilic H₁-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [³H]mepyramine were mirrored by changes in the subcellular distribution of H₁ receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H₁ receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca²⁺ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca²⁺/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H₁-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of G_q protein-coupled receptors is transiently inhibited by Ca²⁺/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.     

Activation of plasma membrane H+-ATPase by ammonium ions in Aspergillus niger

The addition of ammonium ions to Aspergillus niger cells originally growing on another nitrogen source resulted in rapid medium acidification. The addition of glucose or other fermentable sugars to the mycelium growing on glycerol did not have the same effect. The enzyme responsible for acidification seems to be plasma membrane H+-ATPase, which is most probably triggered by phosphorylation. Using specific activators and inhibitors, we tried to figure out which signalling pathway is involved in the process. No activation of H+-ATPase could be detected in the presence of diacylglycerol and other activators of protein kinase C, indicating that the stimulus is transmitted by another signalling chain. In the presence of inhibitors known to suppress the phosphatidyl-inositol signalling pathway, such as neomycin, compound 48/80 and calmidazolium, no increased H+-ATPase activity could be detected after the addition of ammonium ions. However, some tested inhibitors of the cAMP signalling pathway could not prevent activation of the enzyme by the stimulant. These results support the model in which ammonium-induced activation of proton extrusion in A. niger is mediated via the phosphatidyl-inositol signalling pathway, involving Ca2+/calmoduline-dependent protein kinase but not protein kinase C.