Solvent-free glycerolysis of palm and palm kernel oils catalyzed by commercial 1,3-specific lipase from Humicola lanuginosa and composition of glycerolysis products

Glycerolysis of palm and palm kernel oils were conducted using a commercial 1,3-specific lipase from Humicola lanuginosa (trade name: SP 398) as catalyst (500 units lipase g^–1 oil) at 40°C and oil:glycerol (1:2 mol mol^–1) in a solvent-free system. After 24 h, the glycerolysis products of palm and palm kernel oils consisted of 23% triacylglycerols, 18% monoacylglycerols, 38% diacylglycerols and 18% triacylglycerols, 31% monoacylglycerols, 42% diacylglycerols, respectively. The monoacylglycerol fraction of the glycerolysis product of palm oil was enriched in oleic acid. Palmitic acid content of the monoacylglycerol fraction of the same product was less than that of the original oil. Under the same conditions, monacylglycerol fraction of the palm kernel oil glycerolysis product was enriched in palmitic, stearic and oleic acids.     

Immobilisation ofCandida cylindracea lipase on a new range of ceramic supports

Summary Lipase fromC. cylindracea was covalently immobilised to a number of surface-treated ceramic supports (3–10 mg. (g dry wt support)^–1). At room temperature, the immobilised lipase could convert R,S-citronellol and butyric acid to citronellyl butyrate at rates in the range 7–51 mol. (mg lipase.min)^–1. The lipase maintained 90–100% of its initial activity over a period of 150 days.     

Continuous and consecutive conversion of free fatty acid in rice bran oil to triacylglycerol using immobilized lipase

Free fatty acid (FFA), monoacylglycerol (MG) and diacylglycerol (DG) in high-FFA rice bran oil were continuously converted with glycerol (G) to form triacylglycerol (TG), using lipase fromRhizomucor miehei immobilized on anion-exchange resin. The reaction was continued for more than 1 month by a reactor with two circulation loops, each being connected to a fixed-bed reactor and a dehydrator. The reaction of 2 FFA + G DG + 2H₂O appeared to occur until the glycerol was exhausted; the reaction of FFA + DG TG + H₂O then followed. The consecutive esterificaion continued in the presence of 2–8 ppm water and the TG content reached 74%–88%. The industrial feasibility of this process was assessed from the standpoints of enzyme cost and value added by esterification.     

Purification and characterization of mono-and diacylglycerol lipase isolated from Penicillium camembertii U-150

Summary A novel enzyme hydrolysing mono- and diacylglycerol was found in the culture filtrate of an isolated fungus, Penicillium camembertii. The enzyme was separated into two forms (A- and B-enzyme) with almost the same molecular weight (37,000–39,000), amino acid composition and identical N-terminal amino acid sequence. B-Enzyme, a major component, was purified approximately 210-fold with an activity yield of 2.6%. The B-enzyme was specific to mono- and diacylglycerols and hydrolysed long-chain monoacylglycerols most efficiently. Triacylglycerols were completely inert as substrates for the enzyme. The B-enzyme preferred to attack -position to -position of monoacylglycerol, but showed no stereospecificity on mono- and diacylglycerol. Both Fe³⁺ and Hg²⁺ inhibited B-enzyme activity significantly.Offprint requests to: S. Yamaguchi     

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn-1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.     

Inhibition of Glutamate-Induced Intensification of Free Radical Reactions by Gangliosides: Possible Role in Their Protective Effect in Rat Cerebellar Granule Cells and Brain Synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 M) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na⁺, K⁺-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.     

Indigogenic methods for glycosidases

Summary In the histochemical detection of -D-glucosidase the indigogenic method of Pearson et al. was tested and evaluated. 4-Cl-5-Br-3-indolyl--D-glucoside was used as the substrate.Intestinal -D-glucosidase is firmly bound to the structure. About 60% of activity survives 2 hours fixation in cold 4% formaldehyde and some activity can be demonstrated even in paraffin sections after a shortened embedding.The localization obtained with the original method is not correct. Due to a slow oxidation of indoxyl in the acid pH range and to hydrogen peroxide formation indigo is deposited in sites with an active peroxidase or pseudoperoxidase. The addition of horse-radish peroxidase improves the localization but does not entirely prevent diffusion artifacts. A striking improvement of the localization can be achieved by a mixture of ferri-ferrocyanide. 3.1 · 10^–3 M concentration of this oxidation catalyst which is still very effective causes only a 26% inhibition of the enzyme activity as revealed by biochemical assays in homogenates of rat intestine.A new medium was devised consisting of 0.1 M citrate phosphate buffer pH 5.5, 8 · 10^–4 M 4-Cl-5-Br-3-indolyl--D-glucoside and 3.1 · 10^–3 M ferri-ferrocyanide mixture. With this medium a very clear brush border localization of the enzyme activity (activities) in enterocytes of the rat and human intestine was demonstrated. This activity is present in differentiated enterocytes covering the villi. The highest activity resides in the jejunum. Enzyme activity is considerably inhibited by galactonolactone (5 · 10^–3 M) and gluconolactone (4 · 10^–4 M). It is completely inhibited by florizin (5 · 10^–3 M). Cellobiose (8 · 10^–2 M) caused 65%, lactose (8 · 10^–2 M) 48% and glucose (8 · 10^–2 M) 35% inhibition (data were obtained by cytospectrophotometry). In patients with celiac sprue the activity is very much decreased. Its restitution after a gluten-free diet proceeds roughly parallel to that of lactase. The relationship of the demonstrated activity (activities) to florizin hydrolase and lactase is discussed.In the kidney the reaction is very weak and is confined to the cells of proximal convoluted tubules (diffuse staining with some enhancement at the luminal surface of the brush border). The method is also very useful for processing zymograms.     

Gangliosides Attenuate Ethanol-Induced Apoptosis in Rat Cerebellar Granule Neurons

Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 M MK-801 (an NMDA receptor antagonist), and 20–200 mM ethanol for 1–4 days. Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3. Inclusion of 5 M MK-801 or 100 M glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway. Preincubation with 50 M gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 M LIGA₂₀ (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death. Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Changes in mediobasal hypothalamic dopamine and GABA release: A possible mechanism underlying taurine-induced prolactin secretion

Summary Taurine (Tau), a putative inhibitory amino acid neurotransmitter, has been shown to stimulate prolactin (PRL) release. Using ovariectomized, estrogen-replaced adult rats we investigated initially the effect of this amino acid, injected by different routes, on PRL secretion in vivo. Tau (100–500 mg/kg) had no effect on PRL release when given i.p.; 15 min after i.c.v. injection of Tau (3moles), a significant increase in serum PRL levels was observed (78 ± 9 ng/ml over basal levels, p < 0.01 vs. controls). In vitro (cultured anterior pituitary cells) PRL release was not affected by a 5 h incubation with Tau (10^–3–10^–8 M). Basal dopamine (DA) or gamma-aminobutyric acid (GABA) output from superfused mediobasal hypothalamic fragments (MBH) was not affected by Tau (10^–3 M or 10^–5 M). However, during stimulation with KCl (50mM), Tau (10^–3 M) significantly lowered DA release, and increased GABA output. It is concluded that Tau acts at a central level to increase PRL secretion, most probably by modulating the hypothalamic release of neurotransmitters controlling lactotroph function.     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10^–5M and 10^–8M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [³H] and [³⁵S] methionine and [³H]ethanolamine as precursors showed an increased methylation of [³H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [³H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [³H]- and [³⁵S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased³H-and³⁵S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [³H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-³H] label after incubation of neurons with [³H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

In vitro propagation of the bamboo (Bambusa tulda Roxb.) through shoot proliferation

An efficient protocol has been developed for the in vitro propagation of Bambusa tulda through shoot proliferation. Shoots from 3-week-old aseptically grown seedlings were used to initiate cultures. Multiple shoots were obtained on liquid Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (8×10^–6M) and kinetin (4×10^–6M). Continuous shoot proliferation at a rate of 4–5 fold every three weeks was achieved through forced axillary branching. More than 90% of the shoots could be rooted on a modified MS medium containing indoleacetic acid (1×10^–5M) and coumarin (6.8×10^–5M). Following simple hardening procedures, the in vitro raised plants were transferred to the soil with more than 80% success.Abbreviations BAP 6-benzylaminopurine - 2-ip 6-,-dimethylallylaminopurine - Kn kinetin - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - NAA 1-naphthaleneacetic acid     

Distinct Responses of Osphradial Neurons to Chemical Stimuli and Neurotransmitters in Lymnaea stagnalis L.

1. In Lymnaea stagnalis L. (Pulmonata, Basommatophora) the neurons in the osphradium were visualized by staining through the inner right parietal nerve by 5,6-carboxyfluorescein (5,6-CF). Three types of neurons were identified: three large ganglionic cells (GC1-3; 80–100 m), the small putative sensory neurons (SC; 20 m) and very small sensory cells (3–5 m).2. The ganglionic and putative sensory neurons were investigated by whole cell patch-clamp method in current-clamp condition. The three giant ganglionic neurons (GC1-3) located closely to the root of osphradial nerve, had a membrane potential (MP) between –30 and –70 mV and showed tonic or bursting activities. The small putative sensory cells (SCs) scattered throughout the osphradial ganglion, possessed a MP between –25 and –55 mV and showed an irregular firing pattern with membrane oscillations. At resting MP the GC1-3 cells were depolarized and increased the frequency of their firing, while the SCs were hyperpolarized and inhibited by NaCl (10^–2 M) and L-aspartate (10^–5 M) applied to the osphradium.3. 5-Hydroxytryptamine (5HT, 10^–6 M), -aminobutyric acid (GABA; 10^–6 M) and the GABA_B agonist baclofen (10^–6 M) depolarized the neurons GC1-3 and increased their firing frequency. In contrast, on the GC1-3 neurons, acetylcholine (Ach; 10^–6 M) and FMRFamide (10^–6 M) caused hyperpolarization and cessation of the firing activity. The 5HT effect was blocked by mianserin (10^–6 M) but picrotoxin (10^–5 M) failed to block the GABA-induced effect on the GC1-3 cells.4. The small putative sensory neurons (SCs) were excited by Ach (10^–6 M) and 5HT (10^–6 M) but were inhibited by GABA (10^–6 M). FMRFamide (10^–6 M) had a biphasic response. The Ach effect was blocked by hexamethonium (10^–6 M) and tetraethylammonium (10^–6 M), indicating the involvement of nicotinic cholinergic receptors.5. The distinct responses of the two populations of osphradial neurons to chemical stimuli and neurotransmitters suggest that they can differently perceive signals from environment and hemolymph.     

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

Induction of the high-affinity nerve growth factor receptor on embryonic chicken sensory nerve cells by elevated potassium

Culture medium with elevated K⁺ has been shown to enhance the survival of neurons isolated from several different regions of the nervous system. Nerve growth factor binds to binding sites on sensory and sympathetic neurons through two sites, one of high-affinity (K _d13×10^–11 M) and the other of low-affinity (K _d22×10^–9 M). Equilibrium binding data generated on dissociated cells derived from E9 chicken embryo dorsal root ganglia, has shown that there is a two-fold increase in the number of high affinity (type I) receptors, with no effect on the affinity, when cells are incubated for 2 hours in buffer containing 59 mM K⁺. There does not appear to be a significant change in the affinity or the number of low-affinity binding sites. This two-fold increase in type I receptors is dependent on temperature, Ca²⁺, and active protein synthesis. There does not appear to be an intracellular pool of the type I receptor sufficient to account for this increase. The induction is not observed on sensory nerve cells cultured in 59 mM K⁺ for 24 hours, either in the presence or absence of nerve growth factor. Additionally, the induction in the number of type I receptors requires that both nerve growth factor and K⁺ be present simultaneously. Taken in total, this data suggests that there may be a critical period in which the sensory neurons require nerve growth factor exposure to respond. Evidence is presented which indicates that nerve growth factor responsive cells are able to elicit neurites after an acute exposure to nerve growth factor of as little as 4 hours. Finally, there is an approximate two-fold decrease in the concentration of nerve growth factor needed to elicit maximal fiber outgrowth, consistent with the two-fold increase in the number of type I receptors.Abbreviations NGF nerve growth factor - 7S NGF the high molecular weight form of NGF - NGF the -subunit of 7S NGF - ¹²⁵I-NGF ¹²⁵I-labeled NGF - mNGF–_rAb polyclonal rabbit IgG raised against mouse NGF - DRG dorsal root ganglia - K_d the equilibrium dissociation constant - N the maximal number of binding sites for the ligand NGF - NGFR the biologically relevant receptor through which the neurite outgrowth and neuron survival are mediated - GBS Gey's balanced salts - HKGBS high K⁺ GBS - PBG phosphate buffered GBS - HKPBG high K⁺ PBG - CFHKPBG Ca⁺² free high K⁺ PBG - PBG-cyt c PBG containing 2 mg/ml cytochrome c - HKPBG-cyt c HKPBG containing 2 mg/ml cytochrome c - AbU antibody unit - BU biological unit PBS, phosphate buffered saline - HKPBS high K⁺ PBS Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.