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PubMed1980

Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates.

Several enzymes that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates (dNTP's) are present as contaminants when nucleotides are extracted from HeLa cells with 60% methanol. These activities include a nuclease, nucleoside diphosphokinase, and deoxyribonucleoside monophosphokinases which phosphorylate dAMP, dGMP, and dCMP. Collectively, these enzymes are able to degrade and reutilize the DNA template which is used together with DNA polymerase for dNTP assays. This process introduces large errors when dNTP assays are performed in this manner. Attempts to block the enzymatic conversion of deoxyribonucleoside diphosphates to triphosphates by inhibition of nucleoside diphosphokinase were unsuccessful because of the inability to block completely the kinase activity. Acid extraction of nucleotides also results in the presence of an activity that interferes with the enzymatic dNTP assay. The error introduced by this interfering activity is much smaller than that arising from the enzymes present in methanol extracts. All of these interfering activities are removed when cells are first extracted with 60% methanol and the resulting extract is subsequently treated with perchloric acid.

DNA-Directed DNA Polymerase↗
PubMed1998

Fluoride uptake to demineralised enamel: A comparison of sampling techniques.

Fluoride uptake is a recognised way of assessing the potential anticaries efficacy of fluoride (F) treatments. The aim of the present study was to compare an abrasion method of sampling treated enamel, based on that of Weatherell et al. [Caries Res 1985;19:97-102], with the acid-etch method of Raven et al. [Caries Res 1991;25:130-137]. Two adjacent demineralised areas were created on the polished surfaces of bovine incisors using an acid gel system. One artificial lesion from each tooth was subsequently treated for 6 h at 37 degrees C with one of two fluoridated dentifrice slurries (1 part: 3 parts water), whereas the other was treated similarly with a slurry of non-F control dentifrice. One set of treated lesions was then separated, the base of each enamel block polished until planoparallel with the demineralised surface and the lesions isolated by cutting away the adjacent sound enamel. Each block was mounted on the probe of a digital micrometer and the demineralised surface abraded with silicon carbide lapping film until sound material was reached. Abraded material was dissolved in perchloric acid and the buffered solution analysed for fluoride by ion-selective electrode. Each lesion of a second set of treated, demineralised enamel blocks was etched by 20microl acid and the resulting solutions analysed for F. Mean F uptakes [microg cm-2 (SD)] were: abrasion (n = 7/treatment); F dentifrice A = 1.39 (0.89) and B = 0.86 (0.45) relative to non-F controls = 0.11 (0.12), 0.14 (0.06), respectively; and acid-etch (n = 14/treatment); A = 1.27 (0. 49), B = 0.69 (0.23), controls = 0.12 (0.06), 0.12 (0.06), respectively. Significant differences (p<0.05) for both data sets were: A>B> control. The results show good agreement between the sampling methods and demonstrate the ability of the abrasion technique to distinguish between F treatments.

Acid Etching, Dental↗
PubMedDate not supplied

Effect of sustained pyridoxine treatment on seizure susceptibility and regional brain amino acid levels in genetically epilepsy-prone BALB/c mice.

Epilepsy-prone and epilepsy-resistant substrains were selectively bred from a strain of BALB/c mice; audiogenic-sensitive epilepsy-prone animals showed enhanced sensitivity to chemical convulsants. Treatment with pyridoxine (100 mg/L in drinking water) initiated at mating and continued throughout pregnancy and the life of the offspring abolished the enhanced sensitivity to chemical convulsants and reduced the severity of audiogenic seizures. Withdrawal of pyridoxine restored the enhanced seizure sensitivity. [1H] Nuclear magnetic resonance (NMR) spectroscopy of perchloric acid extracts of tissue was used to determine the concentrations of several compounds [N-acetylaspartate (NAA), GABA, glutamate, aspartate, alanine, taurine, creatine, cholines, inositol] in the hippocampus, neocortex, brainstem, and cerebellum of untreated and pyridoxine-treated 6-week-old female animals. The ratios of the concentrations of excitatory to inhibitory putative neurotransmitter amino acids tended to be higher in epilepsy-prone animals, with the most pronounced difference being a significantly elevated glutamate/GABA ratio in every brain region examined. Pyridoxine treatment abolished this imbalance in the hippocampus, brainstem, and cerebellum, but not in the neocortex. Treatment of epilepsy-resistant animals with pyridoxine using the same protocol decreased the glutamate/GABA concentration ratio in the hippocampus, brainstem, and neocortex and resulted in impaired development of the animals. The amino acid imbalance and the accompanying seizure susceptibility in these genetically epilepsy prone mice may originate from an inborn error in pyridoxine metabolism or in a pyridoxine-dependent enzyme system.

Acoustic Stimulation↗
PubMed1981

The similarity of histones from turtle erythrocytes and liver.

Using the fresh-water "red-eared turtle" Pseudomys scriptans elegans, we have confirmed the existence of a minor component H1s among the lysine-rich histones of turtle erythrocytes by three forms of gel electrophoresis and two forms of chromatography. It was separated from the major components by both cation-exchange chromatography and molecular-exclusion chromatography and shown to differ slightly but significantly in content of several amino acids as well as being shorter than the other lysine-rich histones. Although its composition is close to that of the "tissue-specific" F1b of sea turtle erythrocytes, it is present in even greater relative amount in red-eared turtle livers. In most respects it resembles the satellite histone H10 of mammalian tissues. No unusual histones were observed among the perchloric acid insoluble histones of turtle erythrocytes.

Amino Acids↗
PubMed1980

Novel reagent and method for direct determination of chloride in serum with a centrifugal analyzer.

We report a novel reagent containing ferric perchlorate, perchloric acid, and polyoxyethylene (23) lauryl ether (Brij 35) with which the concentration of chloride in serum can be measured. We applied this reagent to use with a centrifugal analyzer (CentrifiChem 400) in a dynamic bichromatic procedure, resulting in broad linearity of the standard curve (0-180 mmol/L), short analysis time (1 min), and little interference from bilirubin, hemoglobin, turbidity, or bromide ions. The reagent is simple, contains no mercury, and the combination of low acid concentration and surfactant prevents serum protein precipitation. Precision is good (for x- = 93 mmol/L, CV = 1.55%), and results correlate well with those obtained by coulometry (r = 0.974).

Bilirubin↗
PubMed1990

Determination of six indolic compounds, including melatonin, in rat pineal using high-performance liquid chromatography with serial fluorimetric-electrochemical detection.

A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, N-acetylserotonin, tryptophan, 5-hydroxytryptamine (serotonin) and melatonin in rat pineal using a buffered aqueous eluent containing acetonitrile and methanol as organic modifiers and an ion-pairing agent to assist in controlling the retention of compounds containing an amine group. Serial fluorimetric-electrochemical detection provided additional assurance of compound identity. Analyte preparation simply involved the sonication of pineals in dilute perchloric acid containing an antioxidant and a chelating agent, followed by centrifugation to clarify. The method simplifies the determination of this range of indolic compounds, which normally would require at least two separate runs with different eluents. Detection limits for melatonin were 60 and 135 pg for fluorimetric and electrochemical detection, respectively. (This represented the "worst case" considering the levels and detection limits of all compounds present.) Using flow programming and a flow-rate varying between 1 and 1.5 ml/min, the analysis time was 27.5 min, which made the determination of ten samples in a working day possible.

5-Hydroxytryptophan↗
PubMed1985

A novel, highly phosphorylated protein, of the high-mobility group type, present in a variety of proliferating and non-proliferating mammalian cells.

The present work describes a perchloric-acid-soluble high-mobility-group (HMG)-like protein present in HeLa and Ehrlich ascites cells, rat and calf liver. The protein is designated P1 and has, depending on the source, a molecular mass 48-53 kDa and an amino acid composition which, like the HMG proteins, is characterized by a high content of acidic and basic residues and of proline. The protein contains about 10 mol serine/100 mol amino acid residues, is highly phosphorylated and has, in contrast to the known HMG proteins, an acidic isoelectric point of 5.0. An estimate suggests that protein P1 in HeLa interphase cells contains 25-30 residues of phosphate. Like HMG 1 and 2 it is distributed between the nucleus and the cytoplasm. In HeLa metaphase cells P1 is further modified, resulting in an increase in apparent molecular mass from 53 kDa to 56 kDa.

Amino Acids↗
PubMed2005

Determination of fluvoxamine and its metabolite fluvoxamino acid by liquid-liquid extraction and column-switching high-performance liquid chromatography.

This study describes a new simultaneous determination of fluvoxamine and fluvoxamino acid by automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1.5 ml of plasma using chloroform-toluene (15:85, v/v), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C18 analytical column for separation. The mobile phase for separation consisted of phosphate buffer (0.02 M, pH 4.6), acetonitrile and perchloric acid (60%) (62.4:37.5:0.1, v/v/v) and was delivered at a flow rate of 0.6 ml/min. The peak was detected using a UV detector set at 254 nm. The method was validated for the concentration range 0.8-153.6 ng/ml for fluvoxamine and 0.6-115.2 ng/ml for fluvoxamino acid, and their good linearity (r > 0.998) were confirmed. Intra-day coefficient variations (CVs) for fluvoxamine and fluvoxamino acid were less than 6.6 and 6.0%, respectively. Inter-day CVs for corresponding compounds were 6.3 and 6.5%, respectively. Relative errors ranged from -18 to 9% and mean recoveries were 96-100%. The limit of quantification was 1.2 and 0.9 ng/ml for fluvoxamine and fluvoxamino acid, respectively. This method shows successful application for pharmacokinetic studies and therapeutic drug monitoring.

Amino Acids↗
PubMed2003

Future role of large neutral amino acids in transport of phenylalanine into the brain.

OBJECTIVE: The treatment of phenylketonuria (PKU) in children and adults has been difficult because of erosion of dietary adherence, leading to poor school performance, impairment of executive functioning, loss of IQ, and deterioration of white matter in the brain. Mutant PKU mice produced by exposure to N-ethyl-N'-nitrosourea (ENU) were used to examine the effect of large neutral amino acid (LNAA) supplementation on brain and blood phenylalanine (Phe). METHODS: Mice with PKU, genotype ENU 2/2 with features of classical PKU, were supplemented with LNAA while on a normal diet. Two dosages of LNAA were given 0.5 g/kg and 1.0 g/kg by gavage. Blood Phe was determined in the experimental, control, and sham-treated mice. Brain Phe was determined by magnetic resonance spectroscopy after perchloric acid extraction. Branched-chain amino acid transferase (BCAT) was determined in brain as a marker for energy metabolism. RESULTS: Blood Phe was reduced in the LNAA-treated mice by an average of 15% (0.5 g/kg) and 50% (1.0 g/kg) in 48 hours. There was a sustained decrease in the blood Phe levels over a 6-week trial. The untreated mice and sham-treated mice maintained high blood Phe throughout the experiments. Brain Phe level determined by magnetic resonance spectroscopy showed a decline of 46% after the LNAA treatment. BCAT levels were lower (33%) in the ENU 2/2 mice compared with wild-type. The BCAT normalized in mice with PKU that were treated with LNAA. CONCLUSION: The results suggest that giving LNAA lowered brain and blood Phe levels in mice with PKU. Energy metabolism generated from BCAT also improved in mice with PKU after treatment with LNAA. Data from the mice suggest that LNAA should be considered among the strategies to treat PKU in humans.

Amino Acids, Neutral↗
PubMed1987

Rapid and sensitive method for measuring norepinephrine, dopamine, 5-hydroxytryptamine and their major metabolites in rat brain by high-performance liquid chromatography. Differential effect of probenecid, haloperidol and yohimbine on the concentrations of biogenic amines and metabolites in various regions of rat brain.

A rapid and sensitive method has been developed for the simultaneous determination of norepinephrine, dopamine, 5-hydroxytryptamine and their respectively metabolites 3-methoxy-4-hydroxyphenylglycol, homovanillic acid, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid in discrete brain regions of rats. The supernatants of tissue homogenates were injected directly into a reversed-phase liquid chromatography system, coupled with electrochemical detection. Each of these compounds gave a linear response over the range 5.5-200 ng/ml cerebellar homogenate (0.11-4.0 ng on column). Analytical recoveries of these compounds, added to the homogenate, were essentially complete when compared with standards dissolved in perchloric acid. The average between-assay coefficients of variation for all these compounds were lower than 6.9% over the range 5.5-200 ng/ml. The within-assay coefficients of variation were lower than 9.7%, measured at 5.5 or 23.6 ng/ml. With the present test parameters and mobile phase conditions, all compounds were readily oxidized at 0.8 V vs. a Ag/AgCl reference electrode. The method was applied to an analysis of the differential activity of biogenic amines in the rat striatum, hypothalamus, and hippocampus, produced by probenecid, haloperidol and yohimbine.

Animals↗
PubMed1991

Sepsis does not impair tricarboxylic acid cycle in the heart.

Sepsis has been reported to cause mitochondrial dysfunction and inhibition of key enzymes that regulate the tricarboxylic acid (TCA) cycle. We investigated the effect of sepsis on high-energy phosphates, glycolytic and TCA cycle intermediates, and specific amino acids that are involved in regulating the size of the TCA cycle pool during changes in metabolic state of the heart. Sepsis was induced in 12 female rats by the cecal ligation and perforation technique under halothane anesthesia; seven control rats underwent cecal manipulation without ligation. At 36-42 h postsurgery, the rats were reanesthetized, the chest was opened, and the hearts were freeze-clamped. Perchloric acid extracts of the hearts were analyzed with fluorometric enzymatic methods and 31P nuclear magnetic resonance spectroscopy. There were no significant differences in the levels of the TCA cycle intermediates or high-energy phosphates between the septic and control rats. The major metabolic changes were the 28% decrease in alanine and the 31% decrease in glutamate in the septic hearts compared with control (P less than 0.05 and P less than 0.005, respectively). Phosphocholine, a component of membrane phospholipids, was increased by 91% in the septic hearts (P less than 0.01). We conclude that sepsis does not impair the TCA cycle or induce significant cellular ischemia in the heart. The increase in phosphocholine may represent significant cellular membrane disruption during sepsis.

Adenosine Triphosphate↗
PubMed2000

Nonhistological diagnosis of human cerebral tumors by 1H magnetic resonance spectroscopy and amino acid analysis.

We describe a multivariate analysis procedure to classify human cerebral tumors nonhistologically in vitro, combining the use of 1H magnetic resonance spectroscopy (MRS) with automatic amino acid analysis of biopsy extracts. Eighty-one biopsies were obtained surgically and classified histologically in eight classes: high-grade astrocytomas (class 1, n = 19), low-grade astrocytomas (class 2, n = 10), normal brain (class 3, n = 9), medulloblastomas (class 4, n = 4), meningiomas (class 5, n = 18), metastases (class 6, n = 8), neurinomas (class 7, n = 9), and oligodendrogliomas (class 8, n = 4). Perchloric acid extracts were prepared from every biopsy and analyzed by high resolution 1H MRS and automatic amino acid analysis by ionic exchange chromatography. Intensities of 27 resonances and ratios of resonances were measured in the 1H MRS spectra, and 17 amino acid concentrations were determined in the chromatograms. Linear discriminant analysis provided the most adequate combination of these variables for binary classifications of a biopsy between any two possible classes and in multiple choice comparisons, involving the eight possible classes considered. Correct diagnosis was obtained when the class selected by the computer matched the histological diagnosis. In binary comparisons, consideration of the amino acid profile increased the percentage of correct classifications, being always higher than 75% and reaching 100% in many cases. In multilateral comparisons, scores were: high-grade astrocytomas, 80%; low-grade astrocytomas, 74%; normal brain, 100%; medulloblastomas, 100%; meningiomas, 94.5%; metastases, 86%; neurinomas, 100%; and oligodendrogliomas, 75%. These results indicate that statistical multivariate procedures, combining 1H MRS and amino acid analysis of tissue extracts, provide a valuable classifier for the nonhistological diagnosis of biopsies from brain tumors in vitro.

Algorithms↗
PubMed2004

Selectivity of effects of redox-active cobalt(III) complexes on tumor tissue.

AIM: To estimate the selectivity of action of cobalt complexes on tumor tissue. MATERIALS AND METHODS: Cobalt(III) complexes containing both the tetradentate Schiff-base ligand derived from acetylacetone and ethylenediamine, and compounds of the vitamin PP series or their synthetic analogs, viz. nicotinamide, isonicotinamide or nicotinic acid, as extra (axial) ligands, were tested in vivo on transplanted mice tumors, namely Lewis lung carcinoma (3LL), melanoma B16, and mammary adenocarcinoma Ca755. concentrations of malondialdehyde in tissue extracts were measured by standard biochemical methods. The rate of DNA unwinding was used to detect DNA damage in tumor cells. Level of tumor hypoxia as well as bioenergetic status were estimated using 31P NMR spectroscopy in perchloric acid extracts of tissue. RESULTS: A significant and selective increase of malondialdehyde in tumor tissue reflecting activation of lipid peroxidation was found after administration of the complexes. The bioenergetic status in tumor was also selectively affected by the complexes: minimization of signals of high-energy phosphates was observed two hours after injection of the complexes. An increase of the number of DNA single-strand breaks was registered in tumor tissue, supporting the suggestion that the complexes may directly affect DNA. A correlation between the above tumor effects and the structure of axial ligands was demonstrated. CONCLUSION: Cobalt(III) complexes affect tumor tissue with a very high level of selectivity; in particular they activate lipid peroxidation, induce DNA single-strand breaks, suppress the bioenergetic status, and enhance hypoxia. It is supposed that the selective action of these complexes on tumor tissue is due to peculiarities of tumor microphysiology, in particular significant tumor hypoxia.

Adenocarcinoma↗
PubMedDate not supplied

Physiological and morphological evidences for the presence acidocalcisomes in Trypanosoma evansi: single cell fluorescence and 31P NMR studies.

A new Ca(2+) intracellular store, the acidocalcisome, has been reported in trypanosomatids. It has been characterized physiologically as a Ca(2+) store sensitive to nigericin. The Ca(2+)/H(+)-ATPase is the system responsible for Ca(2+) accumulation, which depends on a pH gradient formed by ATP- and PPi-dependent proton pumps. In this work we present physiological and morphological evidences for the presence of acidocalcisomes in Trypanosoma evansi. The parasites were purified and loaded with the fluorescent dye Fura 2-AM in order to detect the intracellular changes of Ca(2+) levels in individual cells. The simultaneous incubation of T. evansi cells with ionomycin and nigericin led to large release of Ca(2+) (ca. 200 nM) from intracellular stores, which was not observed with either agent alone. On the other hand, no enhancement of the nigericin-induced Ca(2+) release was observed in the presence of oligomycin. Additionally, the pretreatment with bafilomycin decreases the nigericin-induced Ca(2+) release. These results confirm the presence of an intracellular non-mitochondrial acidic Ca(2+) storage compartment. These results suggest that H(+)-ATPase is involved in the process of Ca(2+) accumulation into the acidocalcisomes. Furthermore, the cells loaded with acridine orange exhibited abundant fluorescent vacuoles, which were sensitive to nigericin or bafilomycin A(1). Electronic transmission microscopy observations demonstrated the presence of electron dense particles in the parasites. High levels of inorganic pyrophosphate and triphosphate were detected in perchloric acid extracts of T. evansi by high resolution 31P NMR. Taken together, these results present the first evidence for the presence of acidocalcisomes in T. evansi.

Acridine Orange↗
PubMed1979

Determination of polyethylene glycols 4000 and 6000 in plasma protein preparations.

Polyethylene glycols 4,000 and 6,000 were determined in different plasma protein preparations by the formation of complexes with barium and iodide ions after precipitation of the proteins with perchloric acid. The method, which is fast and reliable, can be used for the determinations of PEG down to 0.005% (w/w) of the total protein content.

Absorption↗
PubMed1995

Role of hepatic carbonic anhydrase in de novo lipogenesis.

The role of carbonic anhydrase in de novo lipid synthesis was examined by measuring [1-14C]acetate incorporation into total lipids, fatty acids and non-saponifiable lipids in freshly isolated rat hepatocytes. Two carbonic anhydrase inhibitors, trifluoromethylsulphonamide (TFMS) and ethoxozolamide (ETZ) decreased incorporation of 14C into total lipids. Both fatty acid and non-saponifiable lipid components of the total lipid were inhibited to approximately the same extent by 100 microM TFMS (29 +/- 0.3% and 35 +/- 0.3% of control respectively in replicate studies). However, neither drug significantly affected ATP concentrations or the transport activity of Na+/K(+)-ATPase, two measures of cell viability. To establish the site of this inhibition, water-soluble 14C-labelled metabolites from perchloric acid extracts of the radiolabelled cells were separated by ion-exchange chromatography. TFMS inhibited 14C incorporation into citrate, malate, alpha-oxoglutarate and fumarate, but had no effect on incorporation of 14C into acetoacetate. Since ATP citrate-lyase, the cytosolic enzyme that catalyses the conversion of citrate into acetyl-CoA, catalyses an early rate-limiting step in fatty acid synthesis, levels of cytosolic citrate may be rate controlling for de novo fatty acid and sterol synthesis. Indeed citrate concentrations were significantly reduced to 37 +/- 6% of control in hepatocytes incubated with 100 microM TFMS for 30 min. TFMS also inhibited the incorporation of 14C from [1-14C]pyruvate into malate, citrate and glutamate, but not into lactate. This supports the hypothesis that TFMS inhibits pyruvate carboxylation, i.e. since all of the 14C from [1-14C]pyruvate converted into citric acid cycle intermediates must come via pyruvate carboxylase (i.e. rather than pyruvate dehydrogenase). Our findings indicate a role for carbonic anhydrase in hepatic de novo lipogenesis at the level of pyruvate carboxylation.

Adenosine Triphosphate↗
PubMed2000

Determination of 5-aminolevulinic acid (ALA) by HPLC method.

5-Aminolevulinic acid is at present most popular precursor of photosensitizer for photodynamic therapy and diagnostics. The effect of accumulation of PpIX induced by exogenous ALA is based on the avoidance of feedback control in the pathway of heme biosynthesis. For use ALA in medicine, development of analytical methods that will enable precise determination of content and purity is necessary. The ALA content is determined i. a. by titration with perchloric acid. Much greater problems are connected with purity determination by HPLC method. There are rather scarce literature data concerning purity determination of ALA by HPLC.

Aminolevulinic Acid↗
PubMed1990

Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen.

Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second dimensional lithium dodecyl sulfate-PAGE than its neuronal counterpart. As measured by 32Pi incorporation, neuromodulin phosphorylation in oligodendrocytes is dramatically increased after short-term phorbol ester treatments, which activate protein kinase C, and is totally inhibited by long-term phorbol ester treatments, which downregulates protein kinase C, thus confirming its probable specific in vivo phosphorylation by protein kinase C. In primary cultures of neuronal cells, two of the three neuromodulin spots were observed to be phosphorylated with an apparent preferential phosphorylation of the more acid forms.

Animals↗