An LC-MS Approach to Quantitative Measurement of Ammonia Isotopologues.

Ammonia is a fundamental aspect of metabolism spanning all of phylogeny. Metabolomics, including metabolic tracing studies, are an integral part of elucidating the role of ammonia in these systems. However, current methods for measurement of ammonia are spectrophotometric, and cannot distinguish isotopologues of ammonia, significantly limiting metabolic tracing studies. Here, we describe a novel LC-MS-based method that quantitatively assesses both 14N-and 15N-isotopologues of ammonia in polar metabolite extracts. This assay (1) quantitatively measures the concentration of ammonia in polar metabolite isolates used for metabolomic studies, and (2) accurately determines the percent isotope abundance of 15N-ammonia in a cell lysate for 15N-isotope tracing studies. We apply this assay to quantitatively measure glutamine-derived ammonia in lung cancer cell lines with differential expression of glutaminase.

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains.

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3 , the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples.

Human striosomes are enriched in 5-HT2A receptors: autoradiographical visualization with [3H]MDL100,907,[125I](+/-)DOI and [3H]ketanserin.

5-HT2A receptors have been visualized with [3H]MDL100,907 in selected human brain areas by autoradiography. These areas included caudate and putamen, nucleus dentatus of the cerebellum, substantia nigra, nucleus raphe dorsalis, locus coeruleus and inferior olive. In the striatum [3H]MDL100,907 labelling was compared with the pattern obtained with [125I](+/-)DOI and [3H]ketanserin. [3H]MDL100, 907 and [125I](+/-)DOI showed an identical patchy distribution which was hardly observed with [3H]ketanserin. In the remaining regions, [3H]MDL100,907 and [3H]ketanserin autoradiographical signals and percentage of specific binding were compared. Whereas the pattern of distribution was identical for both radioligands, [3H]MDL100,907 presented a much lower percentage of nonspecific binding compared with [3H]ketanserin. These results confirm the presence of 5-HT2A receptors in human striosomes and in those areas where [3H]ketanserin presented a high nonspecific binding, and they highlight the advantage of using [3H]MDL100,907 to visualize these receptors.

A simple assay for measuring cellobiose dehydrogenase activity in the presence of laccase.

The commonly used assay for measuring cellobiose dehydrogenase (CDH) activity, based on the reduction of dichlorophenol-indophenol (DCIP), has been adapted to measure this enzyme activity in the presence of laccase, which is often formed concurrently with CDH by a number of fungi. Laccase interferes with the assay by rapidly reoxidizing the reduced form of DCIP and can mask CDH activity completely. It can be conveniently and completely inhibited by 4 mM fluoride in the assay, while CDH activity is only slightly affected by the addition of this inhibitor. The modified assay enables the detection of low CDH activities even in the presence of very high excesses of laccase. It should be useful for screening culture supernatants of wood-degrading fungi for CDH since the assay is rapid and uses inexpensive and nontoxic reagents. Furthermore, it might be used for the detection of other enzyme activities which are assayed by following the reduction of quinones or analogue compounds such as DCIP.

Selective increases in serotonin 5-HT1B/1D and 5-HT2A/2C binding sites in adult rat basal ganglia following lesions of serotonergic neurons.

Quantitative autoradiography was used to examine possible adaptive changes in serotonin 5-HT1B/1D and 5-HT2A/2C receptor binding sites in adult rat basal ganglia, after partial or severe lesions of serotonergic neurons produced by intraraphe injections of variable amounts of 5,7-dihydroxytryptamine. In controls, the 5-HT1B/1D sites labeled with S-CM-G[125I]TNH2 were evenly distributed in the core and the shell of the nucleus accumbens. The density of 5-HT1B/1D sites was higher in the ventral than dorsal part of the striatum and no regional differences were detected along the rostrocaudal axis of the structure. The 5-HT2A/2C sites labeled with [125I]DOI were preferentially distributed in the mediodorsal striatum and higher densities were detected in the shell than core of the nucleus accumbens. Following 5,7-dihydroxytryptamine injections, there were no changes in binding of either receptor subtype after partial lesions entailing 80-90% 5-HT depletions. After severe 5-HT depletions (over 95%), large increases in 5-HT1B/1D binding were observed in the substantia nigra (78%), but no changes took place in the globus pallidus. Increases in 5-HT1B/1D binding were also detected in the shell of the nucleus accumbens (27%). Similar sized increases in 5-HT2A/2C binding (22%) were restricted to the medial striatum. The present results suggest a preferential association between 5-HT1B/1D receptors and the striatonigral neurons containing substance P, as indicated by the striatal distribution of these receptors and their selective increases in the substantia nigra after severe 5-HT deprivation. We recently proposed a similar relationship between the 5-HT4 receptors and the striatopallidal neurons containing met-enkephalin. Moreover, the increases in 5-HT1B/1D binding in the substantia nigra and in the shell of the nucleus accumbens reinforce the view of an implication of this receptor subtype in motor functions. In contrast, the prominent increases in 5-HT2A/2C binding after severe 5-HT deprivation as restricted to the medial region of the striatum and suggest up-regulation of most probably 5-HT2C receptors in a region implicated in cognitive functions.

NAD(P)H:quinone oxidoreductase expression and mitomycin C resistance developed by human colon cancer HCT 116 cells.

An association between the resistance to mitomycin C (MMC) and a decrease of NAD(P)H:quinone oxidoreductase (NQO1) activity was reported for a MMC-resistant subline, HCT 116-R30A, derived from MMC-sensitive HCT 116 cells. Eight NQO1 cDNA clones were isolated from these two sublines by reverse transcription-PCR. Two clones, pDT9 from HCT 116 and pDT20 from HCT 116-R30A, are the full length of 274 amino acids. These two clones differ by a T to C substitution at nucleotide 464, which results in a replacement of arginine 139 by tryptophan in the enzyme. NQO1 of pDT9 and pDT20 was expressed in Escherichia coli, purified, and shown to have a protein subunit of M(r) 30,000. The change of amino acid 139 resulted in a shift of isoelectric pH from 9.5 to 8.35 and a 60% decrease of activity in reducing MMC. All of the other six clones differ from pDT9 by a deletion of exon 4. On Northern blot, we detected two mRNA species of NQO1 (1.2 and 2.7 kilobases) due to alternative polyadenylation in all sublines. MMC-resistant sublines showed 75-90% mRNA expression relative to HCT 116 cells. Reverse transcription-PCR amplification of cDNA fragment of nucleotide 298-617 revealed two full-length mRNAs in HCT 116 cells but only one full-length mRNA in HCT 116-R30A cells. An exon 4 deletion mRNA was detected in both sublines. The two full-length mRNAs may be from either alleles or chimeras of the same gene and the exon 4 deletion mRNA is a result of alternative splicing. On Western blot, we detected only one M(r) 30,000 protein in all sublines. A substantial decrease of this protein in MMC-resistant sublines (5% of HCT 116) explained the 95% decrease of their NQO1 activity. Transcriptional regulation and posttranscriptional modification may be responsible for the disparity of gene expression of NQO1 and the low concentration of NQO1 protein in MMC-resistant sublines. Reversal of MMC resistance and the recovery of NQO1 in two revertants further supports the hypothesis that cellular control of NQO1 can modulate the cytotoxicity of MMC.

Monoclonal antibody AE-2 modulates carbamate and organophosphate inhibition of fetal bovine serum acetylcholinesterase.

The monoclonal antibody AE-2, raised against the human erythrocyte acetylcholinesterase (AChE) dimer (acetylcholine acetylhydrolase, EC 3.1.1.7), binds to other mammalian AChEs, including the tetramer that occurs in fetal bovine serum (FBS). AE-2 partially inhibited the rate of hydrolysis of the charged substrate acetylthiocholine by FBS AChE, whereas it increased the rate of hydrolysis of the neutral substrate indophenyl acetate. Present results show that AE-2 decreases the rate of inhibition of FBS AChE by the positively charged organophosphate amiton-p-toluene sulfonate and the positively charged carbamates pyridostigmine and neostigmine but accelerates inhibition of FBS AChE by the neutral organophosphates paraoxon and diisopropylfluorophosphate. Results suggest that AE-2 may allosterically modulate an anionic site in the catalytic center of FBS AChE.

Cellobiose oxidase from Phanerochaete chrysosporium. Stopped-flow spectrophotometric analysis of pH-dependent reduction.

Cellobiose oxidase (CBO) from Phanerochaete chrysosporium can utilize dichlorphenol-indophenol (Cl2Ind) and cytochrome c as effective electron acceptors for the oxidation of cellobiose. However, the pH dependencies of activity for these electron acceptors are significantly different. Both compounds act as effective electron acceptors at pH 4.2, whereas only dichlorophenol-indophenol is active at pH 5.9. To explain this discrepancy, the pH dependencies of the reduction rates of FAD and heme, respectively, in CBO by cellobiose have been investigated by stopped-flow spectrophotometry. Both FAD and heme are reduced with a high rate constant at pH 4.2. In contrast, at pH 5.9, only FAD reduction is fast, while the reduction of the heme is extremely slow. As a conclusion, the reduction of cytochrome c by CBO is dependent on heme, which functions at a lower pH range compared to reduction of FAD.

Acetylcholinesterase: inhibition by tetranitromethane and arsenite. Binding of arsenite by tyrosine residues.

Tetranitromethane inhibits acetylcholinesterase with respect to the hydrolysis of both acetylthiocholine and indophenyl acetate. The loss of activity with indophenyl acetate, a poor substrate, is preceded by an increase in enzyme activity. Only 12 of the 21 tyrosine residues/monomer of enzyme are susceptible to nitration. Loss of activity with respect to indophenyl acetate occurs well after no further nitration of tyrosines occurs and must be due to the modification of other residues. Incubation of the enzyme with arsenite before nitration results in the nitration of only 10 tyrosines. This experiment reveals that the structural basis for the binding of arsenite is the formation of a diester with two tyrosine residues.

Cutaneous penetration of some hairdyes in the hairless rat.

The cutaneous penetration of certain number of oxidation dyestuffs for hair [p-aminophenol, 2,4-diaminoanisole, 2,4-diaminophenoxyethanol, resorcinol, 3-acetylamino-2,6-dimethylphenol and the indamine: N-(4'-hydroxyphenyl)-3-amino-6-methylbenzoquinoneimine], either alone or when formulated with other dyestuffs, and under various experimental conditions, has been evaluated in the hairless rat using C14 labeled molecules. It has been found that: Dyestuffs having indamine structures, formed by oxidation of their precursors (bases, couplers), for all practical purposes do not cross the cutaneous barrier. No fixation of those dyestuffs investigated (resorcinol, 2,4-diaminoanisole, 2,4-diaminophenoxyethanol) which do pass through the cutaneous barrier is found in either liver or thyroid of the experimental animals, 4 days after their topical application. The amount of the hairdye 3-acetylamino-2,6-dimethylphenol which penetrates the cutaneous barrier is constant for quantities of hairdye solution greater than or equal to 20 mg/cm2 of skin. The type and composition of the excipient used in the formulation of hairdyes plays an important role in their penetration.

Purification and properties of pyridine nucleotide-independent L-lactate dehydrogenase from Polyporus circinatus.

Cell extracts of Polyporus circinatus grown on lactate catalyze the reduction of 2,6-dichlorophenolindophenol by l-lactate without the participation of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The enzyme has been purified 78-fold and was homogenous by disc gel electrophoresis. The optimal pH was found to be 6.7. The Michaelis constant for l-lactate was 5.9 x 10(-4) M and the oxalate inhibition constant was 1.5 x 10(-4) M. The nature of the prosthetic group is discussed.

Cyanide formation from oxidation of glycine of Pseudomonas species.

With whole cells of a hydrogen cyanide-producing bacterium strain C, of the genus Pseudomonas, it was found that the oxygen necessary for the oxidation of glycine to cyanide could be replaced by various artificial electron acceptors. The order of reactivity was: oxygen > phenazine methosulphate > methylene blue > 2,6-dichlorophenolindophenol > ferricyanide. Cyanide production was inhibited by pyrrolnitrin, a well-known inhibitor of many flavine enzymes. The molar ratio of added glycine to cyanide produced was found to be 1.09. With whole bacteria the apparent K(m) (glycine) for the cyanide production was found to be 5.0 x 10(-4) M.