Cholera toxin-induced PGE(2) activity is reduced by chemical reaction with L-histidine.

Mediators of cholera toxin (CT)-induced fluid secretion include 3',5'-adenosine monophosphate (cAMP), prostaglandin E(2) (PGE(2)), and 5-hydroxytryptamine (5-HT). Administration of L-histidine significantly reduced the net secretory response of the small intestine of mice challenged with CT and reduced the capacity of PGE(2) to stimulate Na+ transport in Ussing chambers. We demonstrated that L-histidine chemically modified the structure of PGE(2) but had no direct effect on cAMP or 5-HT. L-Histidine and imidazole reacted with PGE(2) in vitro in cell-free mixtures incubated at 37 degrees C and pH 7.0 under an atmosphere of N(2) with the formation of PGE(2)-imidazole and PGE(2)-histidine covalent adducts. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analysis of the purified adduct showed that imidazole catalyzed the dehydration of PGE(2). A Michael adduct then was formed between C11 of 11-deoxy-Delta(10) PGE(2) (PGA(2)) and the tau nitrogen in the imidazole ring of L-histidine. Importantly, the isolated PGE(2)-imidazole and PGE(2)-histidine adducts inhibited CT-induced fluid loss and cAMP accumulation in mouse intestinal loops. The protection provided by PGE(2)-imidazole, PGE(2)-histidine, and L-histidine against intestinal fluid loss could provide a basis for future therapy against cholera.

Nerve growth factor effects on pyridine nucleotides after oxidant injury of rat pheochromocytoma cells.

Neurotrophic factors regulate neuronal survival and neurite growth in development and following injury. Oxidative stress produced in neurons as a consequence of primary injury, or during reperfusion following ischemia, may contribute to cell death. Here, the effects of nerve growth factor (NGF) on the response to H2O2 injury were examined in the PC12 rat pheochromocytoma cell line. Specifically, the effect of NGF on cell viability after H2O2 injury was measured. Pretreatment with NGF enhanced survival after H2O2 treatment, as measured by Trypan blue dye exclusion, radiolabeled amino acid incorporation, tetrazolium salt reduction, or cytoplasmic enzyme release. One early event associated with H2O2 treatment was a rapid decrease in NAD+. Although initial decreases in NAD+ levels were similar in control and NGF-treated cells, the latter recovered more rapidly and extensively. The decline in total NAD observed after NGF treatment was almost equal in magnitude to the measured increase in NADP. Inhibition of poly(ADP-ribose) polymerase also enhanced viability following H2O2 injury. Treatment with both NGF and an inhibitor of this enzyme resulted in a greater reduction of H2O2 toxicity than was observed with either agent alone. These data suggest that NGF protection is multifactorial and that a significant component of the NGF effect is due to its regulatory role in the metabolism of pyridine nucleotides.

Mental retardation and hypotonia seen in the knock out mouse for Canavan disease is not due to succinate semialdehyde dehydrogenase deficiency.

Canavan disease (CD) is an autosomal recessive disorder caused by aspartoacylase deficiency leading to accumulation of N-acetylaspartic acid and spongy degeneration of the brain. The mouse model for CD showed low levels of glutamate and gamma-aminobutyric acid (GABA) in the brain. Whether the low levels of glutamate and GABA observed in the CD mouse brain lead to abnormal production of glutamate-GABA associated enzymes and resulting succinate production is not obvious. While glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase complex activities are lower in the cerebellum and brain stem of the CD mouse, alanine aminotransferase and succinate semialdehyde dehydrogenase (SSADH) activities and succinate level are similar to the levels observed in the wild type. Deficiency of SSADH has been suggested to be associated with mental retardation and hypotonia, similar to the clinical features of CD. The normal SSADH activity in the CD mouse brain suggests that mental retardation and hypotonia seen in the CD mouse is not due to SSADH activity and if documented also in patients with CD.

Recognition of D-homoannulation by proton and carbon NMR spectra.

One-and two dimensional proton and carbon NMR spectra of the D-homoannulated rearrangement product of triamcinolone (9 alpha-fluoro-11 beta,16 alpha,17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) establish its structure as that of 9 alpha-fluoro-11 beta,16 alpha,17 alpha-trihydroxy-17 beta-hydroxy-methyl-D-homoandrosta-1,4-diene-3,17 alpha-dione. These methods accord ready recognition of D-homoannulation of C21-17-hydroxy-20-ketosteroids.

On the ozonization of cholesterol 3-acyl esters in protic media.

The structures of cholesterol 3 beta-acyl ester ozonides formed by reaction with ozone in participating alcoholic solvents are established by proton and carbon-13 spectra as a 3 beta-acyloxy-7 alpha-alkoxy-(5R,7R)-5 alpha-B-homo-6-oxacholestane-5-hydroperoxides (7a, 7b), and that of the dimeric cholesterol ozonide formed in nonparticipating solvents with cholesterol acting as alcohol is established as 7 alpha-cholest-5'-en-3'-yloxy-3 beta-hydroxy-(5R,7R)-5 alpha-B-homo-6-oxacholestane-5-hydroperoxide (7c).

17O nuclear magnetic resonance spectra of steroids.

The 17O NMR spectra of cholesterol and 31 other steroid alcohols, esters, ketones, and acids enriched by synthesis with 17O from H2(17)O have been observed under ordinary operating conditions, and correlations between 17O chemical shift and structure have been adduced. Spectra-structure correlations for these steroids are in conformity with those previously adduced with simpler compounds by others.

13C NMR studies of glucose metabolism in human leukemic CEM-C7 and CEM-C1 cells.

Glucose metabolism of human leukemic cell lines CEM-C7 and CEM-C1 was investigated in vivo by 13C NMR using 13C-labeled glucose. Exact knowledge of glucose concentration, cell count, and cell viability of the cell suspensions made it possible to analyze glucose metabolism in detail. In both cell lines aerobic glycolysis accounts for virtually all glucose consumption. The use of D-[13C2]glucose provided a simple method to measure the glucose flux through the pentose phosphate pathway as 9% (CEM-C1) and 11% (CEM-C7) of glucose channeled into glycolysis. The dexamethasone-sensitive CEM-C7 cells consume glucose at a rate about 50% higher than the dexamethasone-resistant CEM-C1 cells. It is shown that this higher consumption correlates with a larger size of the CEM-C7 cells. Therefore in CEM cells the development of drug resistance does not seem to involve related changes in cell energetics.

Catechol-O-methyltransferase-catalyzed rapid O-methylation of mutagenic flavonoids. Metabolic inactivation as a possible reason for their lack of carcinogenicity in vivo.

Quercetin is highly mutagenic in vitro, yet is not carcinogenic when administered chronically at large doses to rodents for 12 months. We hypothesized that catechol-O-methyltransferase-catalyzed O-methylation of quercetin and other mutagenic catechol-containing flavonoids may provide an efficient inactivation in vivo and may therefore prevent tumor induction by these flavonoids. After one intraperitoneal administration of 50 mg/kg quercetin to hamsters, a urinary ether extract contained 2% quercetin and 97% 3'-O-methylquercetin. When the urine was treated first with beta-glucuronidase and sulfatase, 13% quercetin and 87% 3'-O-methylquercetin were recovered. Quercetin was rapidly O-methylated by either porcine liver or hamster kidney catechol-O-methyltransferase, with Km values of 6.1 and 6.9 microM and Vmax values of 14,870 and 200 pmol/mg of protein/min, respectively. S-Adenosyl-L-homocysteine exhibited a potent feedback inhibition of the catechol-O-methyltransferase-catalyzed O-methylation of quercetin by a competitive mechanism with respect to S-adenosyl-L-methionine and by a competitive plus noncompetitive mechanism with respect to the substrate. A comparison of the O-methylation rates and kinetic characteristics (Km, Vmax, and Vmax/Km) demonstrated that rates of O-methylation of quercetin and fisetin were up to three orders of magnitude higher than those of catechol estrogens and catecholamines. In conclusion, the rapid metabolic inactivation of mutagenic flavonoids catalyzed by catechol-O-methyltransferase may be a major reason for the lack of their carcinogenic activities in vivo.

MR microscopy of cobalt-labeled nerve cells and pathways in an invertebrate brain (Sepia officinalis, Cephalopoda).

This article describes a novel application of contrast-enhanced MR microscopy to trace nerve cells and pathways through small invertebrate brains. Using the cuttlefish Sepia officinalis (Cephalopoda) as a model, the cells and pathways of one of the brain nerves were labeled with paramagnetic cobalt(II) ions by conventional centripetal cobalt iontophoresis. In MR microscopy, the cobalt-labeled cell bodies and pathways became hypointense in 9.4 T spin echo images. Their course and distribution were identical with those seen with conventional histological techniques after cobalt sulphide precipitation (with or without subsequent silver intensification). Magn Reson Med 45:575-579, 2001.

Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system.

BACKGROUND: Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N-acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock-out mouse for Canavan disease that can be used for gene transfer. METHODS: Genomic library lambda knock-out shuttle (lambdaKOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid with 10 base pair (bp) deletion of exon four of the murine aspa. Following linearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and confirmed by Southern analysis. Chimeras were generated by injection of ES cells to blastocysts. Germ line transmission was achieved by the birth of heterozygous mice as confirmed by Southern analysis. RESULTS: Heterozygous mice born following these experiments have no overt phenotype. The homozygous mice display neurological impairment, macrocephaly, generalized white matter disease, deficient ASPA activity and high levels of NAA in urine. Magnetic resonance imaging (MRI) and spectroscopy (MRS) of the brain of the homozygous mice show white matter changes characteristic of Canavan disease and elevated NAA levels. CONCLUSION: The newly created ASPA deficient mouse establishes an important animal model of Canavan disease. This model should be useful for developing gene transfer vectors to treat Canavan disease. Vectors for the central nervous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data generated from this animal model will be useful for developing strategies for gene therapy in other neurodegenerative diseases.

Adeno-associated virus-mediated aspartoacylase gene transfer to the brain of knockout mouse for canavan disease.

Canavan disease (CD) is an autosomal recessive leukodystrophy caused by deficiency of aspartoacylase (ASPA). Deficiency of ASPA leads to elevation of N-acetyl-L-aspartic acid (NAA) in the brain and urine. To explore the feasibility of gene transfer to replace ASPA in CD, we generated a knockout mouse and constructed an AAV vector that encodes human ASPA cDNA (hASPA) followed by green fluorescent protein (GFP) after an intraribosomal entry site. We injected CD mice with rAAV-hASPA-GFP in the striatum and thalamus or injected rAAV-GFP identically into control animals. Three to five months after the injection, we determined the presence of ASPA in the CD mouse brain by ASPA activity assay, GFP expression, and Western blot analysis. While rAAV-GFP-injected animals displayed undetectable levels of ASPA, all detection methods revealed significant ASPA levels in rAAV-hASPA-GFP-injected CD mice. We evaluated the functional effects of rAAV-hASPA-GFP-mediated ASPA expression by standard histological methods, magnetic resonance spectroscopy (MRS) for in vivo NAA levels, and magnetic resonance imaging of CD mice. rAAV-hASPA-injected animals displayed a remarkable lack of spongiform degeneration in the thalamus. However, pathology in sites unrelated to the injected areas showed no improvement in histopathology. The improvement in thalamic neuropathology was also detectable via in vivo MRI. MRS revealed that in vivo NAA levels were also reduced. These data indicate that rAAV-mediated ASPA delivery may be an interesting avenue for the treatment of CD.