Avena magna: An Important New Tetraploid Species of Oats.

Avena magna is a new tetraploid species morphologically similar to the hexaploid A. sterilis, having a high concentration of protein, large caryopses, and outstanding resistance to crown rust. One genome in A. magna appears homologous to the As genome present in hexaploid, tetraploid, and one group of diploid species. Avena magna is a possible ancestor of cultivated oats.

A novel mutation in the flavin-containing monooxygenase 3 gene, FM03, that causes fish-odour syndrome: activity of the mutant enzyme assessed by proton NMR spectroscopy.

We have previously shown that primary trimethylaminuria, or fish-odour syndrome, is caused by an inherited defect in the flavin-containing monooxygenase 3 (FMO3) catalysed N-oxidation of the dietary-derived malodorous amine, trimethylamine (TMA). We now report a novel causative mutation for the disorder identified in a young girl diagnosed by proton nuclear magnetic resonance (NMR) spectroscopy of her urine. Sequence analysis of genomic DNA amplified from the patient revealed that she was homozygous for a T to C missense mutation in exon 3 of the FMO3 gene. The mutation changes an ATG triplet, encoding methionine, at codon 82 to an ACG triplet, encoding threonine. A polymerase chain reaction/restriction enzyme-based assay was devised to genotype individuals for the FMO3Thr82 allele. Wild-type and mutant FMO3, heterologously expressed in a baculovirus-insect cell system, were assayed by ultraviolet spectrophotometry and NMR spectroscopy for their ability to catalyse the N-oxidation of TMA. The latter technique has the advantage of enabling the simultaneous, direct and semi-continuous measurement of both of the products, TMA N-oxide and NADP, and of one of the reactants, NADPH. Results obtained from both techniques demonstrate that the Met82Thr mutation abolishes the catalytic activity of the enzyme and thus represents the genetic basis of the disorder in this individual. The combination of NMR spectroscopy with gene sequence and expression technology provides a powerful means of determining genotype-phenotype relationships in trimethylaminuria.

Localization of an ecto-ATPase/cell-CAM 105 (C-CAM) in the rat parotid and submandibular glands.

We investigated an ecto-ATPase/cell-CAM 105 (C-CAM), previously shown to be distinct from the Ca2+ pump, in rat parotid and submandibular glands. Polyclonal antibodies raised against the enzyme were employed using indirect immunofluorescence, peroxidase-anti-peroxidase (PAP), and electron microscopic immunogold labeling procedures to visualize the location of the enzyme. In the PAP-stained sections and with immunofluorescence, labeling was observed on the luminal and lateral surfaces and the intercellular canaliculi of the acinar cells of both glands. The luminal surface of the intercalated ducts was brightly stained, whereas those of the striated and excretory ducts were less prominently labeled. The basal surface of the acinar cells in the parotid gland and the lateral and basal surfaces of the duct cells were not labeled. Apparent labeling was observed on the basal surface of the submandibular acinar cells. Electron microscopy revealed that for both glands the enzyme was primarily localized along the luminal border of the acinar cells, mainly in association with microvilli, with slightly less reactivity along the intercellular canaliculi and lateral borders and relatively little along the basolateral membranes. Gold labeling was also observed on the luminal borders of the intercalated and striated ducts. Possible functions of the C-CAM include breakdown of ATP, stabilization of the microvillar membranes, cell adhesion, and involvement in secretory mechanisms.

Metabolism of extracellular ATP by rat parotid cells.

ATP hydrolysis and the products of ATP metabolism were measured in intact rat parotid acini. The purpose was to determine the contribution of extracellular enzymes in metabolizing ATP and its metabolites. The total enzyme activity accounting for extracellular ATP breakdown was at least 75% dependent on added divalent cations, consistent with the presence of ectoATPase. Approximately 50% of the added ATP was hydrolysed in 1 h by the cells and this percentage was independent of cell protein concentration from 80 to 296 micrograms/ml and independent of ATP concentration from 4 to 80 microM. ADP. AMP and adenosine were identified as metabolites. Cell adenosine uptake was not a factor in controlling the levels of extracellular adenosine. Generation of adenosine was limited under conditions of higher rates of ATP hydrolysis. Studies in parotid cell membranes showed that very little feedback inhibition of ectoATPase was observed. 5' Nucleotidase was present at levels of activity of 0.06-0.19 mumol/mg protein/h in intact acini. The results confirm the presence of ectonucleotidases which can generate ADP, AMP and adenosine. Ectonucleotidase could contribute to reducing the effect of extracellular ATP on the parotid cell.

Fetal programming of hepatic lobular architecture in the rat demonstrated ex vivo with magnetic resonance imaging.

We demonstrate that MRI imaging at sub-millimetre resolution can distinguish between periportal and perivenous zones of the rat liver lobule. This made it possible to measure the hepatic lobular radius in ex-vivo perfused fixed livers using MRI. Comparisons of histomorphometric and MRI measurements of lobular radius were in good agreement, although MRI measurements were significantly smaller (P< 0.001). Male rats whose mothers were fed 40% of the protein of controls during gestation and lactation, had a significantly larger hepatic lobular radius than that of controls [449+/-11 microm vs. 373+/-9 microm (mean +/- SEM), respectively, p<0.001, n = 12; histomorphometry data]. The proton T(2) in periportal and perivenous zones was mapped both before and after antegrade or retrograde perfusion of 10 ml of digitonin (4 mg ml(-1)). Only the T(2) of the hypointense regions increased significantly following antegrade perfusion of digitonin and conversely only that of the intense regions following retrograde perfusion. Digitonin causes permeabilization of cells in specific hepatic zones, determined by the direction of perfusion. The intense and hypointense regions of the hepatic MR images thus arise from the perivenous and periportal zones of the hepatic lobule, respectively.

Lactate supply as a determinant of the distribution of intracellular pH within the hepatic lobule.

When isolated livers from starved rats are perfused with lactate at constant perfusate pH and P(co(2)), there is a marked gradient of cell pH (pH(i)) along the length of the lobular radius, with periportal cells being substantially more alkaline than perivenous cells. In the present studies, the perivenous 21% of the lobular volume was destroyed by retrograde digitonin perfusion, and antegrade perfusion restored. pH(i) was determined by (31)P-NMR. The remaining periportal cells, the site of gluconeogenesis from lactate, had a substantially higher mean pH(i) (7.42) than did the intact liver (7.23). When lactate was removed from the perfusate, mean pH(i) decreased to 7.25. The corresponding concentration of cell bicarbonate fell with a half-time of approximately 5 min. When lactate was re-introduced mean pH(i) rose to 7.34. We conclude that a major contributor to periportal alkalinity under these conditions is proton consumption during gluconeogenesis from lactate ions.

Hepatic intralobular mapping of fructose metabolism in the rat liver.

Detailed mapping of glucose and lactate metabolism along the radius of the hepatic lobule was performed in situ in rat livers perfused with 1.5 mM lactate before and during the addition of 5 mM fructose. The majority of fructose uptake occurred in the periportal region; 45% of fructose taken up in the periportal half of the lobular volume being converted into glucose. Periportal lactate uptake was markedly decreased by addition of fructose. Basal perivenous lactate output, which was derived from glucose synthesized periportally, was increased in the presence of fructose. During fructose infusion there was a small decrease in cell pH periportally, but acidification of up to 0.5 pH units perivenously. The evidence suggests that in situ the apparent direct conversion of fructose into lactate represents, to a substantial extent, the result of periportal conversion of fructose into glucose and the subsequent uptake and glycolysis to lactate in the perivenous zone of some of that glucose. (31)P NMR spectroscopy showed that the cellular concentration of phosphomonoesters changes very little periportally during fructose infusion, but there was an approximate twofold increase perivenously, presumably due to the accumulation of fructose 1-phosphate. It may be inferred that fructokinase activity is expressed throughout the hepatic lobule.

Investigation of human low-density lipoprotein by (1)H nuclear magnetic resonance spectroscopy: mobility of phosphatidylcholine and sphingomyelin headgroups characterizes the surface layer.

The resolution of the trimethyl headgroup resonance of phosphatidylcholine (PC) and sphingomyelin (SM) in the intact human low-density lipoprotein (LDL) (1)H NMR spectrum at 600 MHz enabled the investigation of LDL surface structure and phospholipid-apoB interactions. We have previously shown that a higher proportion of PC headgroups (25-35% of total PC in LDL) compared to SM were tightly bound to apoB and therefore NMR-invisible [Murphy, H. C., et al. (1997) Biochem. Biophys. Res. Commun. 234 (3), 733-737]. In the present study, we have investigated the mobility of phospholipid (PL) headgroups, using (1)H NMR spin-spin (T(2)) relaxation measurements, in LDL isolated from nine volunteers. We show that both PC and SM exist in two additional and distinct environments indicated by the biexponential behavior of the relaxation decays in each case. The data showed that 36% of PC headgroups had a short T(2) component, mean T(2) of 31 ms, and 64% had a longer T(2) component of 54 ms. Approximately 15% of SM headgroups had a short T(2) component (mean T(2) of 27 ms) and 85% had a longer T(2) component of 78 ms. Therefore the majority of SM headgroups (85%) were more mobile than PC (P < 0.001) and since PC headgroups in organic media were more mobile than SM, we conclude that the characteristic high mobility of LDL SM is not an intrinsic property but arises from a high degree of order in molecular packing of the surface PL of human LDL. We suggest that because PC and SM interact differentially with cholesterol and possibly with neighboring phospholipids, this results in the formation of relatively long-lived microdomains of PL in vivo.

Evidence for distinct behaviour of phosphatidylcholine and sphingomyelin at the low density lipoprotein surface.

This study demonstrates that the use of high field 1H NMR spectroscopy permits individual detection of phosphatidylcholine and sphingomyelin molecules at the surface of native low density lipoprotein (LDL) particles. Distinct behaviour was observed for the choline head group -N(CH3)3 resonances of these different phospholipids revealing preferential immobilisation for phosphatidylcholine. This suggests the existence of reversible and irreversible phosphatidylcholine-apolipoprotein B interactions and is consistent with microdomain formation at the surface monolayer of LDL. The novel resonance assignment and results show that 1H NMR can provide efficient and practical means for future studies on the structure and dynamics at the LDL surface.