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Biomedical subjects

T W Fan

Publications and source records attributed to T W Fan.

14 recordsLinked to original sources
PubMed1992

Identification of glycerophosphorylcholine in mussel ovarian extracts by two-dimensional nuclear magnetic resonance.

The abundance of the "phosphodiester" peak in differentiating or proliferating tissues, including reproductive organs and tumors, warrants further investigations of its metabolic role(s), which would require a rigorous confirmation of its identity. The assignment of this peak to glycerophosphorylcholine in 31P NMR spectra of biological samples has been largely based on chemical shift, which can result in ambiguities. We employed a combination of two-dimensional 31P-1H heteronuclear shift correlation and 1H total correlation spectroscopies to trace the spin connectivities of glycerophosphorylcholine and thus to identify its structure directly from crude ovarian extracts of mussels without ambiguities and the need for extensive purification. This approach can be applied generally to the identification of molecules containing heteroatoms in crude tissue extracts.

Animals↗
PubMed1992

Temperature dependence of arginine kinase reaction in the tail muscle of live Sycionia ingentis as measured in vivo by 31P-NMR driven saturation transfer.

We have employed the driven 31P-NMR saturation transfer method to measure in vivo the temperature dependence of the forward and reverse unidirectional fluxes of the arginine kinase reaction in the tail muscle of a live shrimp, Sycionia ingentis. Our results indicated that neither the forward nor the reverse rate constants of this reaction were significantly temperature-dependent between 8 and 16 degrees C, in contrast to the kinetic characteristics of isolated arginine kinases.

Adenosine Triphosphate↗
PubMed1992

Hypoxia does not affect rate of ATP synthesis and energy metabolism in rice shoot tips as measured by 31P NMR in vivo.

The cytoplasmic pH, concentrations of phosphate metabolites, and rate of ATP synthesis were measured in vivo in excised rice shoot tips under normoxic and hypoxic conditions using 31P NMR. When supplied with glucose, the shoot tips grew rapidly and were relatively unaffected by hypoxia. The cytoplasmic pH decreased transiently by only 0.2 units during hypoxia, and the concentration of ATP was maintained to at least 90% of the normoxic level. Most importantly, the unidirectional rate constant of ATP synthesis from free phosphate decreased less than 25% during hypoxia. This is in contrast to other actively growing tissues such as the maize root tip. gamma-Aminobutyrate was the major nonvolatile fermentation end product after 22 h of hypoxia. Other hypoxia-induced changes included a modest increase in [Ala] and [succinate] as well as a substantial decrease in [malate].

Adenosine Triphosphate↗
PubMed1991

Emergence and recovery response of phosphate metabolites and intracellular pH in intact Mytilus edulis as examined in situ by in vivo 31P-NMR.

We employed surface probe-localized 31P-NMR spectroscopy to examine in situ the impact of short-term emergence (hypoxia) and resubmergence on phosphate metabolites and intracellular pH (pHi) in intact mussels. The use of intact organisms ensured that all intrinsic responses remained active while monitoring of individuals minimized uncertainties resulting from stochastic behavior and other individual differences. The use of a photoetched, balanced-match foil probe combined with 1H-NMR images allowed 31P-NMR spectra to be acquired from the posterior adductor muscle with good signal-to-noise. Upon emergence, all mussels exhibited an increase in [Pi], a decline in [phosphoarginine] and pHi, and very little changes in [ATP] with time. The complementary behavior of [phosphoarginine] and [Pi] indicated a precursor-product relationship involved in the maintenance of [ATP] but the similarity between [phosphoarginine] and pHi time-courses cannot be so readily explained. Irregularity in the time-courses of some parameters could have reflected stochastic gaping activity. Resubmergence responses exhibited a reversal of the emergence responses, except that the pHi eventually became supraalkaline with irregular fluctuations. This might be related to the 'oxygen debt' phenomenon and increased oxidative phosphorylation.

Adaptation, Physiological↗
PubMed1991

Sublethal effects of pentachlorophenol in the abalone (Haliotis rufescens) as measured by in vivo 31P NMR spectroscopy.

The sublethal biochemical effects of pentachlorophenol (PCP) were investigated in live, intact red abalones (Haliotis rufescens), using a flow-through exposure system, by in vivo 31P NMR spectroscopy. Based on rangefinding tests (6-hr LC50 = 1.6 mg/L; 6-hr no-observable-effect-level (NOEL) = 0.8 mg/L), three abalones were separately exposed to a sublethal concentration (1.2 mg/L) for 5 hr, followed by a 13 hr recovery period. Effects in foot muscle included both a decrease in phosphoarginine and an increase in inorganic monophosphate concentrations ([PA] and [Pi], respectively); both foot muscle concentrations of adenosine triphosphate [ATP] and intracellular pH (pHi) also declined. Parallel in vitro experiments revealed that concentrations of glycerol 3-phosphate, lactate, citrate, succinate, malate, and alanine (Ala) all increased, while those of glyceraldehyde 3-phosphate and glutamine (Gln) remained stable. Also, these effects were not evident until 2 hr into exposure, possibly the time required for PCP to attain an effective concentration in foot muscle. During recovery, while Pi declined to pre-exposure levels, [PA] completely recovered in only one individual. Also, realkalinization of pHi was similar to recovery of [Pi], and ATP returned to near-initial levels, as did glycerol 3-phosphate, lactate, succinate, malate, and Ala; glyceraldehyde 3-phosphate, citrate, and Gln levels declined. Recovery responses corresponded to the time for PCP clearance from foot muscle. The effects of PCP were similar to those of hypoxia, fatigue, hypersalinity, and arginine kinase inhibitors, and so sublethal PCP concentrations may also inhibit electron transport and arginine kinase as well as uncouple mitochondrial oxidative phosphorylation in intact molluscs. Thus, the effects of pollutants on key biochemical processes may now be measured in intact aquatic organisms as they occur, improving our ability to accurately assess the environmental effects of pollutants in the laboratory.

Animals↗
PubMed1989

Monitoring of metabolic responses of intact Haliotis (abalones) under salinity stress by 31P surface probe localized NMR.

Surface probe localized 31P NMR spectroscopy was employed to record the metabolic responses of the foot of intact Haliotis cracherodii and H. rufescens (black and red abalones) under hyper- and hypoosmotic stresses. Use of the surface probe allowed spectral localization on the foot of intact abalones, facilitated monitoring of different sizes of animals, and minimized constraints on aquatic chamber design normally imposed by homogeneous-field probes. Generally, hyperosmotic stress (51%) elicited more rapid changes of phosphate metabolites than hypoosmotic stress (17%). As with the well-studied hypoxic stress in intact mammalian and excised molluscan tissue, both salinity treatments caused drops in the phosphagen and increases in inorganic phosphate levels. However, osmotic stress was distinct from hypoxic stress in that intracellular pH did not change and nucleotide triphosphate (NTP) concentrations dropped immediately. Although these findings are preliminary, they demonstrate the utility of the surface probe approach for studies of environmental stress in intact marine invertebrates.

Animals↗
PubMed1989

In vivo 23Na and 31P NMR measurement of a tonoplast Na+/H+ exchange process and its characteristics in two barley cultivars.

A Na+ uptake-associated vacuolar alkalinization was observed in roots of two barley cultivars (Arivat and the more salt-tolerant California Mariout) by using 23Na and 31P in vivo NMR spectroscopy. A NaCl uptake-associated broadening was also noted for both vacuolar Pi and intracellular Na NMR peaks, consistent with Na+ uptake into the same compartment as the vacuolar Pi. A close coupling of Na+ with H+ transport (presumably the Na+/H+ antiport) in vivo was evidenced by qualitative and quantitative correlations between Na+ accumulation and vacuolar alkalinization for both cultivars. Prolongation of the low NaCl pretreatment (30 mM) increased the activity of the putative antiport in Arivat but reduced it in California Mariout. This putative antiport also showed a dependence on NaCl concentration for California Mariout but not for Arivat. No cytoplasmic acidification accompanied the antiporter activity for either cultivar. The response of adenosine phosphates indicated that ATP utilization exceeded the capacity for ATP synthesis in Arivat, but the two processes seemed balanced in California Mariout. These comparisons provide clues to the role of the tonoplast Na+/H+ antiport and compensatory cytoplasmic adjustments including pH, osmolytes, and energy phosphates in governing the different salt tolerance of the two cultivars.

Adenine Nucleotides↗
PubMed1988

An in vivo 1H and 31P NMR investigation of the effect of nitrate on hypoxic metabolism in maize roots.

The effect of nitrate on the short-term hypoxic response and recovery of flooded mature maize roots has been investigated in vivo by 1H and 31P NMR and in vitro by 1H NMR and gas chromatography-mass spectrometry. Employing 1H NMR in addition to 31P NMR extended the number of identifiable compounds in vivo from 4 to 15, while in vitro two-dimensional NMR and gas chromatography-mass spectrometry aided rigorous in vivo 1H NMR resonance assignments and quantitation of 24 compounds. In the absence of nitrate, the concentrations of key metabolites including alanine, ethanol, gamma-aminobutyrate, lactate, succinate, and sucrose changed during 8 h of hypoxia in a manner consistent with reduced tricarboxylic acid cycle activity and diversion to glycolytic fermentation. The pH drop in the cytoplasm during hypoxia was rapid, about 0.2 unit, and diminished quickly upon recovery. Rapid recovery of ethanol, succinate, and sucrose levels was also observed, which indicates a return to normal aerobic metabolism. Although the hypoxic response itself, including pH, was not greatly affected by the presence of nitrate, nitrate reduced the amount of fermentation end products produced, helped maintain a higher free NTP concentration during hypoxia, and increased the rate of overall recovery from hypoxia. These findings suggest the presence of a nitrate-induced maintenance-level respiration in hypoxic maize roots, which helps explain the protection imparted by nitrate to flooded hypoxic maize plants.

Energy Transfer↗
PubMed1986

Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments.

Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism.

Animals↗
PubMed1986

Monitoring of hypoxic metabolism in superfused plant tissues by in vivo 1H NMR.

We have used a coaxial superfusion system to obtain physiologically interpretable in vivo 1H NMR spectra at 500 MHz of carrot roots, maize roots, and rice shoots in water (no 2H2O). The superfusion system was constructed from common laboratory parts, required no modification of the probe and sample loading procedure, and was inherently leak resistant. The assignment and quantitation of the in vivo 1H NMR resonances were achieved by performing two-dimensional NMR experiments in vivo, and by in vitro analysis including NMR and gas chromatography-mass spectrometry. The in vivo spectra were dominated by resonances arising from sugars, organic acids, amino acids, and ethanol. In vivo measurements of spin-lattice relaxation times and chemical shifts of beta protons of malate in carrot roots suggested that malate was located in a relatively viscous and acidic compartment. In rice shoots, the hypoxic time courses of 9 metabolites were established in vivo, and 23 in vitro. In both cases, accumulation of lactate, ethanol, Ala, and gamma-aminobutyrate as well as a decrease in Gln and Asn concentrations were observed. These findings are consistent with accelerated glycolysis and decreased tricarboxylic acid cycle activity.

Amino Acids↗
PubMed1985

Isolation and characterization of two cyanogenic beta-glucosidases from flax seeds.

Two cyanogenic beta-glucosidases, linustatinase and linamarase, were isolated and purified from flax seeds (Linum ussitatissimum). They catalyze the sequential hydrolysis of linustatin and neolinustatin to yield acetone and methylethyl ketone cyanohydrins, respectively. The purification procedure for linustatinase involved acetone extraction, precipitation by polyethyleneimine and ammonium sulfate (40-80% saturation), and Red A gel, concanavalin A-Sepharose, and PBE 94 column chromatography; that for linamarase was similar except that polyethyleneimine precipitation was eliminated and DE-52 and Sepharose CL-6B replaced Red A gel column chromatography. The native substrates neolinustatin and linamarin were used for the assay during purification. Both proteins were purified to electrophoretic homogeneity. Linustatinase is an alpha beta dimer (molecular weights of alpha and beta = 39,000 and 19,000, respectively) while linamarase appears to be an alpha 5 beta 5 decamer (molecular weights of alpha and beta = 62,500 and 65,000, respectively). Both enzymes contain mannose or glucose. Linustatinase exists in five different isozymic forms (isoelectric points between 7 and 8) whereas linamarase occurs in one major form (isoelectric point 4 to 5). The kinetic parameters of the two enzymes are similar: acidic pH optima, Km's in the millimolar range, and competitive inhibition by delta-gluconolactone, a transition state analog. The presence of an aglycone structure in the substrates is important for both enzyme activities. In addition, both enzymes are specific towards the beta-glycosidic linkage; linustatinase (a beta-bis-glucosidase) readily hydrolyzes beta-bis-glucosides with 1,6 and 1,3 linkages whereas linamarase (a beta-monoglucosidase) exhibits little activity towards these substrates.

Amygdalin↗