Endoscopic assessment of the duodenum in dogs with inflammatory bowel disease.

BACKGROUND: Endoscopy is performed for direct inspection of the mucosa and acquisition of biopsies in dogs with inflammatory bowel disease (IBD). AIM: To evaluate the interobserver agreement in the endoscopic assessment of duodenal mucosa in dogs with IBD. METHODS: Thirty-five archived endoscopic images of grossly normal (n = 6) and inflamed (n = 29) duodenal mucosa were displayed to 3 expert and 5 trainee endoscopists. Each image was assessed independently by endoscopists for mucosal abnormalities using established indices (of hyperemia, granularity, friability, lymphatic dilatation, and erosions) or interpreted as normal mucosa (trial 1). A repeated trial (trial 2) was performed with the same images presented in random order 1 month later, and accompanied by a visual template. RESULTS: There was slight interobserver agreement in initial mucosal assessment for expert and trainee endoscopists in trial 1 (kappa ≤ 0.02, P > .05). Interobserver agreement improved in trial 2 for both expert and trainee endoscopists (kappa = 0.2, P > .05) for experts and (P < .05) for trainees. There was a significant (P < .01) improvement in trainee endoscopy scores of lesions from trial 1 to trial 2. Regression analysis showed a significant (P < .01) difference between expert versus trainee endoscopy scores in trial 1. Repeat lesion assessment aided by use of a visual template (trial 2) improved the overall scores of trainee endoscopists to near that of expert endoscopists (P = .06). CONCLUSIONS AND CLINICAL IMPORTANCE: Interobserver agreement of IBD mucosal appearance from endoscopic findings benefitted from operator experience.

Carbon Partitioning and Growth of a Starchless Mutant of Nicotiana sylvestris.

We have further characterized the photosynthetic carbohydrate metabolism and growth of a starchless mutant (NS 458) of Nicotiana sylvestris that is deficient in plastid phosphoglucomutase (Hanson KR, McHale NA [1988] Plant Physiol 88: 838-844). In general, the mutant had only slightly lower rates of photosynthesis under ambient conditions than the wild type. However, accumulation of soluble sugars (primarily hexose sugars) in source leaves of the mutant compensated for only about half of the carbon stored as starch in the wild type. Therefore, the export rate was slightly higher in the mutant relative to the wild type. Starch in the wild type and soluble sugars in the mutant were used to support plant growth at night. Growth of the mutant was progressively restricted, relative to wild type, when plants were grown under shortened photoperiods. When grown under short days, leaf expansion of the mutant was greater during the day, but was restricted at night relative to wild-type leaves, which expanded primarily at night. We postulate that restricted growth of the mutant on short days is the result of several factors, including slightly lower net photosynthesis and inability to synthesize starch in both source and sink tissues for use at night. In short-term experiments, increased "sink demand" on a source leaf (by shading all other source leaves) had no immediate effect on starch accumulation during the photoperiod in the wild type or on soluble sugar accumulation in the mutant. These results would be consistent with a transport limitation in N. sylvestris such that not all of the additional carbon flux into sucrose in the mutant can be exported from the leaf. Consequently, the mutant accumulates hexose sugars during the photoperiod, apparently as the result of sucrose hydrolysis within the vacuole by acid invertase.

Evidence for Mitochondrial Regulation of Photosynthesis by a Starchless Mutant of Nicotiana sylvestris.

Mutant NS458 of Nicotiana sylvestris (Speg. et Comes) contains a defective plastid phosphoglucomutase and accumulates only trace amounts of starch. Determinations of carbon partitioning using tracer d-[3-(14)C]glyceric acid showed that the maximal CO(2) assimilation by mature leaves of the mutant at saturating [CO(2)] and light and low [O(2)] was close to the flux for sucrose formation in the wild type. The mutant is characterized by exceptionally slow oscillations in maximal CO(2) assimilation. The postulate that these slow oscillations follow changes in the cytosolic rate of sucrose phosphate synthesis has been investigated. Studies with wild-type and mutant leaf discs subjected to various treatments failed to indicate that any significant activation-inactivation cycle in sucrose-P synthase activity can occur. The rate of sucrose phosphate synthesis, however, might be altered by variations in the supply of uridine UDP-glucose which is controlled by the rate of ATP regeneration (via UTP regeneration). Treating mutant leaf protoplasts and young leaves with oligomycin, an inhibitor of mitochondrial ATP regeneration, reduced photosynthesis by as much as 25 and 40%, respectively. The wild type failed to show inhibition by oligomycin, i.e. its effect is masked when starch and sucrose synthesis can interact. It is concluded that maximal CO(2) assimilation in the mutant is fine tuned by mitochondrial metabolism such that interactions between sucrose synthesis and mitochondrial processes may generate the observed oscillations.

Steady-State and Oscillating Photosynthesis by a Starchless Mutant of Nicotiana sylvestris.

The photosynthetic characteristics of wild type Nicotiana sylvestris (Speg. et Comes) were compared with those of a ;starch-less' mutant NS458 that contains a defective plastid phosphoglucomutase (EC 2.1.5.1) (KR Hanson, NA McHale [1988] Plant Physiol 88: 838-844). The steady-state rate of net CO(2) assimilation (A) was studied as a function of [CO(2)], [O(2)], irradiance, and temperature. At 30 degrees C with saturating light and [CO(2)] and low [O(2)], A for the mutant was half that for the wild type, whereas in normal air it was 90%. The irradiance and [CO(2)] at low [O(2)] required for saturation were lower than the values for the wild type. At 2000 microbars CO(2), 30 degrees C, and saturating irradiance A for both the mutant and wild type was stimulated on going from 4 to 25% O(2) by at least 13%. Slow oscillations in A were readily induced with the mutant but not the wild type, provided irradiance and [CO(2)] were saturating and [O(2)] was low. The period, which was about 5 minutes at 30 degrees C and decreased by about 0.67 minutes per degree, was an order of magnitude slower than periods reported for other plants at corresponding temperatures. To achieve the full oscillation amplitude both irradiance and [CO(2)] had to exceed the minimal levels for steady-state saturation. The slowness and duration of the oscillations and the metabolic simplification introduced by deleting starch synthesis makes the mutant especially suitable for investigating the regulatory processes that generate such oscillations.

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase.

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [gamma-(32)P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P(2), but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [gamma-(32)P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [(32)P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [gamma-(32)P]ATP or [(32)P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.

A Starchless Mutant of Nicotiana sylvestris Containing a Modified Plastid Phosphoglucomutase.

A mutant (NS 458) of Nicotiana sylvestris (Spegazzini and Comes) unable to synthesize leaf starch was isolated in the M(2) generation following ethyl methanesulfonate mutagenesis by testing with iodine. Segregation ratios in reciprocal F(2) progenies showed that the starchless phenotype resulted from a recessive mutation in a single nuclear gene. DEAE-agarose chromatography showed that the mutant is grossly deficient in plastid phosphoglucomutase (EC 2.1.5.1) activity. The structure of the enzyme is changed, as evidenced by increased Michaelis constants and by the prolonged activation period (>40 minutes) observed when the enzyme is assayed in triethanolamine buffer rather than imidazole buffer. The activity of the wild-type enzyme with saturating glucose 6-P alone was 7% of the activity when saturating glucose 1,6-P(2) was also present. The results suggest that glucose 1,6-P(2) is both an effector and a dissociable reaction intermediate. The growth rate of mutant and wild-type plants were not significantly different in continuous light and on an 8-hour dark, 16-hour light cycle and the mutants grew normally under greenhouse conditions. The mutant supports growth during diurnal periods of darkness by vacuolar storage of sugars instead of chloroplast storage of starch. The simplification in metabolism achieved by blocking the diversion of plastid fructose-6-P to starch facilitates the induction of oscillations in CO(2) fixation.

A Nuclear Mutation in Nicotiana sylvestris Causing a Thiamine-Reversible Defect in Synthesis of Chloroplast Pigments.

We report the recovery of a nuclear recessive mutation in Nicotiana sylvestris (Spegazzini and Comes) producing a conditional disruption in the pathway for synthesis of chlorophyll a and b and carotenoids which is fully reversible by exogenous thiamine (0.3 micromolar). In the absence of supplemental thiamine, chlorophyll levels declined by 50% after 5 days, and fell to undetectable levels by 11 days. Mitochondrial (KCN sensitive) respiration rates remained normal in albino leaves (80% loss of chlorophyll), suggesting that chlorosis results primarily from a deficiency of thiamine in the chloroplasts. After thiamine removal, mutant plants produced at least 10 albino leaves with a substantial capacity for growth (0-15 centimeters; 70-fold increase in area), demonstrating sustained operation of many cellular functions in spite of chloroplast disruption. Activities of the plastid isozymes of phosphoglucomutase and phosphoglucoisomerase in albino leaves indicated that the decline in pigment synthesis does not result from a general loss of metabolic activity in chloroplast. Plastid pyruvate dehydrogenase from mutant and wild-type plants displayed a similar affinity for thiamine pyrophosphate, showing that chlorosis does not result from an alteration in this enzyme. Growth of albino leaves and ultrastructural evidence for thylakoid membranes in the chloroplasts suggest that a certain level of fatty acid synthesis is maintained after the interruption of pigment synthesis. Since thiamine deprivation is expected to block production of acetyl-coenzyme A from pyruvate by pyruvate dehydrogenase, acetyl-coenzyme A supporting fatty acid synthesis in albino leaves may be derived solely from mitochondrial acetate.

Photorespiration stoichiometry in leaves estimated by combined physical and stereochemical methods: allowance for isomerase-catalyzed 3H losses in ribulose bisphosphate regeneration.

We showed previously [K.R. Hanson and R.B. Peterson (1986) Arch. Biochem. Biophys. 246, 332-346] that under steady-state photosynthetic conditions the fraction of ribulose bisphosphate oxidized and the fraction of glycolate carbon photorespired (the stoichiometry of photorespiration) may be estimated in leaves by a combination of physical and stereochemical methods. The calculations assumed that when (3R)-D-[3-3H1,3-14C]glyceric acid is supplied to illuminated leaf discs the only loss of 3H from the combined photosynthetic and photorespiratory system is the result of glycolate oxidase action; i.e., the isomerase-catalyzed losses in the regeneration of ribulose bisphosphate are negligible. The present study of tobacco leaf discs under zero-photorespiration conditions (low O2 and high CO2 concentrations), and also of maize leaf discs, shows that some 3H losses occur (between 8 and 13% of the 3H at C-1 of ribulose 5-phosphate). The calculated loss varied moderately with temperature but did not vary when the flux of ribulose bisphosphate formation was altered by changing the irradiance. The calculated loss under zero-photorespiration conditions, therefore, may be used to calculate ribulose bisphosphate and glycolate partitioning under other conditions. Earlier experiments on the influence of O2 and CO2 concentrations of temperature on the partitioning of ribulose bisphosphate and glycolate have been reexamined. The loss corrections decreased all values for the fraction of ribulose bisphosphate oxidized and increased all values for the stoichiometry of photorespiration. Essentially all stoichiometry values were above the theoretical lower limit of 25%. The previous conclusion that the stoichiometry of photorespiration substantially exceeds 25% at higher O2 concentrations and higher temperatures is unchanged. The results with maize leaf discs implied that there is very little oxidation of ribulose 1,5-bisphosphate under normal-air conditions; i.e., photorespiration is indeed suppressed, not merely hidden, by efficient refixation of CO2.

Regulation of photorespiration in leaves: evidence that the fraction of ribulose bisphosphate oxygenated is conserved and stoichiometry fluctuates.

Under steady-state conditions the combined system of the reductive photosynthetic cycle and the oxidative photorespiratory loop may be defined by two partitioning terms: the fraction of ribulose bisphosphate oxygenated and the fraction of glycolate carbon photorespired (the stoichiometry of photorespiration). A combination of physical and stereochemical methods [K.R. Hanson, and R. B. Peterson, (1985) Arch. Biochem. Biophys. 237,300-310] has been used to estimate these partitionings for tobacco leaf discs. Inverted discs, as compared to normally oriented discs, were found to have greater net photosynthesis; their ratio of photorespiration to net photosynthesis was less, and less of their glycolate carbon was photorespired. An eightfold reduction of irradiance below that of full sunlight for inverted discs in normal air at 32 degrees C reduced both photosynthesis and photorespiration about threefold but had little effect on the partitioning of ribulose bisphosphate and glycolate. Increasing the temperature from 22 to 40 degrees C for inverted discs in normal air and 1000 microE m-2 s-1 irradiance had little effect on net photosynthesis but increased the ratio of photorespiration to net photosynthesis almost threefold; ribulose bisphosphate partitioning was little changed but the fraction of glycolate carbon photorespired more than doubled. If field-grown plants respond to temperature in a similar fashion, genetic intervention to reduce the increase in photorespiration stoichiometry with temperature could increase total daily carbon assimilation and hence improve crop yields.

The stoichiometry of photorespiration during C3-photosynthesis is not fixed: evidence from combined physical and stereochemical methods.

The stoichiometry of photorespiration, S, is defined as the fraction of glycolate carbon photorespired. It is postulated that under steady-state conditions there are two determinants of the ratio of photorespiration to net photosynthesis: the partitioning of ribulose bisphosphate between oxidation and carboxylation, and the partitioning of glycolate between reactions leading to complete oxidation to CO2 (S = 100%) and those yielding CO2 plus serine (S = 25%). S may be calculated using two independent probes of the system. The physical probe, using an infrared gas analyzer, measured photorespiration and net photosynthesis, and hence their ratio PR/NPS = pn(phys). The metabolic probe employed tracer (3R)-D-[3-3H1,3-14C]glyceric acid to determine r, the fraction of 3H retained in the triose phosphates leaving the chloroplasts. It is deduced from the postulated model that S = pn(phys) . r/(1 - r). Experiments have been performed with illuminated tobacco leaf discs (inverted) under varying concentrations of O2 and CO2. Increasing O2 at constant CO2 increased pn(phys) and decreased r, whereas increasing CO2 at constant O2 had the opposite effect. S more than doubled at 32 degrees C on going from 16 to 40% O2 (340 microliters CO2/liter) and decreased 40% on going from 200 to 700 microliters CO2/liter (21% O2). For discs in normal air S was somewhat greater than 27%. It is suggested that net photosynthesis, and therefore crop yields, could be increased by selecting for crop plants with reduced photorespiration stoichiometry.

Stereochemical determination of carbon partitioning between photosynthesis and photorespiration in C3 plants: use of (3R)-D-[3-3H1, 3-14C]glyceric acid.

When (3R)-D-[3-3H1,3-14C]glyceric acid is supplied in tracer amounts to illuminated tobacco leaf discs, the acid penetrates to the chloroplasts without loss of 3H, and is phosphorylated there. Subsequent metabolism associated with the reductive photosynthetic cycle fully conserves 3H. Oxidation of ribulose bisphosphate (RuBP) by RuBP carboxylase-oxygenase (EC 4.1.1.39) results in the formation of (2R)-[2-3H1, 14C]glycolic acid which, on oxidation by glycolate oxidase (EC 1.1.3.1), releases 3H to water. Loss of 3H from the combined photosynthetic and photorespiratory systems is, therefore, associated with the oxidative photorespiratory loop. Assuming steady-state conditions and a basic metabolic model, the fraction of RuBP oxidized and the photorespiratory carbon flux relative to gross or net CO2 fixation can be calculated from the fraction of supplied 3H retained in the triose phosphates exported from the chloroplasts. This retention can be determined from the 3H:14C ratio for glucose obtained from isolated sucrose. The dependence of 3H retention upon O2 and CO2 concentrations can be deduced by assuming simple competitive kinetics for RuBP carboxylase-oxygenase. The experimental results confirmed the stereochemical assumptions made. Under conditions of negligible photorespiration 3H retention was essentially complete. The change in 3H retention with O2 and CO2 concentrations were investigated. For leaf discs (upper surface up) in normal air, it was estimated that 39% of the RuBP was oxidized, 32% of the fixed CO2 was photorespired, and the photorespiration rate was 46% of the net photosynthetic CO2 fixation rate. These are minimal estimates, as it is assumed that the only source of photorespired CO2 is glycine decarboxylation.

A suggestion for naming faces of ring compounds.

Discussion of the topology of interaction of ring compounds with macromolecules and receptors requires a system for naming the faces of the cyclic compound. An alpha/beta-face nomenclature is suggested that is based on the clockwise/counterclockwise direction of ascending numbering in the ring with the lowest numbered unshared ring atom. This system is applicable to a very broad range of compounds: sugars, cyclic bases, steroids, cyclitols, porphyrins, etc., and molecules with a single ring, fused rings, encompassing rings, and rings formed by head-to-tail polymerization.

Phenylalanine ammonia-lyase: enzymic conversion of 3-(1,4-cyclohexadienyl)-L-alanine to trans-3-(1,4-cyclohexadienyl)acrylic acid.

The phenylalanine analogue 3-(1,4-cyclohexadienyl)-L-alanine is converted to the hitherto unknown cinnamate analogue trans-3-(1,4-cyclohexadienyl)acrylic acid by L-phenylalanine ammonia-lyase (EC 4.3.1.5) from maize, potato, or Rhodotorula glutinis. The structure assigned to the product is confirmed by its 1H nuclear magnetic resonance spectrum and by the chemical synthesis to be described in a subsequent paper. On comparing the above substrate analogue with L-phenylalanine, the Km was lowered only slightly but kcat was reduced 14--40-fold depending on the source of the enzyme. Because the compounds closely resemble each other in size and hydrophobic properties, this lowering of kcat may be attributed to the electronic effect of replacing the pi electrons of the aromatic system by those of a double bond. Correct alignment at the active site appears to depend upon the space-filling properties of the ring system; open chain analogues that retain the gamma, beta double bond were found to be inhibitors, not substrates.

Reactions at prochiral centers. Interdependence in the estimation of enzyme stereospecificity toward prochiral centers and the configurational purity of labeled substrates.

In many enzymic reactions one or the other hydrogen is released from a prochiral center abCH2. To determine the stereospecificity with respect to such centers under nonreversal conditions a substrate with a chiral center abC1HH is employed (H = 3H or 2H). Assuming the simplest possible model, the R specificity of an enzyme or enzyme system (eR) may be defined in terms of the configurational R purity of the substrate with respect to the labeled center (pR), and the fraction of H released (r) or conserved (l - r) during the reaction by the equation eR = [(l - r) -pR]/[1 - 2pR]. Where r is constant, this is a rectangular hyperbola. The apparent stereospecificity calculated with this equation serves as a characteristic of the experimental system irrespective of the assumed model. The problems arising from direct isotope effects are discussed and the theoretical advantages of using product 3H:14C ratios under low conversion conditions noted. Applications of the equation to the determination of configurational purity at labeled centers, to the study of stereospecificity in enzymes and enzyme systems, and to the study of the biosynthesis of natural products are discussed.