Evaluating a new method to estimate the rate of leaf respiration in the light by analysis of combined gas exchange and chlorophyll fluorescence measurements

Day respiration (R(d)) is an important parameter in leaf ecophysiology. It is difficult to measure directly and is indirectly estimated from gas exchange (GE) measurements of the net photosynthetic rate (A), commonly using the Laisk method or the Kok method. Recently a new method was proposed to estimate R(d) indirectly from combined GE and chlorophyll fluorescence (CF) measurements across a range of low irradiances. Here this method is tested for estimating R(d) in five C(3) and one C(4) crop species. Values estimated by this new method agreed with those by the Laisk method for the C(3) species. The Laisk method, however, is only valid for C(3) species and requires measurements at very low CO(2) levels. In contrast, the new method can be applied to both C(3) and C(4) plants and at any CO(2) level. The R(d) estimates by the new method were consistently somewhat higher than those by the Kok method, because using CF data corrects for errors due to any non-linearity between A and irradiance of the used data range. Like the Kok and Laisk methods, the new method is based on the assumption that R(d) varies little with light intensity, which is still subject to debate. Theoretically, the new method, like the Kok method, works best for non-photorespiratory conditions. As CF information is required, data for the new method are usually collected using a small leaf chamber, whereas the Kok and Laisk methods use only GE data, allowing the use of a larger chamber to reduce the noise-to-signal ratio of GE measurements.     

Application of commercial enzymes to measure the activity of the arginine pathway-urea cycle in intact cells

The activity of the complete arginine pathway-urea cycle was assessed in intact plant cells by employing the commercial enzymes arginase (EC 3.5.3.1) and urease (EC 3.5.1.5) to determine the amount of NaH14CO3 incorporated into [guanido-14C]arginine and/or into [14C]urea during a 3-h labeling period. Recovery of [guanido-14C]arginine was linear from 5 to 1000 nmol/g tissue and averaged 80 +/- 5% (mean +/- SE, N = 3). The procedure is reliable, inexpensive, well suited to the simultaneous analysis of numerous samples, and significantly more sensitive than existing methods. The method is ideally suited for assessing the activity of the complete arginine biosynthetic pathway in intact cells. In addition, the method has the distinct advantage of providing simultaneous measurement of the amount of NaH14CO3 accumulating in arginine relative to the amount accumulating as urea. Evidence is presented demonstrating that both the activity of the arginine pathway and the relative amounts of [guanido-14C]arginine and [14C]urea synthesized from NaH14CO3 were influenced by changes in the level of ornithine, NH+4, or phosphorus available to plant tissues.     

Purification of the human complement control protein C3b inactivator.

An alternative method of isolation from human plasma is described for C3b inactivator, C3bINA, the proteinase that in conjunction with either beta 1H or C4b-binding protein will hydrolyse respectively C3b or C4b, the activation products of the third, C3 and fourth, C4, components of complement. The purification is by chromatography of plasma on columns of QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite and Sephacryl S-200. The yield of C3bINA (6 mg from 500ml of plasma) is severalfold higher than in previously described methods. The sensitivity of the assay for C3bINA has been increased by including optimal amounts of beta 1H, and it was observed that beta 1H was essential for hydrolysis by C3bINA of C3b, whether the C3b was in solution or bound to a cell surface. Native C3 is not hydrolysed by C3bINA + beta 1H, but the haemolytically inactive form that appears on prolonged storage at 4 degrees C or on freezing and thawing is hydrolysed and gives fragments of the alpha-chain of 75000 and 43000 apparent mol.wt. As the alpha'-chain of C3b, which has lost an N-terminal peptide C3a, gives fragments of 67000 and 43000 apparent mol.wt. when incubated with C3bINA + beta 1H, this suggests that the larger fragment is N-terminal and the smaller one C-terminal. The pH optimum of C3bINA with soluble substrates is 6.0, but no clear classification of the type of proteinase to which this enzyme belongs has been obtained.     

Method for the typing of the C3 polymorphism in stored sera by means of isoelectric focusing

A method is described by which the three common phenotypes of C3 can be typed by desialylation and isoelectric focusing in serum samples stored at -20 degrees C for several years.     

Application of simultaneous determination of 3H, 14C, and 22Na by liquid scintillation counting to the measurement of cellular ion-transport.

A liquid scintillation counting method for simultaneous determination of three radioactive nuclides (3H, 14C, and 22Na) of biological interest was studied. By comparing the beta spectra of the three nuclides, their counting energy ranges, A, B, and C, were determined. 22NA was set high enough to avoid any spillover counts from lower-energy nuclides. Region A for 3H was set to maximize the counting efficiency. A good correlation between the counting efficiency for 22Na in region C and the counting efficiency of other nuclides in all regions was obtained. Prior to 3H and 14C dpm calculations, the 22Na counts spilled down in regions A and B were subtracted from the total counts in regions A and B. A simple linear equation was then used to compute 3H and 14C dpm. Findings show that the method presented is adaptable for highly quenched samples up to quenching indices of tSIE = 100. The method is useful for studying the biological transport coupled to Na+.     

Influence of the chain length of chitosan on complement activation

Non-water soluble chito-oligosaccharides (COSs) (molecular weights: 8, 800; 14, 200; 18, 200; and 33, 000) were investigated for complement activation by the single radial immuno-diffusion method. C3 activation was increased in a chain length-dependent manner. On regression analysis, there was significant correlation (P<0.01) between the number of NH₂ group of COSs and the extent of C3 activation after 20 min of incubation. The binding of C3b to chitosan (82% deacetylated chitin) was also investigated by an immunofluorescence method, and binding of C3b to chitosan particles was clearly observed. From these results, number of amino groups, and trapping of C3b are important evidence of complement activation via alternative pathway by chitosan and non-water soluble COSs.     

Rat-liver cholesterol 7 alpha-hydroxylase. 1. Development of a new assay based on the enzymic exchange of the tritium located on the 7 alpha position of the substrate.

A new assay is described to measure the activity of cholesterol 7alpha-hydroxylase and compared to the conventional 14C method used by other investigators. This method is based on the mechanism of the enzymic hydroxylation, i.e. a direct and stereospecific substitution of the 7alpha-hydrogen by a hydroxyl group. [7alpha-3H]Cholesterol is incubated at 37 degrees C and in the presence of molecular O2, in a medium buffered by postassium phosphate at pH 7.4 and containing liver microsomes (or 9000 X g supernatant), NADPH, MgCl2 and cysteamine. Tween-80 (1.5 mg/ml) is used to introduce enough substrate (300 muM) in the incubation mixture to saturate the enzyme (Km = 100 muM). Under these conditions the tritiated water released into the incubation medium reflects accurately the enzymic activity. The results obtained with this method are similar to the one obtained with a [4-14C]cholesterol technique (r = 0.96; P less than 0.001). The main advantage of the [7alpha-3H]cholesterol method is a complete independence from further metabolism of the first enzymic product, the 7alpha-hydroxycholesterol, the tritiated water representing the entire cholesterol 7alpha-hydroxylase activity.     

Pharmacokinetic monitoring during aminoglycoside treatment: the optimal method for individualizing the dosage of tobramycin

The results of tobramycin concentration monitoring in 33 patients with nonspecific pulmonary infections showed a marked individual variability of the antibiotic blood levels and model-independent pharmacokinetic parameters: total clearance, steady-state volume of distribution and mean residence time whose values were distributed log-normally. Adjusting of the tobramycin dosage by the individual values of the clearance (three-point method, by concentrations 1 h (C1), 3 h (C3) and 6 h (C6), after intramuscular single administration of the antibiotic and one-point method, by C3, after repeated administrations of the antibiotic) provided by the end of a 7-day course a 1.7-fold decrease in the individual ranges of the antibiotic concentration as compared to those without the dosage adjusting. Retrospective analysis revealed that reliable individual dosing of tobramycin was provided with the simplest one-point method when the only blood specimen was collected 3 hours after the injection, i.e. the time interval inversed to the elimination rate constant. According to this method individual doses Dind were calculated by the equation Dind = DpopCpop/Cind, where pop was the population value of D and C. The values of Dind estimated in such a way did not practically differ from those estimated with the more complicated two-point (by C1 and C6) and three-point methods. Application of the equation to the tobramycin "maximum" concentration C1 or the "minimum" one (toward the end of the dosing interval, C6) resulted in less accurate and unbiased estimation of Dind.     

Concerted evolution of the primate immunoglobulin alpha-gene through gene conversion.

We determined four nucleotide sequences of the hominoid immunoglobulin alpha (C alpha) genes (chimpanzee C alpha 2, gorilla C alpha 2, and gibbon C alpha 1 and C alpha 2 genes), which made possible the examination of gene conversions in all hominoid C alpha genes. The following three methods were used to detect gene conversions: 1) phenetic tree construction; 2) detection of a DNA segment with extremely low variability between duplicated C alpha genes; and 3) a site by site search of shared nucleotide changes between duplicated C alpha genes. Results obtained from method 1 indicated a concerted evolution of the duplicated C alpha genes in the human, chimpanzee, gorilla, and gibbon lineages, while results obtained from method 2 suggested gene conversions in the human, gorilla, and gibbon C alpha genes. With method 3 we identified clusters of shared nucleotide changes between duplicated C alpha genes in human, chimpanzee, gorilla, and gibbon lineages, and in their hypothetical ancestors. In the present study converted regions were identified over the entire C alpha gene region excluding a few sites in the coding region which have escaped from gene conversion. This indicates that gene conversion is a general phenomenon in evolution, that can be clearly observed in non-functional regions.     

Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver

1. Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-(3)H]- or [1,3-(3)H]-glycerol and [1-(14)C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from (14)C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-(3)H]glycerol and [1-(14)C]glycerol into lipid occurred with little or no change in the (3)H/(14)C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-(3)H]- and sn-[1-(14)C]-glycerol 3-phosphate with a resultant increase in the (3)H/(14)C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a (3)H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-(3)H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-(3)H]glycerol and [1-(14)C]glycerol for various times. 6. By using this method it was shown that 40-50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50-60% was incorporated via dihydroxyacetone phosphate.     

3H]tryptophan-[14C]proline dual label method for the simultaneous determination of collagen and noncollagen protein production

A new method for the simultaneous determination of newly synthesized collagen and noncollagen proteins has been developed. Because tryptophan is not found in collagen noncollagen proteins were specifically labeled with [3H]tryptophan. [14C]Proline was used to label both groups of proteins. To calculate the 14C-labeled noncollagen protein the 3H radioactivity of the protein mixture was divided by the ratio of 3H:14C in noncollagen protein of a representative sample. This value was obtained by collagenase digestion. The remaining 14C radioactivity in the protein mixture was attributed to [14C]collagen. There was a very good correlation between the dual label method and the widely used collagenase digestion method for the measurement of collagen and noncollagen protein production and for the calculation of the relative rate of collagen synthesis. This new method provides a simple and accurate analysis of collagen production, and it is suitable for rapid processing of a large number of biological samples.     

Improving the Performance of Outbreak Detection Algorithms by Classifying the Levels of Disease Incidence

We evaluated a novel strategy to improve the performance of outbreak detection algorithms, namely setting the alerting threshold separately in each region according to the disease incidence in that region. By using data on hand, foot and mouth disease in Shandong province, China, we evaluated the impact of disease incidence on the performance of outbreak detection algorithms (EARS-C1, C2 and C3). Compared to applying the same algorithm and threshold to the whole region, setting the optimal threshold in each region according to the level of disease incidence (i.e., high, middle, and low) enhanced sensitivity (C1: from 94.4% to 99.1%, C2: from 93.5% to 95.4%, C3: from 91.7% to 95.4%) and reduced the number of alert signals (the percentage of reduction is C1∶4.3%, C2∶11.9%, C3∶10.3%). Our findings illustrate a general method for improving the accuracy of detection algorithms that is potentially applicable broadly to other diseases and regions.     

The separation of functionally distinct forms of the third component of human complement (C3).

Complement component C3 prepared by the method of Tack & Prahl [(1976) Biochemistry 15, 4513-4521] was found to contain the following trace contaminants: C3b, haemolytically inactive C3 with intact alpha- and beta-chains (C3u) and degraded C3 (apparent mol.wt. 140000) with an intact beta-chain but with a fragmented alpha-chain. The proportion of C3u in the C3 is increased on standing and by freezing and thawing. These contaminants could be separated from each other and from native C3 by chromatography on sulphated Sepharose. They have been characterized by their susceptibility to C3b inactivator in the presence of beta 1H, their ability to be cleaved by C3 convertase and their ability to form alternative-pathway C3 convertase in solution. Incubation of C3b or C3u with beta 1H and C3b inactivator resulted in cleavage of the C3 species; the alpha'-chain of C3b was cleaved to fragments of apparent mol.wts. 67000 and 43000, the alpha-chain of C3u was cleaved to fragments of apparent mol.wt. 75000 and 43000. Native C3 and degraded C3 were unaffected by incubation with beta 1H and C3b inactivator. C3u, unlike C3, was not cleaved to C3b by the classical- or alternative-pathway C3 convertase in solution. When C3b or C3 was incubated with factors B and D, forming C3 convertase, the initial rate of factor-B cleavage was several order of magnitude lower in the presence of C3 than in the presence of C3b. The slow rate observed for C3 could be decreased by preincubation with beta 1H and C3b inactivator or by rechromatography of the C3. The degraded C3 did not support factor-B cleavage by factor D.     

A method for the synthesis of [14C]-kynurenine

A new method is described for the synthesis of [14C]-labelled L-kynurenine from [14C]-L-tryptophan, using extracts of tryptophan-adapted cells of Pseudomonas marginalis. It is based on the selective, rapid inactivation of kynureninase by a newly discovered inhibitor of this enzyme, 3-chloro-L-alanine. The yield of [14C]-kynurenine produced in this manner is 76% theoretical.     

A simplified procedure for the in vitro assay of the initial linear rate of the reaction of lecithin-cholesterol acyltransferase in human serum

A simple sensitive method for the determination of the initial rate of the reaction of lecithin-cholesterol acyltransferase by equilibrating [3H]cholesterol with unesterified cholesterol of human serum is described. The resulting serum is incubated for various time periods at 37 degrees C and the increase of the label in the cholesterol ester fraction is measured. The labeling is effected by a filter paper method in which a paper strip containing the labeled cholesterol is placed in serum at 4 degrees C, thereby preventing the formation of labeled cholesterol esters by the action of the enzyme. The rate of the reaction was linear up to 30 min.     

高等植物碳循环基因工程研究进展

高等植物根据其CO2同化方式的不同, 可分为C3植物、C4植物和CAM植物。由于C4植物特殊的光合作用方式, 其光合能力明显高于C3植物。然而, 大多数农作物都是C3植物。为了改善C3植物的光合能力, 人们试图通过转基因的方法来改造C3作物, 以提高主要农作物如水稻(Oryz a sativa)、小麦(Tri ticum aestivum)和大豆(Glycine max)等的光合生产力, 并在这些方面做了很多有益的尝试。该文主要综述了通过转基因方法改善碳循环能力的一些进展, 并对一些尚需深入研究的问题进行了探讨。     

高等植物碳循环基因工程研究进展

高等植物根据其CO2同化方式的不同,可分为C3植物、C4植物和CAM植物。由于C4植物特殊的光合作用方式,其光合能力明显高于C3植物。然而,大多数农作物都是C3植物。为了改善C3植物的光合能力,人们试图通过转基因的方法来改造C3作物,以提高主要农作物如水稻（Oryza sativa）、小麦（Triticum aestivum）和大豆（Glycine max）等的光合生产力,并在这些方面做了很多有益的尝试。该文主要综述了通过转基因方法改善碳循环能力的一些进展,并对一些尚需深入研究的问题进行了探讨。     

Evidence for substrate channeling in the early steps of cholesterogenesis

We have directly tested the ability of acetoacetate, upon activation to the CoA thioester, to channel into the cholesterogenic pathway prior to scrambling of its carbon skeleton with the acetate pool. The approach relies upon trapping [3-13C]acetoacetate-derived hydroxymethylglutaryl-CoA, hydrolyzing this metabolite, and esterifying the resulting hydroxymethylglutaric acid to allow gas chromatography/mass spectrometry analysis of the dimethyl esters for the 13C enrichment and labeling pattern. 99% enriched [3-13C] and [1,3,5-13C]hydroxymethylglutaric acid samples were synthesized, providing standards against which physiological samples could be compared. Cytosolic extracts from brain and liver of cholestyramine-fed rats were incubated with [3-13C]acetoacetate (2 mM) or with [1-13C]acetate (5 mM). In contrast to [13C]acetate-derived hydroxymethylglutarate, which shows the expected triple labeling pattern, [13C]acetoacetate-derived hydroxymethylglutarate from both liver and brain extracts is predominantly monolabeled. These data suggest that, after acetoacetate is activated to the CoA thioester, cytosolic hydroxymethylglutaryl-CoA synthase effectively commits much of this acetoacetyl-CoA to cholesterogenesis before thiolase can scramble the carbon skeleton of the acetoacetyl moiety into the acetate pool. This chemical approach represents an alternative method for testing the channeling of metabolites through sequential steps in a metabolic pathway. Such a method may be useful when physical or kinetic techniques prove to be unsuitable.     

The purification and properties of the second component of guinea-pig complement.

A method has been developed for the purification to homogeneity of guinea-pig complement component C2. Contrary to previous reports, guinea-pig C2 is a single polypeptide chain with apparent mol.wt. of 102000, the same as human C2. It is cleaved by C1s to yield fragments C2a (apparent molwt. 74000) and C2b (apparent mol.wt. 34000). The amino acid composition and N-terminal sequences of these fragments are similar to those of human C2a and C2b. Human and guinea-pig C2 show more extensive sequence homology to Factor B than previously identified. The known homology around the sites of cleavage by C1s and Factor D has now been extended by a stretch of ten identical or conservatively substituted residues. Sequence homology has now been identified at the N-terminal of C2b and Factor Ba. The properties of the classical-pathway C3 convertases assembled from human C4b, C1s and human or guinea-pig C2 have been compared. The rates of cleavage of human and guinea-pig C2 by C1s (and therefore the rates of assembly of the C3 convertases) are similar. The rate of decay of the activity of the C3 convertase formed from guinea-pig C2 is 10-fold lower than for human C2. This greater stability reflects a higher affinity of guinea-pig C2a for human C4b. The presence of C2b is not necessary for C3 convertase activity.     

A validated method for the quantification of pimarane and trachylobane diterpenes in the leaves of Croton zambesicus by capillary gas chromatography

A sensitive and accurate method, combining Soxhlet extraction, solid-phase extraction and capillary gas chromatography, is described for the quantitative determination of four new diterpenes (ent-trachyloban-3beta-ol, ent-18-hydroxy-trachyloban-3-one, ent-trachyloban-3-one and isopimara-7,15-dien-3beta-ol) from the leaves of Croton zambesicus. This is the first method describing the quantification of trachylobane diterpenes in a crude extract. It has been fully validated in order to be able to compare the diterpene composition in other samples of C. zambesicus, which is an important source of trachylobanes.