~(13)C-methacetin breath test parameter S for liver diseases diagnosis

The mechanism of 13C-methacetin breath test is set forth clearly with the analysis of pharmacokinetics mode, and the measuring method of 13C-methacetin breath test and its clinical applications in the diagnosis of liver diseases are reported in detail. On the basis of comprehensive analysis of the clinical test data, the advanced diagnostic parameter S is of important significance for the application and development of breath test.     

Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

 The chemical shifts of several ¹³C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D_4h symmetry. The additional contributions to the paramagnetic shifts of ¹³C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental ¹³C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from ¹³C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on ¹³C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from ¹³C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available. Received: 27 December 1995 / Accepted: 17 April 1996     

Cloning single-stranded DNA

DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning sites. A chronology of the original concepts and experiments is reviewed.     

Compartmentation and flux characteristics of nitrate in spruce

The radiotracer¹³N was used to undertake compartmental analyses for NO ₃ ^– in intact non-mycorrhizal roots ofPicea glauca (Moench) Voss. seedlings. Three compartments were defined, with half-lives of exchange of 2.5 s, 20 s, and 7 min. These were identified as representing surface adsorption, apparent free space, and cytoplasm, respectively. Influx, efflux, and net flux as well as cytoplasmic and apparent-free-space nitrate concentrations were estimated for three different concentration regimes of external nitrate. After exposure to external NO ₃ ^– for 3 d, influx was calculated to be 0.09 mol·g^–1·h^–1 (at 10 M [NO ₃ ^– ]_o), 0.5mol·g^–1·h^–1 (at 100 M [NO _inf3 ^sup– ]_o), and 1.2 mol · g^–1· h^–1 (at 1.5 mM [NO ₃ ^– ]_o). Efflux increased with increasing [NO ₃ ^– ]_o, constituting 4% of influx at 10 M, 6% at 100 M, and 21% at 1.5 mM. Cytoplasmic [NO ₃ ^– ] was estimated to be 0.3 mM at 10 uM [NO ₃ ^– ]_o, 2mM at 100 M [NO ₃ ^– ]_o, and 4mM at 1.5 mM [NO ₃ ^– ]_o, while free-space [NO ₃ ^– ] was 16 M, 173 M, and 2.2 mM, respectively. A series of experiments was carried out to confirm the identity of the compartments resolved by efflux analysis. Pretreatment at high temperature or application of 2-chloro-ethanol, sodium dodecyl sulphate or hydrogen peroxide made it possible to distinguish the metabolic (cytoplasmic) phase from the remaining two (physical) phases. Likewise, varying [Pi] of the medium altered efflux and thereby [NO ₃ ^– ]_cyt, but did not affect [NO ₃ ^– ]_{free space}.Abbreviations and Symbols [NO ₃ ^– ]_cyt cytoplasmic NO ₃ ^– concentration - [NO ₃ ^– ]_{free space} apparent-free-space NO ₃ ^– concentration - [NO ₃ ^– ]_o concentration of NO ₃ ^– in the external solution - NO ₃ ^– flux - _co efflux from the cytoplasm - _oc influx to the cytoplasm - _net net flux - _xylem flux to the xylem - _red/vac combined flux to reduction and the vacuole The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia Campus for providing¹³NO ₃ ^– , Drs. R.D. Guy and S. Silim for providing plant material, and Dr. M.Y. Wang, Mr. J. Mehroke and Mr. P. Poon for assistance in experiments and for helpful discussions.     

URF13, a ligand-gated,pore-forming receptor for T-toxin in the inner membrane ofcms-T mitochondria

URF13 is the product of a mitochondrial-encoded gene (T-urfl3) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility ofcms-T maize to the fungal pathogensBipolaris maydis race T andPhyllosticta maydis. Mitochondria isolated fromcms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed inEscherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning -helices, two of which are amphipathic and can contribute to pore formation. Chemical crosslinking of URF13 was used to demonstrate the existence of URF13 oligomers incms-T mitochondria andE. coli cells. The ability of the carboxylate-specific reagent,N,N-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four--helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor incms-T mitochondria.     

Carbon isotope composition of N2-fixing and N-fertilized legumes along an elevational gradient

Dinitrogen-fixing legumes are frequently assumed to be less water-use efficient than plants utilizing soil mineral N, because of the high respiratory requirements for driving N₂ fixation. However, since respiration is assumed not to discriminate against ¹³C, any differences in water-use efficiency exclusively due to respiration should not be apparent in carbon isotope discrimination () values. Our objective was to determine if the source of N (N₂ fixation versus soil N) had any effect on of field-grown grain legumes grown at different elevations. Four legume species, Glycine max, Phaseolus lunatus, P. vulgaris, and Vigna unguiculata, were grown on five field sites spanning a 633 m elevational gradient on the island of Maui, Hawaii. The legumes were either inoculated with a mixture of three effective strains of rhizobia or fertilized weekly with urea at 100 kg N ha^-1 in an attempt to completely suppress symbiotic N₂-fixing activity. In 14 of 20 analyses of stover and 12 of 15 analyses of seed values were significantly higher (p=0.10) in the inoculated plants than the N-fertilized plants. Nitrogen concentrations were generally higher in the fertilized treatments than the inoculated treatments. The different values obtained depending on N-source may have implications in using as an indicator of water-use efficiency or yield potential of legumes.     

Changes in the origin and quality of soil organic matter after pasture introduction in Rondônia (Brazil)

We examined the effects of the conversion of tropical forest to pasture on soil organic matter (SOM) origin and quality along a chronosequence of sites, including a primary forest and six pastures. Bulk soil samples received a physical size-fractionation treatment to assess the contribution of each compartment to total SOM pool. Besides a general increase in total C and N stocks along the chronosequence, we observed a reduction of the relative contribution of the coarser fractions to total soil C content, and an increased concentration in the finer fractions. The origin of the C in each size fraction was established from measurements of¹³C abundance. After 80 years about 93% of the C in the least humified fraction of the top 10 cm of soil was of pasture origin, while in the most humified it was 82%. Chemical analyses indicated that the fine silt and coarse clay fractions contained the most refractory carbon.     

Effects of pasture introduction on soil CO2 emissions during the dry season in the state of Rondônia,Brazil

Soil CO₂ evolution rates, soil temperatures and moisture were measured during the dry season in two forest-to-pasture chronosequences in Rondônia, Brazil. The study included pastures ranging from 3 to 80 years-old. Mean dry-season CO₂ evolution from the forest in chronosequence 1, 88.8 mg CO₂-C m^–2h^–1 was lower than from the pastures which ranged from 111 to 158 mg CO₂-C m^–2h^–1. We found that temperature was not a good predictor of CO₂ emissions from pasture but that there was a significant relationship (r = 0.72,p < 0.05) between soil moisture and pasture emissions. The ¹³C of the soil CO₂ emissions also was measured on chronosequence I; ¹³C of the CO₂ emitted from the C3 forest was –29.43%. Pasture¹³CO₂ values increased from –17.91%. in the 3 year-old pasture to –12.86% in the 80 year-old, reflecting the increasing C₄ inputs with pasture age. Even in the youngest (3 year-old) pasture, 70 percent of the CO₂ evolved originated from C₄ pasture-derived carbon.     

The porcine intestinal receptor for Escherichia coli K88ab,K88ac: regional localization on chromosome 13 and influence of IgG response to the K88 antigen

The loci encoding the porcine intestinal receptors for Escherichia coli K88ab and K88ac (K88abR and K88acR) were firmly assigned to chromosome 13 by linkage analysis using a three-generation pedigree. The linear order of these loci and seven other markers on chromosome 13 was determined by multipoint analyses. The K88abR and K88acR loci were tightly linked with the K88abR locus localized 7·4 cM (sex average) proximal to the transferrin locus. The results, together with previous reports from two other groups, provide an unequivocal assignment of the K88 receptor loci to chromosome 13, and reject a previous assignment to chromosome 4. Pigs possessing the receptor had a slightly higher specific IgG response to the K88 antigen after an intramuscular immunization with an E. coli vaccine.