Novel FMN-containing rotenone-insensitive NADH dehydrogenase from Trypanosoma brucei mitochondria: isolation and characterization

A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.     

IMpRH server: an RH mapping server available on the Web

SUMMARY: The INRA-Minnesota Porcine Radiation Hybrid (IMpRH) Server provides both a mapping tool (IMpRH mapping tool) and a database (IMpRH database) of officially submitted results. The mapping tool permits the mapping of a new marker relatively to markers previously mapped on the IMpRH panel. The IMpRH database is the official database for submission of new results and queries. The database not only permits the sharing of public data but also semi-private and private data.     

Evaluating Evolutionary Divergence with Microsatellites

We report the use of microsatellites (MS) to track the recent evolution of swine. Allelic frequencies for nine MS loci linked on swine chromosome 6 (SSC6) representing four western and one Chinese swine breeds were used to estimate genetic distances and times of breed divergence. A phylogenetic tree was constructed which partitioned into western and Meishan breed branches. Yorkshire and Hampshire breeds exhibited the most recent divergence with a calculated distance of 391 years. The oldest divergence, of 2,227 years, was between Meishan and Hampshire swine. Estimates of breed divergence are consistent with historical records. Additional analysis suggests that polymorphic MS linked on a single chromosome are sufficient to determine evolutionary relationships within a single species. Received: 23 September 1996 / Accepted: 26 April 1997     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

A second-generation linkage map of the sheep genome

A genetic map of Ovis aries (haploid n = 27) was developed with 519 markers (504 microsatellites) spanning ∼3063 cM in 26 autosomal linkage groups and 127 cM (female specific) of the X Chromosome (Chr). Genotypic data were merged from the IMF flock (Crawford et al., Genetics 140, 703, 1995) and the USDA mapping flock. Seventy-three percent (370/504) of the microsatellite markers on the map are common to the USDA-ARS MARC cattle linkage map, with 27 of the common markers derived from sheep. The number of common markers per homologous linkage group ranges from 5 to 22 and spans a total of 2866 cM (sex average) in sheep and 2817 cM in cattle. Marker order within a linkage group was consistent between the two species with limited exceptions. The reported translocation between the telomeric end of bovine Chr 9 (BTA 9) and BTA 14 to form ovine Chr 9 is represented by a 15-cM region containing 5 common markers. The significant genomic conservation of marker order will allow use of linkage maps in both species to facilitate the search for quantitative trait loci (QTLs) in cattle and sheep. Received: 20 September 1992 / Accepted: 18 November 1997     

Mechanical analysis of heterogeneous, atherosclerotic human aorta.

An experimental technique was developed to determine the finite strain field in heterogeneous, diseased human aortic cross sections at physiologic pressures in vitro. Also, the distributions within the cross sections of four histologic features (disease-free zones, lipid accumulations, fibrous intimal tissue, and regions of calcification) were quantified using light microscopic morphometry. A model incorporating heterogeneous, plane stress finite elements coupled the experimental and histologic data. Tissue constituent mechanical properties were determined through an optimization strategy, and the distributions of stress and strain energy in the diseased vascular wall were calculated. Results show that the constituents of atherosclerotic lesions exhibit large differences in their bilinear mechanical properties. The distributions of stress and strain energy in the diseased vascular wall are strongly influenced by both lesion structure and composition. These results suggest that accounting for heterogeneities in the mechanical analysis of atherosclerotic arterial tissue is critical to establishing links between lesion morphology and the susceptibility of plaque to mechanical disruption in vivo.     

Innate Killing of Leishmania donovani by Macrophages of the Splenic Marginal Zone Requires IRF-7

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells.     

Characterization of the osmoprotectant transporter OpuC from Pseudomonas syringae and demonstration that cystathionine-beta-synthase domains are required for its osmoregulatory function

The plant pathogen Pseudomonas syringae may cope with osmotic stress on plants, in part, by importing osmoprotective compounds. In this study, we found that P. syringae pv. tomato strain DC3000 was distinct from most bacterial species in deriving greater osmoprotection from exogenous choline than from glycine betaine. This superior osmoprotection was correlated with a higher capacity for uptake of choline than for uptake of glycine betaine. Of four putative osmoregulatory ABC transporters in DC3000, one, designated OpuC, functioned as the primary or sole transporter for glycine betaine and as one of multiple transporters for choline under high osmolarity. Surprisingly, the homolog of the well-characterized ProU transporter from Escherichia coli and Salmonella enterica serovar Typhimurium did not function in osmoprotection. The P. syringae pv. tomato OpuC transporter was more closely related to the Bacillus subtilis and Listeria monocytogenes OpuC transporters than to known osmoprotectant transporters in gram-negative bacteria based on sequence similarity and genetic arrangement. The P. syringae pv. tomato OpuC transporter had a high affinity for glycine betaine, a low affinity for choline, and a broad substrate specificity that included acetylcholine, carnitine, and proline betaine. Tandem cystathionine-beta-synthase (CBS) domains in the ATP-binding component of OpuC were required for transporter function. The presence of these CBS domains was correlated with osmoregulatory function among the putative transporters examined in DC3000 and was found to be predictive of functional osmoregulatory transporters in other pseudomonads. These results provide the first functional evaluation of an osmoprotectant transporter in a Pseudomonas species and demonstrate the usefulness of the CBS domains as predictors of osmoregulatory activity.