Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.     

From the ground up: biotic and abiotic features that set the course from genes to ecosystems

Spatial variation in cone serotiny in Rocky Mountain lodgepole pine (Pinus contorta ssp. latifolia) across Yellowstone National Park influences initial pine recruitment after stand‐replacing fire with tremendous population, community, and ecosystem consequences. A previous study showed that much of the spatial variation in serotiny results from the balance of selection arising from high frequencies of fire favoring serotiny countered by opposing selection exerted by American red squirrels (Tamiasciurus hudsonicus) as seed predators. This earlier study, however, assumed stable local red squirrel densities over multiple generations of pines. Here, we examine environmental properties that might contribute to long‐term stability in the densities of red squirrels among sites. We found that the amount of clay in the soil, an indicator of plant and fungal growth—the latter an important food resource for red squirrels—and the coefficient of variation (CV) in diameter at breast height (DBH) of forest trees together account for a substantial amount of variation in red squirrel density. Soil development occurs over very long time scales, and thus, intersite variation in the amount of clay is unlikely to shift across pine generations. However, CV of DBH and squirrel density increase with stand age, which acts to amplify selection against serotiny with increasing interfire interval. Regardless, much of the variation in the CV of DBH is accounted for by soil bulk density, mean annual temperature, and surface curvature, which are unlikely to vary in their relative differences among sites over time. Consequently, these soil and abiotic attributes could contribute to consistent spatial patterns of red squirrel densities from one pine generation to the next, resulting in consistent local and spatial variation in selection exerted by red squirrels against serotiny.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

The physiological basis of seed dormancy in Avena fatua. II. On the involvement of alternative respiration in the stimulation of germination by sodium azide

Chromatography on DEAE-cellulose of an extract from etiolated leaves of sorghum ( Sorghum vulgare Pers. cv. INRA 450), a C₄ plant, gave only one form of phosphoenol pyruvate carboxylase with functional and regulatory properties of a C₃ type plant enzyme. Greening of the leaves resulted in a significant increase in activity. This increase was due to the appearance of a new form of the enzyme, which eluted at lower ionic strength and exhibited new properties. This form was glucose-6-P activated and showed a sigmoidal curve response to the concentration of the substrate phosphoerralpyruvate. These kinetic properties are typical of a C₄ plant enzyme.     

Experimental and clinical applications of fibrin glue

A 2-year experience with laboratory and clinical applications of fibrin glue is presented. An autologous technique, which eliminates the danger of multidonor preparations, has been developed in our blood bank. While one can obtain different fibrinogen concentrations from the same amount of a patient's blood, in vitro mechanical testing demonstrated that at higher fibrinogen concentrations there is an increase in shear adhesive strength. Evaluation of skin-graft take in 16 Sprague-Dawley rats did not demonstrate significant differences in healing when adhesive use was compared with suture technique. In a clinical study, four different groups of patients (facial burns, hand burns, difficult graft sites, and miscellaneous surgical applications) benefited from autologous or single-donor fibrin glue for a total of 82 cases. There are several distinct advantages to the use of fibrin adhesive: The autologous technique eliminates the risk of transmissible viral diseases (AIDS, hepatitis); it can be used as a sealant in the treatment of seromas, dural leaks, and lymphoceles; and it improves hemostasis and early graft adherence. Face and hands are resurfaced with sheet grafts in a single procedure, obtaining a better aesthetic result with complete graft take and immediate start of physical therapy. Neither sutures nor pressure dressings are required. The minimal postoperative care associated with early return to normal activities seems to increase the satisfaction of patients and nurse personnel.     

Interaction of the peptide CF3-Leu-Ala-NH-C6H4-CF3 (TFLA) with porcine pancreatic elastase. X-ray studies at 1.8 A

The peptide trifluoroacetyl-Leu-Ala-(p-trifluoromethylanilide), is a reversible inhibitor of pancreatic porcine elastase and is characterized by a Km of 2.5 x 10(-8) M. Co-crystals of the 1:1 complex were obtained in an acetate buffer + dimethylformamide solution at pH 5.7. Diffraction data were recorded on films at the LURE synchrotron facility. The inhibitor was localized on difference Fourier maps, and the refinement of the structure was performed by simulated annealing (XPLOR). The current agreement factor is R = 19% (for 13224 observed structure factors and 1.8 A effective resolution). The RMS deviations from ideality of bond distances and angles are 0.02 A and 2 degrees, respectively. The inhibitor molecule was found in the active site, bent around the side chain of Phe-215 in a geometry that resembles the previously reported structure of the CF3-Lys-Ala complex at 2.5 A, in a parallel beta-sheet association with the loop 214-216. The analysis of the close contacts (less than 3.5 A) indicates that the trifluoromethylamide bond interacts with the active site and not the Leu-Ala or Ala-anilide bonds. The two fluorinated groups of the inhibitor exhibit different specificities: the trifluoroacetyl group (N terminus) is tightly stacked between the two chain loops 191-195 and 213-215, while the trifluoromethylanilide (C terminus) shows less specificity and only a single contact.     

Robustness of activity of Calvin cycle enzymes after high light and low temperature conditions in Antarctic vascular plants

Only two vascular plants have naturally colonized the Maritime Antarctic: Colobanthus quitensis and Deschampsia antarctica. We propose that one of the reasons of their success in this environment is the robustness of their CO₂ assimilation machinery. In order to understand the mechanisms involved in the positive photosynthetic rates under stressful conditions, we analyzed changes in the activity of two key Calvin cycle enzymes: Ribulose bisphosphate carboxylase oxygenase (Rubisco) and stromal Fructose-1,6-bisphosphatase after high light and low temperature treatments. Our results show that the activity of both enzymes does not decrease after 48 h high light/low temperature treatments, a feature usually observed in plants adapted to harsh environments. The activation state of both enzymes remained high throughout the treatments. This feature has been related to the redox state of the chloroplast, suggesting that both plants maintain their redox balance under high light and/or low temperature conditions assayed. Both plants differed in their responsiveness to cold acclimation as observed by gas exchange and enzymatic measurements. We propose that these differences may be related to microclimate adaptations to the environment they naturally develop in the Maritime Antarctic.