The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases

Calcium efflux and EGTA-induced calcium release from an internal platelet membrane fraction have been studied after the oxalate-supported calcium uptake had reached steady state. Increasing external calcium concentrations stimulate the calcium efflux velocity, with an apparent half-maximal stimulation at about 5 microM outside calcium concentration and a maximal velocity of calcium efflux of 4.66 +/- 2.32 nmol X min-1 X mg-1. Moreover, the ratio of the liberated calcium on the loaded calcium seems to be independent of the increasing external calcium concentration. Increasing the calculated internal calcium concentration by varying the oxalate potassium concentration from 10 mM to 1 mM results in an increase of the liberated calcium from the membrane vesicles from 7.4% to 63%, respectively, without changing the calcium efflux velocity. Similar conclusions can be drawn from the observation of results from the calcium efflux and EGTA-induced calcium release methods. Moreover, calcium pump reversal does not seem to be responsible for the calcium efflux or calcium release. All these different points added to the previously described regulation of calcium efflux by the catalytic subunit of cAMP protein kinase suggest us that the mechanism of calcium liberation by the platelet membranes is different from the calcium uptake.     

Within-species correlations in leaf traits of three boreal plant species along a latitudinal gradient

Evergreen boreal plant species express high variability in their leaf traits. It remains controversial whether this within-species variability is constrained to the same leaf trait relationships as has been observed across species. We sampled leaves of three boreal evergreen woody species along a latitudinal gradient (from 57o56′N to 69o55′N). Leaf longevity (LL) of Pinus sylvestris L. and Vaccinium vitis-idaea L. correlated negatively with mean annual air temperature (MAT), whereas the LL of Ledum palustre L. was not affected by MAT. V. vitis-idaea and L. palustre had a negative relationship between leaf mass per area (LMA) and MAT. In P. sylvestris, the LMA–MAT relationship was positive. A negative correlation between LL and LMA was significant only for P. sylvestris. Leaf nitrogen concentration was positively related to leaf phosphorus concentration in all three species. Leaf potassium concentration was related to nitrogen concentration only in L. palustre, and to phosphorus concentration in P. sylvestris and L. palustre. Our results demonstrate that although within the studied species the variation in some of the leaf traits may have the same degree as interspecific variation, there is no such intercorrelation of leaf traits within the studied species as has been observed across species.     

Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prognosis of non-Hodgkin's lymphomas in the Dresden district with special reference to combined radio-/chemotherapy

The total population affected with non-Hodgkin's lymphoma and treated by means of radiotherapy or combined radio-chemotherapy between 1960 and 1985 at the Medical Academy Dresden was analysed as to prognosis. 247 patients were classified according to the previous German scheme, 79 were subdivided on the basis of the recommendations laid down in the Kiel classification. The remission rates and survival curves achieved will stand out the comparison with international literature (remission rates of the low malignancy group amounted to 85.3 p.c. and those of the high malignancy group to 80.0 p.c.; the 5-years survival rates of the low malignancy group amounted to 61.9 p.c. and those of the high malignancy group to 41.7 p.c.). The influence of histology, clinical stage and involvement of organs is discussed on the basis of our results and informations obtained in literature. Our analysis confirms the high importance which must be attached to a common radiologic-internal outpatient-department for co-ordinating the diagnostic and therapeutical programme.     

Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria.

D-beta-Hydroxybutyrate dehydrogenase of bovine heart mitochondria has been purified to apparent homogeneity. The membrane-bound enzyme is first released by phospholipase A digestion of the mitochondria. Lithium bromide, 0.4 M, is used to aid release, and dithiothreitol is required to stabilize the enzyme. The membranous material is removed by centrifugation, and the apoenzyme is recovered in the supernatant and precipitated with ammonium sulfate to 50 percent of saturation. The main purification (100-fold) is achieved by selective adsorption and elution on controlled pore glass beads. The purified enzyme has been purified approximately 250-fold from the mitochondria. The purified enzyme is homogeneous as shown by poly-acrylamide gel electrophoresis in sodium dodecyl sulfate or acid-urea systems; a sharp band is obtained which is equivalent to a subunit molecular weight of 31,500. The apoenzyme is devoid of lipid and is completely inactive as isolated. It can be reactivated by adding aqueous microdispersions of lecithin or phospholipids containing lecithin. The apoenzyme is stable, i.e. it has a half-life of about 450 hours at 0-2 degrees in 0.4 M lithium bromide, containing 5 mM dithiothreitol at pH 7, and is soluble at these conditions, existing mainly as a monomer and dimer in dilute solution. It has a tendency to associate into larger aggregates when the salt concentration is lowered. The enzyme does not have a distinctive amino acid composition as compared with other proteins or soluble dehydrogenases. The purified apodehydrogenase is well suited for study of specific protein-lipid interaction, as well as the molecular basis for the role of phospholipid in this lipid-requiring enzyme.     

Prioritizing Tiger Conservation through Landscape Genetics and Habitat Linkages

Even with global support for tiger (Panthera tigris) conservation their survival is threatened by poaching, habitat loss and isolation. Currently about 3,000 wild tigers persist in small fragmented populations within seven percent of their historic range. Identifying and securing habitat linkages that connect source populations for maintaining landscape-level gene flow is an important long-term conservation strategy for endangered carnivores. However, habitat corridors that link regional tiger populations are often lost to development projects due to lack of objective evidence on their importance. Here, we use individual based genetic analysis in combination with landscape permeability models to identify and prioritize movement corridors across seven tiger populations within the Central Indian Landscape. By using a panel of 11 microsatellites we identified 169 individual tigers from 587 scat and 17 tissue samples. We detected four genetic clusters within Central India with limited gene flow among three of them. Bayesian and likelihood analyses identified 17 tigers as having recent immigrant ancestry. Spatially explicit tiger occupancy obtained from extensive landscape-scale surveys across 76,913 km² of forest habitat was found to be only 21,290 km². After accounting for detection bias, the covariates that best explained tiger occupancy were large, remote, dense forest patches; large ungulate abundance, and low human footprint. We used tiger occupancy probability to parameterize habitat permeability for modeling habitat linkages using least-cost and circuit theory pathway analyses. Pairwise genetic differences (F _ST) between populations were better explained by modeled linkage costs (r>0.5, p<0.05) compared to Euclidean distances, which was in consonance with observed habitat fragmentation. The results of our study highlight that many corridors may still be functional as there is evidence of contemporary migration. Conservation efforts should provide legal status to corridors, use smart green infrastructure to mitigate development impacts, and restore habitats where connectivity has been lost.     

Efficacy-Driven Dose Finding with Toxicity Control in Phase I Oncology Studies

Statistics in Biosciences - Traditionally, dose-finding process for oncology compound is carried out in phase I dose escalation study and is driven by safety in order to find maximum tolerated dose...     

Inhibitors of the Candida albicans Major Facilitator Superfamily Transporter Mdr1p Responsible for Fluconazole Resistance

ObjectiveTo identify a novel class of inhibitors of fungal transporters involved in drug resistance.MethodsA series of structurally-related low molecular mass compounds was synthesized using combinatorial chemistry of a cyclobutene-dione (squarile) core. These compounds were screened for their inhibition of plasma membrane Major Facilitator Superfamily (MFS) and ATP-binding cassette (ABC) transporters responsible for efflux pump-mediated drug resistance in the fungal pathogen Candida albicans. Strains of Saccharomyces cerevisiae that specifically overexpress the MFS pump CaMdr1p or the ABC transporter CaCdr1p were used in primary screens and counterscreens, respectively, and to detect inhibition of glucose-dependent Nile Red efflux. Efflux pump inhibition, activity as pump substrates and antifungal activity against yeast and clinical isolates expressing efflux pumps were determined using agarose diffusion susceptibility assays and checkerboard liquid chemosensitization assays with fluconazole.ResultsThe screen identified five structurally-related compounds which inhibited CaMdr1p. Two compounds, A and B, specifically chemosensitized AD/CaMDR1 to FLC in a pH-dependent fashion and acted synergistically with FLC in checkerboard liquid MIC assays but compound B had limited solubility. Compound A chemosensitized to FLC the azole-resistant C. albicans strain FR2, which over-expresses CaMdr1p, inhibited Nile Red efflux mediated by CaMdr1p but not CaCdr1p and was not toxic to cultured human cells. A minor growth-inhibitory effect of B on AD/CaMDR1, but not on AD/CaCDR1 and AD/CaCDR2, indicated that compound B may be a substrate of these transporters. The related compound F was found to have antifungal activity against the three pump over-expressing strains used in the study.ConclusionsCompound A is a ‘first in class’ small molecule inhibitor of MFS efflux pump CaMdr1p.