Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

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.     

Identification of rho as a substrate for botulinum toxin C3-catalyzed ADP-ribosylation

Recombinant Aplysia rho and a GTP-binding protein purified from human neutrophil membranes (G22K) were ADP-ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively. Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP-ribosylated, (iii) G22K was not recognized by an anti-rho antiserum, and (iv) antibody 142-24E05 recognized G22K effectively but only poorly cross reacted with rho. ADP-ribosylation had no effect on the ability of rho to bind or hydrolyse GTP. Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.     

Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets.

Recent studies have shown that ligand-activated growth factor receptors as well as transforming versions of nonreceptor protein-tyrosine kinases physically associate with phosphatidylinositol-3 kinase (PI-3 kinase). Reasoning that PI-3 kinase might also play a role in the normal functions of nonreceptor kinases, we sought to determine whether association with PI-3 kinase might serve as a measure of nonreceptor protein-tyrosine kinase activation under physiological conditions. We found that p60c-src as well as p59fyn, the product of another member of the src family of proto-oncogenes, physically associated with a PI kinase activity within 5 s after exposure to thrombin. Furthermore, PI kinase reaction products generated in p60v-src, p60c-src or p59fyn containing immunoprecipitates were indistinguishable, demonstrating the identity of the associated enzyme as PI-3 kinase. These findings demonstrate a thrombin-dependent interaction between p60c-src or p59fyn and PI-3 kinase and suggest a role for nonreceptor protein-tyrosine kinases in human platelet signal transduction.     

The problem of the acts duration measurement in relation to the “focal-animal” technique of behavioral recording

The focal-animal technique assumes the continuous recording of the behavior of an individual during a certain time interval. The length of this interval (sampling unit) can be problematic when estimating the duration of behavioral acts. Two acts from the behavioral repertoire of the ant Leptothorax fuenteiwere focused on in this work at different ranges of temporal scales. Analyzing these acts we show the possibility of existence of a sampling artifact, in such a way that the estimates of the durations of the acts would be forced to depend upon the length of the sampling unit that is being used.     

Guaianolides and lignans from Centaurea solstitialis subs Schouwii

Aerial parts of Centaurea solstitialis subs schouwii afforded the guaianolides cynaropicrin and aguerin B and the lignans arctigenin and matairesinol. The structure of a third guaianolide previously found also in Centaurea behen was revised.     

Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Intracellular location of T200 and Mo1 glycoproteins in human neutrophils

Mo1 (CD11b), a glycoprotein heterodimer that is involved in cellular adhesion processes and functions as the C3bi receptor of human myeloid cells, and T200 (CD45), a panleukocyte glycoprotein family whose function is still not well understood, increased their expression in the plasma membrane of human neutrophils after exposure to various stimuli which induce degranulation, such as formylmethionylleucylphenylalanine or calcium ionophore A23187. This increment in the expression of both molecules shows a good correlation with the release to the extracellular environment of gelatinase, a marker for an intracellular organelle named "tertiary granule" (Mollinedo, F., and Schneider, D. L. (1984) J. Biol. Chem. 259, 7143-7150). Flow cytometry studies indicate that at least 50% of the total Mo1 and T200 molecules are located in intracellular organelles. Furthermore, the subcellular distribution of Mo1 and T200 glycoproteins in resting human neutrophils was investigated by immunoprecipitation of the radiolabeled membrane proteins obtained from the distinct subcellular fractions. Both Mo1 and T200 were mainly localized in tertiary or specific intracellular granules, which were resolved from the azurophilic granules as well as from the cell membrane fraction. These findings suggest that the mobilization of intracellular Mo1 and T200 to the plasma membrane may regulate early events occurring upon neutrophil activation.     

Ras p21 proteins with high or low GTPase activity can efficiently transform NIH/3T3 cells

We sought to determine whether decreased in vitro GTPase activity is uniformly associated with ras p21 mutants possessing efficient transforming properties. Normal H-ras p21-[Gly12-Ala59] as well as an H-ras p21-[Gly12-Thr59] mutant exhibited in vitro GTPase activities at least fivefold higher than either H-ras p21-[Lys12-Ala59] or H-ras p21-[Arg12-Thr59] mutants. Microinjection of as much as 6 X 10(6) molecules/cell of bacterially expressed normal H-ras p21 induced no detectable alterations of NIH/3T3 cells. In contrast, inoculation of 4-5 X 10(5) molecules/cell of each p21 mutant induced morphologic alterations and stimulated DNA synthesis. Moreover, the transforming activity of each mutant expressed in a eukaryotic vector was similar and at least 100-fold greater than that of the normal H-ras gene. These findings establish that activation of efficient transforming properties by ras p21 proteins can occur by mechanisms not involving reduced in vitro GTPase activity.     

Characterization of a membrane-specific tubulin isoform by peptide mapping

A membrane-specific tubulin-like protein, found in preparations of synaptic plasma membranes and brain mitochondria, was analyzed by chemical and proteolytic peptide mapping to determine which part of the molecule was different from cytoplasmic tubulin. The membrane polypeptide was identical to alpha tubulin in the first two-thirds of the molecule containing the amino terminal, as found by peptide mapping. However, some differences were observed in the peptide maps of the carboxy terminal one third of the molecule which includes a domain that is important in the regulation of tubulin self-assembly.