Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication.

The six MCM (minichromosome maintenance) proteins are essential DNA replication factors that each contain a putative ATP binding motif and together form a heterohexameric complex. We show that these motifs are required for viability in vivo and coordinated ATP hydrolysis in vitro. Mutational analysis discriminates between two functionally distinct MCM protein subgroups: Mcm4p, 6p, and 7p contribute canonical ATP binding motifs essential for catalysis, whereas the related motifs in Mcm2p, 3p, and 5p serve a regulatory function. Reconstitution experiments indicate that specific functional interactions between these two subgroups are required for robust ATP hydrolysis. Our observations show parallels between the MCM complex and the F1-ATPase, and we discuss how ATP hydrolysis by the MCM complex might be coupled to DNA strand separation.     

Genes with expression levels correlating to drip loss prove association of their polymorphism with water holding capacity of pork

Six genes that were known to exhibit expression levels that are correlated to drip loss BVES, SLC3A2, ZDHHC5, CS, COQ9, and EGFR have been for candidate gene analysis. Based on in silico analysis SNPs were detected, confirmed by sequencing, and used for genotyping. The SNPs were genotyped in about 1,800 animals from six pig populations including commercial herds of Pietrain (PI) and German Landrace (DL), different commercial herds of Pietrain × (German Large White × German Landrace) (PIF1(a/b/c)), and one experimental F₂-population Duroc × Pietrain (DUPI). Comparative and genetic mapping established the location of BVES on SSC1, of SLC3A2 and ZDHHC5 on SSC2, of CS on SSC5, of COQ9 on SSC6 and of EGFR on SSC9, respectively, coinciding with QTL regions for carcass and meat quality traits. BVES, SLC3A2, and CS revealed association at least with drip loss and with several other measures of water holding capacity (WHC). Moreover, COQ9 and EGFR were associated with several meat quality traits such as meat color and/or thawing loss. This study reveals statistic evidence in addition to the functional relationship of these genes to WHC previously evidenced by expression analysis. This study reveals positional and genetic statistical evidence for a link of genetic variation at these loci or close to them and promotes those six candidate genes as functional and/or positional candidate genes for meat quality traits.     

An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.     

Red-breasted goose colonies on the Taimyr Peninsula: Factors responsible for the proximity of goose nests to nests of peregrine falcons,rough-legged buzzards,and snowy owls

Red-breasted goose colonies have been studied near Medusa Bay (73°21′N, 80°32′E), on the northwestern Taimyr Peninsula, and along the Agapa River (70°11′N, 86°15′E) down to its mouth (71°26′N, 89° 13′E), in the central Taimyr Peninsula. Red-breasted geese nesting near peregrine falcons are protected by the falcons from arctic foxes; however, they are sometimes attacked by the falcons themselves. In the colonies near peregrine falcon nests, the vast majority of goose nests were situated no farther than 100 m from the falcon nest. When food is abundant, falcons protect a larger area around their nest. The distance between the falcon nest and the surrounding goose nests is inversely related to the falcon’s activity. In years of higher falcon activity, falcons prevent red-breasted geese from nesting as close to their nest as in years of lower falcon activity. Additional stimuli are required for red-breasted geese to form colonies near rough-legged buzzard nests. The distance between snowy owl nests and red-breasted goose nests was smaller when arctic foxes were abundant than when they were scarce.     

Contribution to the study of the biology of soil-dwelling Dipteran larvae (insecta, diptera) of the desert zone

The composition of seven dipteran families from the complex of larvae dwelling in sandy soils of the southeastern Karakum Desert, represented by both free-living soil forms and endobionts ecologically dependent mainly on herbaceous plants, is analyzed. In the surface horizons of the soil, at a depth of up to 20 cm, free-living soil-dwellers form a stable complex, which includes saprophagous coleopteran larvae and their constant companions, the obligatorily predaceous dipteran larvae. Previously unknown data on the lifestyle of the larvae of five therevid genera are given.     

Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.     

31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition

31P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. Our results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (Pi) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP gamma saturation and were used in combination with ST data to determine Pi consumption rates. 13C NMR and O2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O2 consumption, respectively, under the various metabolic conditions examined. Our results suggest that GAPDH/PGK-catalyzed Pi-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.