Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6–32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8–48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment.     

Brain‐derived neurotrophic factor selectively regulates dendritogenesis of parvalbumin‐containing interneurons in the main olfactory bulb through the PLCγ pathway

Molecular mechanisms of neurotrophin signaling on dendrite development and dynamics are only partly understood. To address the role of brain‐derived neurotrophic factor (BDNF) in the morphogenesis of GABAergic neurons of the main olfactory bulb, we analyzed mice lacking BDNF, mice carrying neurotrophin‐3 (NT3) in the place of BDNF, and TrkB signaling mutant mice with a receptor that can activate phospholipase Cγ (PLCγ) but is unable to recruit the adaptors Shc/Frs2. BDNF deletion yielded a compressed olfactory bulb with a significant loss of parvalbumin (PV) immunoreactivity in GABAergic interneurons of the external plexiform layer. Dendrite development of PV‐positive interneurons was selectively attenuated by BDNF since other Ca²⁺‐binding protein‐containing neuron populations appeared unaffected. The deficit in PV‐positive neurons could be rescued by the NT3/NT3 alleles. The degree of PV immunoreactivity was dependent on BDNF and TrkB recruitment of the adaptor proteins Shc/Frs2. In contrast, PLCγ signaling from the TrkB receptor was sufficient for dendrite growth in vivo and consistently, blocking PLCγ prevented BDNF‐dependent dendrite development in vitro. Collectively, our results provide genetic evidence that BDNF and TrkB signaling selectively regulate PV expression and dendrite growth in a subset of neurochemically‐defined GABAergic interneurons via activation of the PLCγ pathway. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. ¹³C nuclear magnetic resonance (NMR) analysis of [1-¹³C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by ¹³C NMR by following the simultaneous metabolism of labeled [¹³C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH₂ of succinate and the CH₃ of acetate when the resting cells were metabolizing [1-¹³C]glucose and unlabeled glycogen were precisely quantified by using ¹³C-filtered spin-echo difference ¹H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.     

Sequestration revisited: integrating traditional electron microscopy, de novo assembly and new results

Electron microscopy analysis of the autophagic sequestration membrane (SM) in various metazoan cell types after different fixation methods shows that: (1) the growing SM cannot derive from preformed rough surfaced endoplasmic reticulum (RER) membranes by transformation; (2) the empty cleft between the two layers of the SM after aldehyde fixation is an artifact of sample preparation; (3) the SM emerges from and grows de novo in cytoplasmic areas where membranous precursors cannot be identified by traditional electron microscopy; (4) the growing SM consists of two tightly packed membrane layers with a sharp bend at the edge; (5) changes in the environment of the growing SM participate in the determination of the size and shape of the autophagosome. We suggest that expansion as well as regression takes place at the edge of the growing SM. Stabilization and irreversibility of formation of the SM is achieved by closure. The immediate source of lipids for the SM must be in the cytoplasmic matrix, supposedly in the form of special phospholipid carrying vesicles that might involve the transmembrane Atg9 protein. To explain the apparent lack of such vesicles by electron microscopy we suggest that they are too small, have a similar density to other frequently occurring structures, or are destroyed during sample preparation.     

Differential proteomics of plant development

In this mini-review, recent advances in plant developmental proteomics are summarized. The growing interest in plant proteomics continually produces large numbers of developmental studies on plant cell division, elongation, differentiation, and formation of various organs. The brief overview of changes in proteome profiles emphasizes the participation of stress-related proteins in all developmental processes, which substantially changes the view on functional classification of these proteins. Next, it is noteworthy that proteomics helped to recognize some metabolic and housekeeping proteins as important signaling inducers of developmental pathways. Further, cell division and elongation are dependent on proteins involved in membrane trafficking and cytoskeleton dynamics. These protein groups are less prevalently represented in studies concerning cell differentiation and organ formation, which do not target primarily cell division. The synthesis of new proteins, generally observed during developmental processes, is followed by active protein folding. In this respect, disulfide isomerase was found to be commonly up-regulated during several developmental processes. The future progress in plant proteomics requires new and/or complementary approaches including cell fractionation, specific chemical treatments, molecular cloning and subcellular localization of proteins combined with more sensitive methods for protein detection and identification.     

Do terrestrial gastropods use olfactory cues to locate and select food actively?

Having been investigated for over 40 years, some aspects of the biology of terrestrial gastropod’s olfactory system have been challenging and highly contentious, while others still remain unresolved. For example, a number of terrestrial gastropod species can track the odor of food, while others have no strong preferences toward food odor; rather they find it by random encounter. Here, while assessing the most recent findings and comparing them with earlier studies, the aspects of the food selection based on olfactory cues are examined critically to highlight the speculations and controversies that have arisen. We analyzed and compared the potential role of airborne odors in the feeding behavior of several terrestrial gastropod species. The available results indicate that in the foraging of most of the terrestrial gastropod species odor cues contribute substantially to food finding and selection. The results also suggest, however, that what they will actually consume largely depends on where they live and the species of gastropod that they are. Due to the voluminous literature relevant to this object, this review is not intended to be exhaustive. Instead, I selected what I consider to be the most important or critical in studies regarding the role of the olfaction in feeding of terrestrial gastropods.     

Between‐male variation in sperm size,velocity and longevity in sand martins Riparia riparia

Sperm mobility is known to be an important determinant of a male's sperm competitive ability. Although more debated, sperm length and its relation to sperm swimming ability has also been proposed to determine a male's fertilisation potential. Furthermore, both mobility and length may covary with a male's phenotype, either positively (the phenotype‐linked fertility hypothesis) or negatively if, for instance, low‐quality males have less access to females but invest more in sperm production. Using dummy females, we collected sperm samples from wild sand martins Riparia raparia males. We investigated the relationship between sperm length and sperm swimming speed as measured by sperm straight line velocity (VSL), and determined whether sperm traits are correlated with male body size and condition. We found that total sperm length is repeatable within‐ejaculate and shows substantial inter‐male variation. Sperm length was associated with sperm velocity: males with short sperm have sperm that swim initially faster but die sooner, whereas males with longer sperm have sperm that swim more slowly but for a longer time. Smaller males produced sperm with higher overall velocity. This correlation between male size and sperm behaviour may reflect alternative fertilisation strategies where small males having less mating opportunities invest more in sperm competitive ability. The existence of such alternative strategies would participate in maintaining variation in sperm length and velocity in this species.     

No evidence of genetic benefits from extra-pair fertilisations in female sand martins (Riparia riparia)

Genetic parentage studies of socially monogamous birds reveal a widespread prevalence of extra-pair paternity. Variation in extra-pair paternity among individuals may depend on how different individuals benefit from extra-pair fertilisations and on the opportunity to pursue extra-pair copulations. A long-term study of sand martins (Riparia riparia) in Hungary allowed us to examine patterns of extra-pair fertilisations in a large colony of over 3,000 breeding pairs with many known age individuals. We used multi-locus DNA fingerprinting to determine whether extra-pair fertilisations occur when females are paired to (1) presumably low quality mates, or (2) genetically similar or dissimilar mates, and whether extra-pair fertilisations result in offspring of higher quality. Extra-paternal young were found in 38% of 47 broods and comprised 19% of 190 offspring. Males that lost paternity did not differ significantly from others in age or body condition. Social mates of broods containing extra-pair offspring did not differ in genetic similarity from pairs without extra-pair offspring. Furthermore, there was no significant difference in body condition between extra-pair young and their maternal half-siblings. We were unable to assign paternity and therefore cannot exclude the possibility that extra-pair males differed from the within-pair males they cuckolded, in age, body condition or genetic similarity with the female. We found a positive relationship between paternity losses and breeding density, suggesting that low breeding density may constrain opportunities for seeking extra-pair copulations.     

Major histocompatibility complex controls susceptibility and dominant inheritance,but not the severity of the disease in mouse models of rheumatoid arthritis

Collagen-induced arthritis (CIA) in DBA/1 and proteoglycan-induced arthritis (PGIA) in BALB/c mice are the most frequently used mouse models for studying clinical, immunological and genetic factors contributing to rheumatoid arthritis. DBA/1 ( H2(q)) mice are susceptible to CIA but resistant to PGIA, whereas BALB/c mice ( H2 (d)) are susceptible to PGIA and resistant to CIA. To gain insight into the mechanisms of how the major clinical (disease susceptibility, severity and onset of arthritis) and immunological traits (antigen-specific T- and B-cell responses) are influenced by the major histocompatibility complex (MHC), we have generated a unique intercross of BALB/c and DBA/1 parent strains, and the F1 and F2 hybrids were immunized for either CIA or PGIA. The major clinical and immunological traits were identified as either binary (qualitative) or quantitative traits on Chromosome 17 with a peak at MHC when the entire population was analyzed. In contrast, when only arthritic (susceptible) mice were selected and analyzed, the major clinical traits (severity and onset) 'lost' the linkage to MHC. Thus, MHC dictates disease susceptibility, but not the severity of arthritis. This was even more evident in the case of the H2(q) allele, which was clearly responsible for the dominant inheritance of arthritis in F2 hybrids (either CIA or PGIA). In conclusion, while certain MHC alleles strongly affect disease susceptibility and determine the mode of inheritance of a polygenic autoimmune disease, neither the type of inheritance (dominant vs recessive) nor other MHC components have evident effects upon the clinical symptoms of arthritis.