Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA.

This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

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.     

Life Cycles of Ground Beetles (Coleoptera,Carabidae) from the Mountain Taiga and Mountain Forest-Steppe in the Eastern Sayan

Seasonal dynamics and demographic structure was studied in 15 dominant ground beetle species in the mountain taiga and mountain forest-steppe belts of the Eastern Sayan (Okinskoe Plateau). Life cycles of the dominant ground beetle species were classified by developmental time, seasonal dynamics, and intrapopulation groups with different reproduction timing. The strategies of carabid life cycles adapted to severe mountain conditions of the Eastern Sayan were revealed.     

Accumulation of DNA structural damages in human lymphocytes during aging

Elastoviscosometric parameters of DNA from normal subjects of different age and patients with Down syndrome were assessed. Characteristics of DNA isolated from lymphocytes trisomic for chromosome 21 were studied to compare normal and pathological rates of ageing. Increased elastoviscosity was observed in normal subjects above 60. Similar changes in this parameter were noted in aberrant lymphocytes isolated from patients above 10. The established dependence of elastoviscosity on ethidium bromide concentration led to the assumption that an increase in hydrodynamic DNA volume in human leukocytes during ageing was due to accumulation of spontaneous irreparable DNA lesions.     

Two remarkable new species of scuttle-fly (Diptera: Phoridae) that parasitize termites (Isoptera) in Sulawesi

ABSTRACT. Diplonevra mortimeri sp.nov. and D.watsoni. sp.nov. are described from specimens caught at Nasutitermitinae termite colonies in Sulawesi. The female of both species has a remarkable pre-oviposition behaviour in which she lures a worker termite into following her away from the colony. She then oviposits in the termite's abdomen. Pupation takes place within the remains of the host's abdomen. Both species only mature a single egg at a time. The female probably guards the comatose host termite during the period of larval development. The morphology of the male hypopygia of the new species allows reinterpretation of the peculiar anal tube in typical male Diplonevra. Furthermore the suggested homologies reinforce the view that the epandrium (tergite 9) has not been replaced by a periandrium (fused gonocoxites) in the Cyclorrhapha and consequently the latter cannot be derived from the Empidoidea.     

Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm. typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium , as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.