Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, α, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).     

Activities of Various Peptidases in the First Leaf of Wheat

In the first leaf of wheat (Triticum aestivum L. cv. Capelle) the content of soluble protein diminished to about 50% of the initial value between the 7th and the 19th day after sowing. In order to understand proteolysis in the leaves, the activities of several peptidases were measured in extracts from leaves at four different ages. The carboxypeptidase activities increased during the growth of the leaves, and then began to decrease. The activities of the alkaline peptidases increased, while that of the benzoyl-DL-arginine-p-nitroanilide (BAPA) hydrolysing enzyme decreased during the whole period studied. The “naphthylamidase” activities showed first a slow rise, but then leveled off. Two bands with naphthylamidase activity could be detected after disc electrophoresis. All the peptidases studied were present in the leaves at rather high concentrations. This indicates that they all may participate in the hydrolysis of leaf proteins into free amino acids.     

INSULIN PRODUCES A BIPHASIC RESPONSE INTETRAHYMENA THERMOPHILABY STIMULATING CELL SURVIVAL AND ACTIVATING PROLIFERATION IN TWO SEPARATE CONCENTRATION INTERVALS

Cells ofTetrahymenamay produce autocrine signal molecules with effects on survival and proliferation. Here we have tested the effects of human recombinant and bovine insulin, and the B22–B30 fragment of bovine insulin over a wide range of concentrations (10^?5–10^?18m ) on cell survival and proliferation in a synthetic nutrient medium. The cells were grown in conical flasks at low initial cell densities (40 and 400cells/ml). Insulin prevented rapid cell death and/or promoted cell proliferation over two separate concentration ranges: down to nanomolar levels and again in the low pico- and femtomolar range. At an initial population density of 400cells/ml the cells multiplied at both concentration intervals. At 40 or fewer organisms/ml the cells multiplied in the high concentration interval, whereas in the low interval they survived for about four times longer than those in the control cultures. B22–B30 added to cultures of 40 initial cells/ml produced a stimulation of cell survival in the low pico- and high femtomolar range. In the presence of hemin (50nm ) cells at 400 initial organisms/ml multiplied at insulin concentrations down to about 3nm and again from 300am to 10pm . In some cases, hemin plus insulin activated cell proliferation between the two concentration intervals as well. At 40cells/ml the cells not only survived but proliferated in the femtomolar range. Cells in cultures supplemented with both hemin and B22–B30 multiplied at the low concentration interval (from about 100fm to 10pm ).     

Sixty simple sequence repeat markers for use in the Festuca–Lolium complex of grasses

So far only very few simple sequence repeat (SSR) markers developed from grass species have had their primer sequences published. To make more markers available to the scientific community, we isolated and sequenced 256 microsatellite‐containing clones from four genome libraries of a Lolium multiflorum×Festuca glaucescens F₁ hybrid following enrichment in (TC)_n, (TG)_n, or both repeats. In this work, we report the primer sequences of 60 SSRs including preliminary results of polymorphism for mapping.     

Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O₂ evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O₂ evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.Microalgae are able to efficiently convert sunlight, water, and CO₂ into a variety of products suitable for renewable energy applications, including H₂, carbohydrates, and lipids (11, 12, 16, 21, 38, 41, 44). The unicellular green alga Chlamydomonas reinhardtii has emerged as a model organism for studying algal physiology, photosynthesis, metabolism, nutrient stress, and the synthesis of bioenergy carriers (12, 15, 19, 24, 32). During acclimation to nitrogen deprivation, C. reinhardtii cells accumulate significant quantities of starch and form lipid bodies (4, 5, 8, 26, 28, 30, 34, 43, 46, 48). Despite the significance of these products in algal physiology and in biofuels applications, the metabolic, enzymatic, and regulatory mechanisms controlling the partitioning of metabolites into these distinct carbon stores in algae are poorly understood. Several C. reinhardtii starch mutants with various phenotypic changes in starch content and structure have been isolated (2,–4). Two of these, the sta6 and sta7 mutants, contain single-gene disruptions that result in “starchless” phenotypes with severely attenuated levels of starch granule accumulation (2, 4, 34, 39, 40, 48).The disrupted loci in the two isolated starchless mutants are distinct and each mutant has a unique phenotype (7, 40). In the sta6 mutant, the small, catalytic subunit of ADP-glucose pyrophosphorylase (AGPase-SS) is disrupted (2, 4, 48), and this mutant accumulates less than 1% of the starch observed in wild-type (WT) cells under conditions of nitrogen deprivation. The sta7 mutant contains a disrupted isoamylase gene (7, 8, 10, 39, 40) and also has severely attenuated levels of starch, but it accumulates a soluble glycogen-like product (4, 9). In this study, we conducted an examination of the unique physiological acclimations that are utilized by these mutants to adapt to the loss of starch synthesis. As the genetic lesions in these two mutants are distinct and block starch synthesis via two very different mechanisms, we investigated the physiological consequences of starch inhibition in both of these mutants from a holistic bioenergy perspective, which included photosynthetic parameters and the overall yields of lipids and carbohydrates, the two primary bioenergy carriers in C. reinhardtii. Specifically, we examined whether the inability to synthesize starch would result in the accumulation of additional lipid, alter cellular growth or cell size, affect acetate utilization, and/or influence photosynthetic O₂ evolution. Our data indicate that both the sta6 (BAFJ5) and sta7 (sta7-10) mutants accumulate more lipid than the CC124 control during nitrogen deprivation. However, the additional lipid does not completely offset the loss of starch synthesis from a complete energetic perspective. Increased lipid accumulation during nitrogen stress has also been reported for a variety of starch mutants in recent papers (26, 27, 46). A significant feature in both of the starchless mutants studied here is that O₂ evolution and acetate utilization are diminished during nitrogen stress, which is undesirable from an overall bioenergy perspective. Remarkably, complementation of sta7-10 with genomic DNA encoding the wild-type isoamylase gene resulted in cells that were larger than those of the sta6, sta7-10, and CC124 strains, exhibited the highest total lipid levels during nitrogen deprivation, and overaccumulated starch even in nutrient-replete medium.     

Site selection by swans wintering in Britain and Ireland; the importance of habitat and geographic location

Monthly surveys of Bewick's Swans Cygnus columbianus bewickii , Whooper Swans Cygnus cygnus and Mute Swans Cygnus olor in Britain and Ireland were made during the 1990–1991 winter to determine factors affecting the swans' selection of feeding sites. Geographic location and habitat both influenced site selection. Whooper Swans occurred in greatest numbers at sites in Scotland, northeastern England and Northern Ireland, whereas Bewick's Swans had a more southerly distribution, reflecting differences in the migratory routes used by these two species. The resident Mute Swans were more widespread, with large flocks occurring in southeastern England and in parts of Scotland. Whooper and Mute Swans were found mainly on permanent inland waters (68% and 61%, respectively), but the majority of Bewick's Swans (60%) were on arable land. The percentage of Bewick's Swan flocks found on permanent inland waters (42%) was higher than that found on arable fields (23%), indicating that the large number recorded on arable land was a result of the birds congregating at a comparatively small number of sites. Overall, less than 15% of Whooper Swans and 3% of Mute Swans were on arable crops during the winter, but the largest flocks were associated with arable land for all three species. Thus, although the occurrence of large flocks at particular arable sites may give an impression that swans feed mainly on farmland, the swans are in fact more widely dispersed. Regional variation in the percentage of juveniles present was recorded for all three species. Changes during the winter in the distribution of juveniles, and of the swans as a whole, are considered in relation to food supply and to migratory routes for the Bewick's and Whooper Swans.     

Survival and Body Condition of Raccoons at the Edge of the Range

ABSTRACT We investigated the influence of intrinsic and extrinsic variables on overwinter survival of raccoons (Procyon lotor; n = 114) at the northern edge of their distribution. A Cox proportional hazard model identified winter severity as the variable with the greatest influence on raccoon survival (β = 1.08). Autumn body condition estimates (20.5 ± 0.46% total body fat) were relatively stable across years even though we observed large differences in autumn food indices. Variations in autumn body condition did not explain heterogeneity observed in overwinter survival nor the spring condition in which raccoons emerged. Relatively constant autumn body condition suggests reliable availability of anthropogenic food resources may negate variations observed in natural food items on which raccoons rely during hyperphagia. Conversely, spring body condition did vary among years and was highly correlated with winter severity. Accordingly, we also observed a strong inverse relationship with overwinter survival and winter severity. Our findings indicate winter climatic constraints are important factors governing the northern limit of raccoon distribution and changes in winter severity could have important implications in further range expansion of this species.     

Growth,development, and life‐history strategies in an unpredictable environment: case study of a rare hoverfly Blera fallax (Diptera,Syrphidae)

1. Development in organisms can vary in response to fluctuating environments. In holometabolous insects, variation in adult phenotypic traits is strongly influenced by growth conditions experienced by larvae. The main aim of this study was to assess how much environmental insight can be gained from analysis of the phenotypic changes in an insect's life history parameters in response to realistic food limitations. 2. This investigation was motivated by a need for more information about the developmental requirements of the endangered pine hoverfly Blera fallax (Linnaeus) (Diptera, Syrphidae) in Scotland. Blera fallax depends on a scarce and often ephemeral habitat, rot holes of Scots pine Pinus sylvestris L. stumps. We studied how rearing conditions affected growth in captive larvae, and compared these responses with a wild population. 3. The growth curve observed in the field was similar to that in resource‐limited, lab conditions, suggesting that resources are limiting in nature. The effects of resource availability on development time and body size depended on sex. Adult females were larger but had more variable size at maturity compared with males. In contrast, males typically were not smaller in resource‐limited conditions, but rather continued to develop for another year. Between 2% and 20% of larvae extended development over 2 years regardless of growth conditions, perhaps indicating a semivoltine strategy to circumvent extinction during years with a low breeding success. 4. These results identify life history traits that may be important for other saproxylic Diptera in rot holes, and organisms that experience food restrictions during growth.