Seasonal adaptations of Daphnia pulicaria swimming behaviour: the effect of water temperature

Daphnia swimming behaviour is controlled by a variety of external factors, including light, presence of food and predators. Temperature represents a key driver in the dynamics of Daphnia populations, as well as on their motion. In this study, we have investigated the behavioural adaptations of adult Daphnia pulicaria to two different temperatures, representative of the mean winter (3°C) and summer (22°C) temperatures to which these organisms are exposed to in the real environment. Video observations were conducted both in the presence and in the absence of light to investigate possible day/night modifications in the motion strategy. Analyses of mean speed, velocity power spectral density and trajectory fractal dimension point out specific adaptations that allow D. pulicaria to successfully adjust to the changing conditions of the environment. Independently of the light conditions, in cold waters D. pulicaria swim almost vertically with defined motional frequencies, likely to increase the encounter with food items diluted in the fluid. A similar behaviour is displayed by the animals at summertime temperatures in the presence of light; however, in this case the vertical swimming is coupled with the absence of peaks in the power spectra and might be exploited to avoid predators. In contrast, at 22°C in dark conditions D. pulicaria move horizontally with lateral motions to take advantage of possible patches of phytoplankton. This information sheds new light into the complex and dynamic adaptations of D. pulicaria in response to external stimuli.     

Copepod grazing in turbulent flow: elevated foraging behavior and habituation of escape responses

When exposed to non-turbulent followed by turbulent conditions,the copepod Centropages hamatus initially responded with numerousescape reactions and increased foraging behavior. However, whenthe cycle of non-turbulent followed by turbulent flow was repeatedfor several consecutive cycles, the two behaviors followed distinctlydifferent patterns. Foraging effort increased during the firsttwo cycles, and then remained at high levels during both turbulentand non-turbulent periods (period durations of 12.5 and 25 min).In contrast, escape behavior habituated rapidly during eachturbulent period and dishabituated during each non-turbulentperiod. These response patterns are suited to the strongly intermittentnature of oceanic turbulence and allow C.hamatus to utilizethe benefits of enhanced encounter rate, while minimizing theexpense of unnecessary escape responses.     

Affinity labeling of steroid binding sites. Study of the active site of 20beta-hydroxysteroid dehydrogenase with 2alpha-bromoacetoxyprogesterone and 11alpha-bromacetoxyprogesterone.

To further characterize the active site of 20beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyced hydrogenans we synthesized 2alpha-bromoacetoxyprogesterone, a substrate for the enzyme in 0.05 M phosphate buffer at 25 degrees, pH 7.0, with Km and Vmax values of 1.90 X 10(-5) M and 6.09 nmol/min/mg of enzyme, respectively. This affinity labeling steroid inactivates 20beta-hydroxysteroid dehydrogenase in an irreversible and time-dependent manner which follows pseudo-first order kinetics with a t1/2 value of 4.6 hours. 2alpha-[2-3H]Bromoacetoxyprogesterone was synthesized and used to radiolabel the enzyme active site. Amino acid analysis of the acid hydrolysate of the radiolabeled enzyme supports a mechanism whereby the steroid moiety delivers the alkylating group to the steroid binding site of the enzyme where it reacts with a methionyl residue. Both 2alpha- and 11alpha-bromoacetoxyprogesterone alkylate a methionyl residue at the active site of 20beta-hydroxysteroid dehydrogenase. The enzyme was inactivated with a mixture containing both 2alpha-[2-3H]Bromoacetoxyprogesterone and 11alpha-2[2-14C]bromoacetoxyprogesterone. Following degradation of separate aliquots of the radiolabeled enzyme by cyanogen bromide or trypsin, the protein fragments were separated by gel filtration and ion exchange chromatography. Resolution of peptides carrying the 3H label from those possessing the 14C label demonstrates that 2alpha-bromoacetoxyprogesterone and 11alpha-bromoacetoxyprogesterone each label a different methionine at the steroid binding site of 20beta-hydroxysteroid dehydrogenase.     

Conservation of Glycerolated Cultures of Ochromonas danica and O. malhamensis at −10 C

SYNOPSIS. Ochromonas danica in a complex natural growth medium dies at 6–10 C in 4 days; O. malhamensis in ∼2 days. O. danica grown in the medium supplemented with 4.0% glycerol survived at −10±2 C for 35 days, and with 8% glycerol 29 days. O. malhamensis lasted only to 5 days in these media supplemented with 4% glycerol. Ethylene glycol and dimethylsulfoxide were too toxic to be effective. Difficulties in freeze-preservation of certain other phagocytic cells, notably blood granulocytes having comparatively simple flexuous outer membranes, add interest to use of O. danica and O. malhamensis as test organisms for preservation methods, especially in the convenient, inexpensive -10 to -20 C range. Biphasic media with an overlay of distilled water serve for conservation at room temperature. Problems of mutational erosion of these photosynthetic phagotrophs are discussed.     

Trypanosoma brucei: Nearest neighbor analysis on the major variable surface coat glycoprotein—Crosslinking patterns with intact cells

Cleavable Crosslinking reagents were used to study interactions among proteins of the surface coat of Trypanosoma brucei. The proteins were resolved by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When intact cells were treated with dithiobis(succinimidylpropionate), we obtained extensive intermolecular Crosslinking of major variable surface coat glycoprotein (VSCG) molecules. This reagent generated no apparent crosslinks between VSCG and other membrane-associated proteins. Complete conversion to oligomers equal to or greater than octamers occurred within 20 min. When purified VSCG in solution was treated with dithiobis(succimidylpropionate), dimers were found. A complex of Cu²⁺ and 1,10-phenanthroline was used to catalyze air oxidation of adjacent sulfhydryls to disulfide bonds; however, no crosslinking among VSCG molecules nor between VSCG and other proteins was observed. The results presented indicate that VSCG in solution exists predominately in the form of dimers. Whether VSCG in situ also occurred as dimers could not be determined; however, since we observed trimeric and tetrameric forms of VSCG when untreated cells were analyzed, it is likely that weak interactions occur among the protein molecules. These interactions are less stable than the dimer association observed with purified VSCG. Finally, the analysis indicated that VSCGs of this stock of T. brucei, derived from UGANDA/ 60/TREU/164[ETat3], contained at least one intramolecular disulfide bond. We examined T. brucei stocks 427 and EATRO 110 and obtained similar results. Thus, it appears that intramolecular disulfide bonding is a general feature of T. brucei VSCGs.     

Photoactivated zinc silicate in thin layer chromatography plates: A potential cause for error in liquid scintillation counting

Thin layer chromatography plates impregnated with fluorescent indicator permit easy identification of many compounds by exposure to ultraviolet light. Exposure of zinc silicate treated plates to ultraviolet light (254 nm) for 30 seconds induced photoluminescence which persisted at significant levels for longer than 24 hours. This artifact significantly interfered with radioactivity quantitation in three commercially available liquid scintillation solutions. Chromatography plates impregnated with calcium silicate demonstrated insignificant ultraviolet light induced photoluminescence. The use of ¹⁴C rather than ³H-radiolabeled compounds, protecting the plate with aluminum foil when visualizing reference compounds with ultraviolet light, and heating the photoactivated plate to accelerate disappearance of the photoluminescence minimized the error in liquid scintillation counting.     

To fuse or not to fuse: What is your purpose?

Since the dawn of time, or at least the dawn of recombinant DNA technology (which for many of today''s scientists is the same thing), investigators have been cloning and expressing heterologous proteins in a variety of different cells for a variety of different reasons. These range from cell biological studies looking at protein-protein interactions, post-translational modifications, and regulation, to laboratory-scale production in support of biochemical, biophysical, and structural studies, to large scale production of potential biotherapeutics. In parallel, fusion-tag technology has grown-up to facilitate microscale purification (pull-downs), protein visualization (epitope tags), enhanced expression and solubility (protein partners, e.g., GST, MBP, TRX, and SUMO), and generic purification (e.g., His-tags, streptag, and FLAG™-tag). Frequently, these latter two goals are combined in a single fusion partner. In this review, we examine the most commonly used fusion methodologies from the perspective of the ultimate use of the tagged protein. That is, what are the most commonly used fusion partners for pull-downs, for structural studies, for production of active proteins, or for large-scale purification? What are the advantages and limitations of each? This review is not meant to be exhaustive and the approach undoubtedly reflects the experiences and interests of the authors. For the sake of brevity, we have largely ignored epitope tags although they receive wide use in cell biology for immunopreciptation.     

Genetic differentiation between two host “races” and two species of cleptoparasitic bees and between their two hosts

In this paper we test the following two hypotheses: (1) that apparently conspecific samples of the cleptoparasitic beeCoelioxys funeraria, differing markedly in size and reared from different host species, do indeed represent one panmictic population; (2) that bees that nest in holes in wood or twigs have higher levels of genetic variation than those nesting in the ground. Based upon 41 loci, the genetic differences between the two samples ofC. funeraria could be explained entirely in terms of sampling error. In contrast, the sympatricC. moesta showed 16 fixed allelic differences from theC. funeraria samples. Similarly, the two hosts ofC. funeraria, Megachile relativa andM. inermis, had 21 fixed allelic differences between them out of 42 presumptive gene loci. Heterozygosities among the wood-nesting bees were not particularly high for Hymenoptera, ranging from 0.045 to 0.054. Comparisons of heterozygosity estimates among bees remain ambiguous as to whether soil nesting confers sufficient environmental buffering effects to reduce possible advantages of heterosis in ground-nesting species.     

Reactivation of human placental 17β, 20α-hydroxysteroid dehydrogenase: Affirmation of affinity labeling principles

Human placental 17β, 20α-hydroxysteroid dehydrogenase was completely inactivated by the affinity alkylator, 3-bromoacetoxy-1,3,5(10)-estratrien-17-one (estrone 3-bromoacetate). The inactivated enzyme was then reactivated to 100% of the enzyme activity by base-catalyzed hydrolysis of the steroidalester-enzyme conjugate. After the reactivated enzyme was repurified by dialysis, re-inactivation studies were performed on it. The reactivated enzyme could not be re-inactivated by the original alkylator, estrone 3-bromoacetate. However, 16α-bromoacetoxyestradiol-17β 3-methyl ether caused a loss of reactivated enzyme activity at a rate comparable to that for the native enzyme. These observations demonstrate that a specific amino acid modification within the enzyme active site was produced by estrone 3-bromoacetate alkylation and suggest that the conformation of the active center was essentially unaltered. Thus, these successful reactivation studies of 17β, 20α-hydroxysteroid dehydrogenase affirm the specificity of affinity labeling. This methodology also offers a new tool to investigate the steroid binding regions of macromolecular proteins.