Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pK_as of 14 ionizable residues. An all-atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pK_as in the protein, in contrast to multiple simulations for the free energy perturbation method. pK_int shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson-Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long-range interactions on the pK_int shifts is discussed. © 1994 Wiley-Liss, Inc.     

On the use of the quenching of luminol luminescence to evaluate sod activity

Addition of horseradish peroxidase to a luminol solution (pH = 9.4) produces a burst of light followed by a steady luminescence that lasts for several minutes. This steady-state luminescence is readily quenched by SOD, with a concentration (the additive concentration needed to decrease by one-half the emitted luminescence intensity) of c.a. 4 ng/ml (14 mU/ml). The luminescence intensity decrease can then be employed to evaluate SOD activity in SOD-containing samples. However, the light intensity can also be quenched by additives, such as Trolox, that are able to trap luminol-derived intermediates. It is proposed that double quenching experiments must be performed in order to be able to relate the observed effect of an additive to its SOD-like activity.     

Distribution of microtubules during the growth of tobacco pollen tubes

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α-, β- and tyrosinated α-tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer-assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.     

Environmental tracking by females

Human females are generally reserved in their sexuality, in keeping with their heavy investment in reproduction. Males tend to be less reserved. Relative to males, however, females demonstrate more variability in sexuality and are more likely to inhibit or express high levels of sexuality. The heightened variability may in part originate with genetic mechanisms that predispose females toward greater variability (the Lyon hypothesis). Menarche, menstrual cycles, menopause, food reactions, responses to living conditions, reactions to cultural factors, and responses to sexual stimuli and potential mates are unique to or are more variable among females than males. There is a correlation between the variation expressed and female reproductive potential—females tend to shift dramatically from sexual inhibition to sexual expression. Females apparently track the quality of the environment and link their sexuality to reproductive opportunities. Successful male reproduction depends less on quality environments and more on the availability of females. In short, females track the environment; males track the females.     

Study of β-1,3-glucanase activity during autolysis of Aspergillus nidulans by FPLC ion-exchange chromatography

The behaviour of β-1,3-glucanase activity during Aspergillus nidulans autolysis was studied in a basal medium and in the same medium supplemented with 0.5 g l^-1 of microcrystalline cellulose, laminarin, pectin, seedling of Lycopersicum esculentum extract, chitin and xylan respectively. In any case β-1,3-glucanase activity was detected in the culture fluid before the onset of the autolysis, but afterwards a progressive increase of β-1,3-glucanase activity took place with incubation time. In the media supplemented with pectin and seedling of Lycopersicum esculentum extract higher activity in the first days of autolysis was found. The activity at the end of the studied process by sample was 2.5, 2.1, 2.5, 1.9, 2.2, 2.3 and 2.3 U, and the specific activity 83, 53, 85, 55, 64, 90 and 53 mU mg^-1 of protein for each medium respectively. The β-1,3-glucanase activity in Aspergillus nidulans seems to be related to autolysis and not to the presence of different substances in the culture medium. The behaviour of β-1,3-glucanase activity during the degradative process was followed by FPLC ion-exchange chromatography. Three proteins (I, II, III) with β-1,3-glucanase activity were separated and quantified. These proteins have similar behaviour in all the media. Proteins I and II increase progressively with incubation time but protein III is only present at the first and last days of autolysis.     

Ecological and behavioral consequences of digestion in frugivorous animals

The postingestional effect of seed size and mass and nutrient composition on fruit profitability are reviewed. It is emphasized that profitability results from the interaction between fruit characteristics and the physiological and morphological traits of frugivores. The processes by which frugivores regurgitate or defecate seeds and the differential processing of the nutrient rich pulp and ballast in fruit are strongly dependent on the interaction between frugivore gut morphology and seed size. Euphonias that lack a functional gizzard defecate seeds, whereas tanagers that have a delete a functional gizzards regurgitate seeds. Some frugivores separate seeds from pulp and exocarp in the gizzard. It is hypothesized that the gizzard plays an important role in determining the postigestional fate of different pulp components. Although fruit nutrient content is often invoked as a determinant of frugivore feeding choices, studies that rely on proximal nutrient analysis, have often failed to find clear nutrient composition-preference correlations. It is argued that a partial reason for this failure is that proximal nutrient analysis ignores the complexities of fruit digestion. The ecological and physiological correlates of lipid and sugar assimilation are used to identify the limitations of traditional proximal nutrient analyses in fruit-frugivore studies. We suggest that recognizing the intricacies of the digestive characteristics of frugivores may reveal a much richer patterning in the interaction of frugivores with plants than has been previously hypothesized.     

The ability of rufous hummingbirds Selasphorus rufus to dilute and concentrate urine

Most terrestrial animals face the challenge of having to conserve water in a desiccating environment. Not surprisingly, the ability to produce concentrated urine has been relatively well studied in birds. Nectar-feeding birds are unusual among terrestrial animals in that they often ingest and excrete prodigious water volumes to obtain adequate energy. Thus, they confront the unusual challenge of having to conserve electrolytes. The diluting abilities of birds and the renal mechanisms that may correlate with them have been relatively neglected. To elucidate diluting and concentrating abilities in nectar-feeding birds, we fed rufous hummingbirds Selasphorus rufus an electrolyte-free nectar and a nectar containing a range of NaCl concentrations. Hummingbirds had a spectacular (and possibly unique) diluting ability: when fed on electrolyte-free food they produced excreta containing less than 0.5 mM l⁻¹ each of sodium and potassium. Hummingbirds also had a poor concentrating ability, retaining sodium and chloride when their food (0.632 M l⁻¹ sucrose) contained more than 35 mM l⁻¹ of NaCl. The kidneys of hummingbirds do not appear to be suited for concentrating urine, and possibly contain structural features that give them a unique diluting ability compared with those of birds that do not feed on nectar.     

Morphogenetic movements during axolotl neural tube formation tracked by digital imaging

During neurulation in vertebrate embryos, epithelial cells of the neural plate undergo complex morphogenetic movements that culminate in rolling of the plate into a tube. Resolution of the determinants of this process requires an understanding of the precise movements of cells within the epithelial sheet. A computer algorithm that allows automated tracking of epithelial cells visible in digitized video images is presented. It is used to quantify the displacement field associated with morphogenetic movements in the axolotl (Ambystoma mexicanum) neural plate during normal neural tube formation. Movements from lateral to medial, axial elongations and area changes are calculated from the displacement field data and plotted as functions of time. Regional and temporal differences are identified. The approach presented is suitable for analyzing a wide variety of morphogenetic movements.