Functional characterization of a Kar3/Vik1-like Kinesin-14 heterodimer from the filamentous multinucleate fungus Ashbya gossypii

Kinesins are motor proteins that convert chemical energy from ATP hydrolysis into mechanical energy used to generate force along microtubules, transporting organelles, vesicles, and proteins within the cell. Kar3 kinesins are microtubule minus-end-directed motors with pleiotropic functions in mating and mitosis of budding and fission yeast. In Saccharomyces cerevisiae, Kar3 is multifunctionalized by two non-catalytic companion proteins, Vik1 and Cik1. A Kar3-like kinesin and a single Vik1/Cik1 ortholog are also expressed by the filamentous fungus Ashbya gossypii, which exhibits different nuclear movement challenges and unique microtubule dynamics from its yeast relatives. We hypothesized that these differences in A. gossypii physiology could translate into interesting and novel differences in its versions of Kar3 and Vik1/Cik1. Presented here is a structural and functional analysis of recombinantly expressed and purified forms of these motor proteins. Compared to the previously published S. cerevisiae Kar3 motor domain structure (ScKar3MD), AgKar3MD displays differences in the conformation of the ATPase pocket. Perhaps it is not surprising then that we observed the maximal microtubule-stimulated ATPase rate (kcat) of AgKar3MD to be approximately 3-fold slower than ScKar3MD, and that the affinity of AgKar3MD for microtubules (Kd,MT) was lower than ScKar3MD. This may suggest that elements that compose the ATPase pocket and that participate in conformational changes required for efficient ATP hydrolysis or products release work differently for AgKar3 and ScKar3. There are also subtle structural differences in the disposition of the secondary structural elements in the small lobe (B1a, B1b, and B1c) at the edge of the motor domain of AgKar3 that may reflect the enhanced microtubule-depolymerization activity that we observed for this motor, or they could relate to its interactions with a different regulatory companion protein than its budding yeast counterpart. Although we were unable to gain experimentally determined high-resolution information of AgVik1, the results of Phyre2-based bioinformatics analyses may provide a structural explanation for the limited microtubule-binding activity we observed. These and other fundamental differences in AgKar3/Vik1 could explain divergent functionalities from the ScKar3/Vik1 and ScKar3/Cik1 motor assemblies.

Terminal nerve-derived neuropeptide Y modulates physiological responses in the olfactory epithelium of hungry axolotls (Ambystoma mexicanum).

The vertebrate brain actively regulates incoming sensory information, effectively filtering input and focusing attention toward environmental stimuli that are most relevant to the animal's behavioral context or physiological state. Such centrifugal modulation has been shown to play an important role in processing in the retina and cochlea, but has received relatively little attention in olfaction. The terminal nerve, a cranial nerve that extends underneath the lamina propria surrounding the olfactory epithelium, displays anatomical and neurochemical characteristics that suggest that it modulates activity in the olfactory epithelium. Using immunocytochemical techniques, we demonstrate that neuropeptide Y (NPY) is abundantly present in the terminal nerve in the axolotl (Ambystoma mexicanum), an aquatic salamander. Because NPY plays an important role in regulating appetite and hunger in many vertebrates, we investigated the possibility that NPY modulates activity in the olfactory epithelium in relation to the animal's hunger level. We therefore characterized the full-length NPY gene from axolotls to enable synthesis of authentic axolotl NPY for use in electrophysiological experiments. We find that axolotl NPY modulates olfactory epithelial responses evoked by L-glutamic acid, a food-related odorant, but only in hungry animals. Similarly, whole-cell patch-clamp recordings demonstrate that bath application of axolotl NPY enhances the magnitude of a tetrodotoxin-sensitive inward current, but only in hungry animals. These results suggest that expression or activity of NPY receptors in the olfactory epithelium may change with hunger level, and that terminal nerve-derived peptides modulate activity in the olfactory epithelium in response to an animal's changing behavioral and physiological circumstances.

A functionally atypical amidating enzyme from the human parasite Schistosoma mansoni

Many neuropeptide transmitters require the presence of a carboxy-terminal alpha-amide group for biological activity. Amidation requires conversion of a glycine-extended peptide intermediate into a C-terminally amidated product. This post-translational modification depends on the sequential action of two enzymes (peptidylglycine alpha-hydroxylating monooxygenase or PHM, and peptidyl-alpha-hydroxyglycine alpha-amidating lyase or PAL) that in most eukaryotes are expressed as separate domains of a single protein (peptidylglycine alpha-amidating monooxygenase or PAM). We identified a cDNA encoding PHM in the human parasite Schistosoma mansoni. Transient expression of schistosome PHM (smPHM) revealed functional properties that are different from other PHM proteins; smPHM displays a lower pH-optimum and, when expressed in mammalian cells, is heavily N-glycosylated. In adult worms, PHM is found in the trans-Golgi network and secretory vesicles of both central and peripheral nerves. The widespread occurrence of PHM in the nervous system confirms the important role of amidated neuropeptides in these parasitic flatworms. The differences between schistosome and mammalian PHM suggest that it could be a target for new chemotherapeutics.

Ultraviolet oscillations in the chromosphere of the quiet Sun

Quiet-Sun oscillations in the four Transition Region and Coronal Explorer (TRACE) ultraviolet passbands centered on 1700, 1600, 1216, and 1550 Angstrom are studied using a wavelet-based technique. Both network and internetwork regions show oscillations with a variety of periods and lifetimes in all passbands. The most frequent network oscillation has a period of 283 s, with a lifetime of 2-3 cycles in all passbands. These oscillations are discussed in terms of upwardly propagating magnetohydrodynamic wave models. The most frequent internetwork oscillation has a period of 252 s, again with a lifetime of 2-3 cycles, in all passbands. The tendency for these oscillations to recur in the same position is discussed in terms of "persistent flashers." The network contains greater oscillatory power than the internetwork at periods longer than 300 s in the low chromosphere. This value is shown to decrease to 250 s in the high chromosphere. The internetwork also displays a larger number of short-lifetime, long-period oscillations than the network, especially in the low chromosphere. Both network and internetwork regions contain a small number of nonrecurring long-lifetime oscillations.

More nitrogen rich B-type stars in the SMC cluster, NGC 330

High resolution spectra of seven early B-type giant/supergiant stars in the SMC cluster NGC330 are analysed to obtain their chemical compositions relative to SMC field and Galactic B-type stars. It is found that all seven stars are nitrogen rich with an abundance approximately 1.3 dex higher than an SMC main- sequence field B-type star, AV304. They also display evidence for deficiencies in carbon, but other metals have abundances typical of the SMC. Given the number of B-type stars with low projected rotational velocities in NGC330 (all our targets have v sin i <50 km s(-1)), we suggest that it is unlikely that the stars in our sample are seen almost pole-on, but rather that they are intrinsically slow rotators. Furthermore, none of our objects displays any evidence of significant Balmer emission excluding the possibility that these are Be stars observed pole-on. Comparing these results with the predictions of stellar evolution models including the effects of rotationally induced mixing, we conclude that while the abundance patterns may indeed be reproduced by these models, serious discrepancies exist. Most importantly, models including the effects of initially large rotational velocities do not reproduce the observed range of effective temperatures of our sample, nor the currently observed rotational velocities. Binary models may be able to produce stars in the observed temperature range but again may be incapable of producing suitable analogues with low rotational velocities. We also discuss the clear need for stellar evolution calculations employing the correct chemical mix of carbon, nitrogen and oxygen for the SMC.

Kinetics of phase transformations in a model with metastable fluid-fluid separation: A molecular dynamics study

By molecular dynamics (MD) simulations we study the crystallization process in a model system whose particles interact by a spherical pair potential with a narrow and deep attractive well adjacent to a hard repulsive core. The phase diagram of the model displays a solid-fluid equilibrium, with a metastable fluid-fluid separation. Our computations are restricted to fairly small systems (from 2592 to 10368 particles) and cover long simulation times, with constant energy trajectories extending up to 76x10(6) MD steps. By progressively reducing the system temperature below the solid-fluid line, we first observe the metastable fluid-fluid separation, occurring readily and almost reversibly upon crossing the corresponding line in the phase diagram. The nucleation of the crystal phase takes place when the system is in the two-fluid metastable region. Analysis of the temperature dependence of the nucleation time allows us to estimate directly the nucleation free energy barrier. The results are compared with the predictions of classical nucleation theory. The critical nucleus is identified, and its structure is found to be predominantly fcc. Following nucleation, the solid phase grows steadily across the system, incorporating a large number of localized and extended defects. We discuss the relaxation processes taking place both during and after the crystallization stage. The relevance of our simulation for the kinetics of protein crystallization under normal experimental conditions is discussed. (C) 2002 American Institute of Physics.

Biotransformation of maximakinin, a bradykinin-related nonadecapeptide from toad venom, by mammalian kallikrein and salivary proteases

Maximakinin is an N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the venom of a Chinese toad (Bombina maxima) that displays highly selective activity at mammalian arterial smooth muscle receptors. In this study, we report that incubation of maximakinin with either kallikrein or human saliva generates catabolites with enhanced bioactivity that retain the tissue selective effects of the parent molecule. In addition, we have observed that kallikrein rapidly cleaves the C-terminal arginyl residue of both maximakinin and bradykinin – a cleavage hitherto considered to be performed by a carboxypeptidase that facilitates selective bradykinin receptor targeting. Maximakinin has thus evolved as a `smart' defensive weapon in the toad with inherent resistance to the signal-terminating protease hardware in the potential predator. Thus, natural selection of amphibian skin peptides for antipredator defence, through interspecies delivery by an exogenous secretory mode, produces subtle structural stabilization modifications that can potentially provide new insights for the design of orally active and selectively targeted peptide therapeutics.

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Novel Porphyrin-Incorporated Hydrogels for Photoactive Intraocular Lens Biomaterials

Novel surface-modified hydrogel materials have been prepared by binding charged porphyrins TMPyP (tetrakis-(4-N-methylpyridyl)porphyrin) and TPPS (tetrakis(4-sulfonatophenyl)porphyrin) to copolymers of HEMA (2-hydroxyethyl methacrylate) with either MAA (methacrylic acid) or DEAEMA (2-(diethylamino)ethylmethacrylate). The charged hydrogels display strong electrostatic interactions with the appropriate cationic or anionic porphyrins to give materials which are intended to be used to generate cytotoxic singlet oxygen (1O2) on photoexcitation and can therefore be used to reduce postoperative infection of the intraocular hydrogel-based replacement lenses that are used in cataract surgery. The UV/vis spectra of TMPyP in MAA:HEMA copolymers showed a small shift in the Soret band and a change from single exponential (161 ���­s) triplet decay lifetime in solution to a decay that could be fitted to a biexponential fit with two approximately equal components with ���´ ) 350 and 1300 ���­s. O2 bubbling reduced the decay to a dominant (90%) component with a much reduced lifetime of 3 ���­s and a minor, longer lived (20 ���­s) component. With D2O solvent the 1O2 lifetime was measured by 1270 nm fluorescence as 35 ���­s in MAA:HEMA, compared to 67 ���­s in solution, although absorbance-matched samples showed similar yield of 1O2 in the polymers and in aqueous solution. In contrast to the minor perturbation in photophysical properties caused by binding TMPyP to MAA:HEMA, TPPS binding to DEAEMA:HEMA copolymers profoundly changed the 1O2 generating ability of the TPPS. In N2-bubbled samples, the polymer-bound TPPS behaved in a similar manner to TMPyP in its copolymer host; however, O2 bubbling had only a very small effect on the triplet lifetime and no 1O2 generation could be detected. The difference in behavior may be linked to differences in binding in the two systems. With TMPyP in MAA:HEMA, confocal fluorescence microscopy showed significant penetration of the porphyrin into the core of the polymer film samples (>150 ���­m). However, for TPPS in DEAEMA:HEMA copolymers, although the porphyrin bound much more readily to the polymer, it remained localized in the first 20 ���­m, even in heavily loaded samples. It is possible that the resulting high concentration of TPPS may have cross-linked the hydrogels to such an extent that it significantly reduced the solubility and/or diffusion rate of oxygen into the doped polymers. This effect is significant since it demonstrates that even simple electrostatic binding of charged porphyrins to hydrogels can have an unexpectedly large effect on the properties of the system as a whole. In this case it makes the apparently promising TPPS/DEAEMA:HEMA system a poor candidate for clinical application as a postoperative antibacterial treatment for intraocular lenses while the apparently equivalent cationic system TMPyP/MAA:HEMA displays all the required properties.

Studies on polypyrrole film in room temperature melt

A bluish-black shining free standing polypyrrole film (PPy) of electronic conductivity 130 S cm-1 has been prepared by electrochemical oxidative polymerization of pyrrole on Pt/transparent glass conducting electrode resistance 15 O cm-1, using a room temperature melt as an electrolyte, composed of 1:3 stoichiometric ratio of cetyl pyridinium chloride and anhydrous aluminum chloride at 0.58 V versus Al wire as a reference electrode. The film possessed a charge transfer resistance of 132 O, and showed two absorption peaks at 457 and 1264 nm in the UV-vis–NIR diffused reflectance spectra. The morphology of the film was hexagonal. The potential step technique suggested a layered structure. This thin film can easily be peeled off from the electrode surface after three cycles and can be used for various applications like dissipation of electrostatic charge, battery electrode materials, solid electrolytic capacitor, electrochromic windows and displays, microactuators etc. It was also characterized by IR, thermal and SEM studies.

Ion Association in [bmim][PF6]/Naphthalene Mixtures: An Experimental and Computational Study

Mixtures of room temperature ionic liquids (IL) with neutral organic molecules provide a valuable testing ground to investigate the interplay of the ionic and molecular-dipolar state in dense Coulomb systems at near ambient conditions. In the present study, the viscosity eta and the ionic conductivity a of 1-n-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6])/naphthalene mixtures at T = 80 degrees C have been measured at 10 stoichiometries spanning the composition range from pure naphthalene to pure [bmim][PF6]. The viscosity grows nearly monotonically with increasing IL mole fraction (x), whereas the conductivity per ion displays a clear peak at x approximate to 15%. The origin of this maximum has been investigated using molecular dynamics simulations based on a classical force field. Snapshots of the simulated samples show that the conductivity maximum is due to the gradual transition in the IL component from an ionic state at high x to a dipolar fluid made of neutral ion pairs at low x. At concentrations x <0.20 the ion pairs condense into molecular-thin filaments bound by dipolar forces and extending in between nanometric droplets of IL. These results are confirmed and complemented by the computation of dynamic and transport properties in [bmim][PF6]/naphthalene mixtures at low IL concentration.

Evolution of the nervous system in Paraphanostoma (Acoela)

According to recent molecular studies, the Acoela are the earliest extant bilaterian group. Their nervous system displays a striking variety of patterns. The aim of the present investigation was to study the variability of the nervous system in a monophyletic group of the Acoela. Six species of Paraphanostoma were chosen for the study. Using immunocytochemical methods and confocal scanning laser microscopy, the immunoreactive patterns of serotonin (5-HT) and the neuropeptide GYIRFamide were described in detail. The study has demonstrated that the brains in Paraphanostoma species, although diverse in detail, still follow the same general pattern. 18S rDNA sequences were used to generate a hypothesis of the phylogeny within the group. Characters of the nervous system revealed in this study were coded and analysed together with 18S rDNA data. Several synapomorphies in the nervous system characters were identified. However, numerous parallelisms in the nervous system evolution have occurred. Data obtained demonstrate that the genus Paraphanostoma is closely related to Childia and should belong to the same family, Childiidae.

The importance of reproductive strategies in population genetic approaches to conservation: an example from the marine angiosperm genus Zostera

Knowledge of the levels of genetic diversity maintained in natural populations can play a central role in conservation programmes, particularly in threatened habitats or species. Fluctuations in population size can lead to loss of variation and, consequently, increase the risk of extinction. We have examined whether such a genetic bottleneck has occurred in populations of two species in the seagrass genus Zostera, which are believed to have been affected by an outbreak of wasting disease at the start of the last century. A test for heterozygote excess at five nuclear microsatellite loci did not suggest the occurrence of a genetic bottleneck, but analysis of seven chloroplast microsatellite loci and sequence data from two regions did suggest a bottleneck in the chloroplast genome. Extremely low levels of between-population diversity suggest that all subpopulations can be treated as a single management unit for each species. Comparable levels of nuclear genetic diversity were found in the three populations of the primarily sexual Zostera marina var. angustifolia studied but a wider range of within-population diversity was found in Zostera noltii, which displays both. sexual and vegetative reproductive strategies. This may be due to an increase in sexual recruitment due to localised fresh water inflow into the study site near to the most diverse population. Such populations should be prioritised as source material for any replanting or remediation due to natural or anthropogenic loss of Zostera beds in the area.

The dichotomy in the DNA-binding behaviour of ruthenium(II) complexes bearing benzoxazole and benzothiazole groups

The substituted tris(bipyridine)ruthenium(II) complexes {[Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) [where bpy = 2,2'-bipyridine and bbob = bis(benzoxazol-2-yl)-2,2'-bipyridine] have been prepared and compared to the previously studied complex [Ru(bpy)(2)(4,4'-bbtb)](2+) [where bbtb = bis(benzothiazol-2-yl)-2,2'-bipyridine]. From the UV/VIS titration studies, Delta-[Ru(bpy)(2)(4,4'bbob)](2+) displays a stronger association than the Lambda-isomer with calf-thymus DNA (ct-DNA). For [Ru(bpy)(2)(5,5'-bbob)](2+), there appears to be minimal interaction with ct-DNA. The results of fluorescence titration studies suggest that [Ru(bpy)(2)(4,4'-bbob)](2+) gives an increase in emission intensity with increasing ct-DNA concentrations, with an enantiopreference for the A isomer, confirmed by membrane dialysis studies. The fluorescent intercalation displacement studies revealed that [Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru.(bpy)(2)(5,5'bbob)](2+) display a preference for more open DNA structures such as bulge and hairpin sequences. While Delta-[Ru(bpy)(2)(4,4'-bbtb)](2+) has shown the most significant affinity for all the oligonucleotides sequences screened in previous studies, it is the A isomer of the comparable benzoxazole ruthenium(II) complex (Delta-[Ru(bpy)(2)(4,4'-bbob)](2+)) that preferentially binds to DNA.