Isolation and characterization of nine microsatellite loci from the Hawaiian grouper Epinephelus quernus (Serranidae) for population genetic analyses

The availability of variable genetic markers for groupers (Serranidae) has generally been limited to mitochondrial DNA. For studies of population genetic structure, more loci are usually required; particularly useful are those that are nuclear in origin such as microsatellites. Here, we isolated and characterized 9 microsatellite loci from the endemic Hawaiian grouper Epinephelus quernus using a biotin-labeled oligonucleotide-streptavidin-coated magnetic bead approach. Of the 20 repeat-containing fragments isolated, 15 had sufficient flanking region in which to design primers. Among these, 9 produced consistent polymerase chain reaction product, and 6 were highly variable. These 6 loci were all composed of dinucleotide repeats, with the number of alleles ranging from 6 to 18, and heterozygosities from 33.3% to 91.7%. The high levels of variability observed should make these markers useful for population genetic studies of E. quernus, and potentially other epinephelines.

Species richness in agamid lizards: chance, body size, sexual selection or ecology?

Why does species richness vary so greatly across lineages? Traditionally, variation in species richness has been attributed to deterministic processes, although it is equally plausible that it may result from purely stochastic processes. We show that, based on the best available phylogenetic hypothesis, the pattern of cladogenesis among agamid lizards is not consistent with a random model, with some lineages having more species, and others fewer species, than expected by chance. We then use phylogenetic comparative methods to test six types of deterministic explanation for variation in species richness: body size, life history, sexual selection, ecological generalism, range size and latitude. Of eight variables we tested, only sexual size dimorphism and sexual dichromatism predicted species richness. Increases in species richness are associated with increases in sexual dichromatism but reductions in sexual size dimorphism. Consistent with recent comparative studies, we find no evidence that species richness is associated with small body size or high fecundity. Equally, we find no evidence that species richness covaries with ecological generalism, latitude or range size.

Allowance for thermodynamic non-ideality in the characterization of protein self-association by frontal exclusion chromatography: hemoglobin revisited

This investigation re-examines theoretical aspects of the allowance for effects of thermodynamic non-ideality on the characterization of protein self-association by frontal exclusion chromatography, and thereby provides methods of analysis with greater thermodynamic rigor than those used previously. Their application is illustrated by reappraisal of published exclusion chromatography data for hemoglobin on the controlled-pore-glass matrix CPG-120. The equilibrium constant of 100/M that is obtained for dimerization of the (02 species by this means is also deduced from re-examination of published studies of concentrated hemoglobin solutions by osmotic pressure and sedimentation equilibrium methods. (C) 2003 Elsevier Science B.V. All rights reserved.

Quantum dynamical characterization of unimolecular resonances

We give a selective review of quantum mechanical methods for calculating and characterizing resonances in small molecular systems, with an emphasis on recent progress in Chebyshev and Lanczos iterative methods. Two archetypal molecular systems are discussed: isolated resonances in HCO, which exhibit regular mode and state specificity, and overlapping resonances in strongly bound HO2, which exhibit irregular and chaotic behavior. Future directions in this field are also discussed.

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

Systematic characterization of mutations in yeast acetohydroxyacid synthase: Interpretation of herbicide-resistance data

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.

Physicochemical and structural characterization of mesoporous aluminosilicates synthesized from leached saponite with additional aluminum incorporation

A thorough investigation was performed on the physical (mechanical, thermal, and hydrothermal stability) and chemical (ion exchange capacity and silanol number) characteristics of aluminosilicate FSMs, synthesized via a new successful short-time synthesis route using leached saponite and a low concentration of CTAB. Moreover, the influence of an additional Al incorporation, utilizing different aluminum sources, on the structure of the FSM derived from saponite is studied. A mesoporous aluminosilicate with a low Si/Al ratio of 12.8 is synthesized, and still has a very large surface area of 1130 m(2)/g and pore volume of 0.92 cm(3)/g. The aluminum-containing samples all have a high cation exchange capacity of around 1 mmol/9 while they still have a silanol number of about 0.9 OH/nm(2); both characteristics being interesting for high-yield postsynthesis modification reactions. Finally, a study is performed on the transformation of the aluminosilicates into their Bronsted acid form via the exchange with ammonium ions and a consecutive heat treatment.

Characterization of the acidic properties of mesoporous aluminosilicates synthesized from leached saponite with additional aluminum incorporation

The acidic properties of hexagonal mesoporous aluminosilicates synthesized via a new successful short time synthesis route using leached saponite and a low concentration of surfactant are thoroughly investigated. The resulting aluminosilicate mesoporous materials with high Si/Al ratios of around 11 have a maximal surface area of 1130 m(2)/g, a pore volume of 0.92 cm(3)/g, and a narrow pore size distribution at around 3 nm. The replacement of the sodium ions, present as counterions in the synthesized aluminosilicates, with protons imparts useful catalytic acidity. This acidity is extensively studied with FTIR spectroscopy after adsorption of ammonia and cyclohexylamine, while deuterated acetonitrile differentiates between Bronsted and Lewis acidity. Al-27 NMR spectroscopy determined the coordination of the aluminum in the FSM materials. Simultaneously the effect of an additional Al incorporation, utilizing sodium aluminate, aluminum nitrate, and aluminum isopropoxide is studied. From an acidic point of view, the incorporation with Al(NO3)(3) appears to be the most optimal, as the sample has a very high amount of acid sites (1.3 mmol/g). Investigating the nature of the acid sites it is found that in all samples except the one incorporated with Al(NO3)(3), more Bronsted than Lewis sites are present, both sites being quite acidic as they resist desorption temperatures up to 300 degreesC. Probing the coordination and location of the Al atoms, all the catalysts appeared to have mostly tetrahedral aluminum, up to 95% of the total Al amount for the proton exchanged AI(NO3)(3) incorporated sample.

Signal trait sexual dimorphism and mutual sexual selection in Drosophila serrata

The evolution of sexual dimorphism may occur when natural and sexual selection result in different optimum trait values for males and females. Perhaps the most prominent examples of sexual dimorphism occur in sexually selected traits, for which males usually display exaggerated trait levels, while females may show reduced expression of the trait. In some species, females also exhibit secondary sexual traits that may either be a consequence of a correlated response to sexual selection on males or direct sexual selection for female secondary sexual traits. In this experiment, we simultaneously measure the intersex genetic correlations and the relative strength of sexual selection on males and females for a set of cuticular hydrocarbons in Drosophila serrata. There was significant directional sexual selection on both male and female cuticular hydrocarbons: the strength of sexual selection did not differ among the sexes but males and females preferred different cuticular hydrocarbons. In contrast with many previous studies of sexual dimorphism, intersex genetic correlations were low. The evolution of sexual dimorphism in D. serrata appears to have been achieved by sex-limited expression of traits controlled by genes on the X chromosome and is likely to be in its final stages.

Characterization of E-cadherin endocytosis in isolated MCF-7 and Chinese hamster ovary cells - The initial fate of unbound E-cadherin

The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.

The development of chromatography for the characterization of protein interactions: a personal perspective

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

Sexual dysfunction in the Australian population

AIM: To describe the prevalence and forms of sexual dysfunction experienced by Australians, and compare these with people in the United States. METHODS: A cross sectional, telephone interview survey of a randomly selected sample of men (n = 876) and women (n = 908) aged between 18 and 59 years on the electoral roll in all states and territories of Australia (response rate = 61%) was conducted between November 1999 and April 2000. RESULTS: Large proportions of Australian men (55.0%) and women (60.5%) reported at least one sexual problem within the preceding year. More serious sexual dysfunction (> 3 symptoms) was observed in 13.2% of men and 19.7% of women. DISCUSSION: Australia and the USA are similar with regard to the high rate of symptoms of sexual dysfunction observed in the population. The low rate of treatment points to both patient and doctor reluctance to discuss sexual performance and practices.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.