The Measurement of Sexual Selection Using Bateman's Principles: An Experimental Test in the Sex-Role-Reversed Pipefish Syngnathus typhle

Angus J. Bateman's classic study of sexual selection in Drosophila melanogaster has had a major influence on the development of sexual selection theory. In some ways, Bateman's study has served a catalytic role by stimulating debate on sex roles, sexual conflict and other topics in sexual selection. However, there is still considerable disagreement regarding whether or not "Bateman's principles" are helpful in the study of sexual selection. Here, we test the idea that Bateman's principles provide the basis for a useful method to quantify and compare mating systems. In this study, we focus on the sex-role-reversed pipefish Syngnathus typhle as a model system to study the measurement of sexual selection. We set up artificial breeding assemblages of pipefish in the laboratory and used microsatellite markers to resolve parentage. Three different sex-ratio treatments (female-biased, even and male-biased) were used to manipulate the expected intensity of sexual selection. Measures of the mating system based on Bateman's principles were calculated and compared to the expected changes in the intensity of sexual selection. We also compare the results of this study to the results of a similar study of Bateman's principles in the rough-skinned newt, a species with conventional sex roles. The results of this experiment show that measures of the mating system based on Bateman's principles do accurately capture the relative intensities of sexual selection in the different treatments and species. Thus, widespread use of Bateman's principles to quantify mating systems in nature would facilitate comparative studies of sexual selection and mating system evolution.     

Calcium triggers folding of lipoprotein lipase into active dimers

The active form of lipoprotein lipase (LPL) is a noncovalent homodimer of 55-kDa subunits. The dimer is unstable and tends to undergo irreversible dissociation into inactive monomers. We noted that a preparation of such monomers slowly regained traces of activity under assay conditions with substrate, heparin, and serum or in cell culture medium containing serum. We therefore studied the refolding pathway of LPL after full denaturation in 6 M guanidinium chloride or after dissociation into monomers in 1 M guanidinium chloride. In crude systems, we identified serum as the factor promoting reactivation. Further investigations demonstrated that Ca2+ was the crucial component in serum for reactivation of LPL and that refolding involved at least two steps. Studies of far-UV circular dichroism, fluorescence, and proteolytic cleavage patterns showed that LPL started to refold from the C-terminal domain, independent of calcium. The first step was rapid and resulted in formation of an inactive monomer with a completely folded C-terminal domain, whereas the N-terminal domain was in the molten globule state. The second step was promoted by Ca2+ and converted LPL monomers from the molten globule state to dimerization-competent and more tightly folded monomers that rapidly formed active LPL dimers. The second step was slow, and it appears that proline isomerization (rather than dimerization as such) is rate-limiting. Inactive monomers isolated from human tissue recovered activity under the influence of Ca2+. We speculate that Ca2+-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity.     

Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.     

Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase

We investigated why treatment of cells with dual aurora A and B kinase inhibitors produces phenotypes identical to inactivation of aurora B. We found that dual aurora kinase inhibitors in fact potently inhibit cellular activities of both kinases, indicating that inactivation of aurora B bypasses aurora A in mitosis. RNAi experiments further established that inactivation of aurora B indeed bypasses the requirement for aurora A and leads to polyploidy. Inactivation of aurora A activates checkpoint kinase BubR1 in an aurora B-dependent manner. Our results thus show that aurora B is responsible for mitotic arrest in the absence of aurora A.     

Complexes of horseradish peroxidase with formate, acetate, and carbon monoxide

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the "hydrated" form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP-acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463-468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron-ligand distances of 2.3 and 1.8 A, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 A from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.     

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.     

Molecular determinants of vanilloid sensitivity in TRPV1

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.