Teleost fish spermatozoa contain a cytosolic protein factor that induces calcium release in sea urchin egg homogenates and triggers calcium oscillations when injected into mouse oocytes

Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCgamma). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCzeta). Homologues of PLCzeta have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.     

High-throughput expression,purification, and characterization of recombinant Caenorhabditis elegans proteins

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.     

Testing of the additivity-based protein sequence to reactivity algorithm

The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.     

Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin

The sodium/proton exchanger type 1 (NHE-1) plays an important role in the proliferation of vascular smooth muscle cells (VSMC). We have examined the regulation of NHE-1 by two potent mitogens, serotonin (5-HT, 5-hydroxytryptamine) and angiotensin II (Ang II), in cultured VSMC derived from rat aorta. 5-HT and Ang II rapidly activated NHE-1 via their G protein-coupled receptors (5-HT(2A) and AT(1)) as assessed by proton microphysiometry of quiescent cells and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Activation of NHE-1 was blocked by inhibitors of phospholipase C, CaM, and Jak2 but not by pertussis toxin or inhibitors of protein kinase C. Immunoprecipitation/immunoblot studies showed that 5-HT and Ang II induce phosphorylation of Jak2 and induce the formation of signal transduction complexes that included Jak2, CaM, and NHE-1. The cell-permeable Ca(2+) chelator BAPTA-AM blocked activation of Jak2, complex formation between Jak2 and CaM, and tyrosine phosphorylation of CaM, demonstrating that elevated intracellular Ca(2+) is essential for those events. Thus, mitogen-induced activation of NHE-1 in VSMC is dependent upon elevated intracellular Ca(2+) and is mediated by the Jak2-dependent tyrosine phosphorylation of CaM and subsequent increased binding of CaM to NHE-1, similar to the pathway previously described for the bradykinin B(2) receptor in inner medullary collecting duct cells of the kidney [Mukhin, Y. V., et al. (2001) J. Biol. Chem. 276, 17339-17346]. We propose that this pathway represents a fundamental mechanism for the rapid regulation of NHE-1 by G(q/11) protein-coupled receptors in multiple cell types.     

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.     

Regulated expression of osteopontin in the peri-implantation rabbit uterus

Blastocyst attachment to the lining of the mammalian uterus during early implantation involves the initial apposition of the trophoblast to the uterine epithelial surface. Osteopontin (OPN) is a glycoprotein component of the extracellular matrix that is secreted by the glandular epithelium of mammalian uteri at the time of implantation. This protein is recognized by several members of the integrin family and promotes cell-cell attachment and adhesion. In the present study, rabbit uteri were examined using Northern and in situ hybridization to evaluate the temporal and spatial distribution of OPN mRNA during early pregnancy. Northern blot analysis demonstrated a dramatic increase in OPN expression on Days 4-7 of pregnancy, corresponding to the rise in circulating progesterone and the time of initial embryo attachment in this species. In situ hybridization analysis revealed OPN mRNA expression on Day 6.75 of pregnancy, which was most prominent on endometrial epithelium. Using immunofluorescence, OPN protein was present on the glandular epithelium on Day 6.75 of pregnancy, but was absent on blastocysts. Further, no expression of OPN mRNA or protein was found in the nonpregnant endometrium. Induction of endometrial OPN expression was observed in unmated rabbits treated with progesterone alone and was prevented by cotreatment with the antiprogestin ZK137.316. Estradiol-17beta had no effect on OPN expression by itself, and estrogen priming was not necessary to demonstrate the stimulatory effect of progesterone. In The rabbit uterus, as in other mammalian species studied, OPN is expressed in a stage-specific manner by the endometrial glands during the peri-implantation period and is regulated by progesterone.     

Testing for spatial correlation in nonstationary binary data, with application to aberrant crypt foci in colon carcinogenesis

In an experiment to understand colon carcinogenesis, all animals were exposed to a carcinogen, with half the animals also being exposed to radiation. Spatially, we measured the existence of what are referred to as aberrant crypt foci (ACF), namely, morphologically changed colonic crypts that are known to be precursors of colon cancer development. The biological question of interest is whether the locations of these ACFs are spatially correlated: if so, this indicates that damage to the colon due to carcinogens and radiation is localized. Statistically, the data take the form of binary outcomes (corresponding to the existence of an ACF) on a regular grid. We develop score-type methods based upon the Matern and conditionally autoregressive (CAR) correlation models to test for the spatial correlation in such data, while allowing for nonstationarity. Because of a technical peculiarity of the score-type test, we also develop robust versions of the method. The methods are compared to a generalization of Moran's test for continuous outcomes, and are shown via simulation to have the potential for increased power. When applied to our data, the methods indicate the existence of spatial correlation, and hence indicate localization of damage.     

Effects of the eukaryotic pore-forming cytolysin Equinatoxin II on lipid membranes and the role of sphingomyelin

Equinatoxin II (EqtII), a protein toxin from the sea anemone Actinia equina, readily creates pores in sphingomyelin-containing lipid membranes. The perturbation by EqtII of model lipid membranes composed of dimyristoylphosphatidycholine and sphingomyelin (10 mol %) was investigated using wideline phosphorus-31 and deuterium NMR. The preferential interaction between EqtII (0.1 and 0.4 mol %) and the individual bilayer lipids was studied by (31)P magic angle spinning NMR, and toxin-induced changes in bilayer morphology were examined by freeze-fracture electron microscopy. Both NMR and EM showed the formation of an additional lipid phase in sphingomyelin-containing mixed lipid multilamellar suspensions with 0.4 mol % EqtII. The new toxin-induced phase consisted of small unilamellar vesicles 20-40 nm in diameter. Deuterium NMR showed that the new lipid phase contains both dimyristoylphosphatidycholine and sphingomyelin. Solid-state (31)P NMR showed an increase in spin-lattice and a decrease in spin-spin relaxation times in mixed-lipid model membranes in the presence of EqtII, consistent with an increase in the intensity of low frequency motions. The (2)H and (31)P spectral intensity distributions confirmed a change in lipid mobility and showed the creation of an isotropic lipid phase, which was identified as the small vesicle structures visible by electron microscopy in the EqtII-lipid suspensions. The toxin appears to enhance slow motions in the membrane lipids and destabilize the membrane. This effect was greatly enhanced in sphingomyelin-containing mixed lipid membranes compared with pure phosphatidylcholine bilayers, suggesting a preferential interaction between the toxin and bilayer sphingomyelin.