Stimulatory effects of bradykinin on the ovulatory process in the in vitro-perfused rat ovary

The role of bradykinin in the ovulatory process was investigated using an in vitro-perfused rat ovary model. Stimulation with LH (0.1 micrograms/ml) resulted in 2.6 +/- 0.5 (mean +/- SEM) ovulations per ovary, whereas no ovulations occurred in the nonstimulated control group. Bradykinin (5 microM) added to the perfusion system hourly for 10 h induced 2 of 5 ovaries to ovulate, with 2 and 3 ovulations, respectively. When bradykinin (5 microM) was given as a single dose at 5 or 10 h after LH, the ovulation rate was significantly increased to 11.0 +/- 2.8 and 8.6 +/- 2.0 ovulations per ovary, respectively. A competitive bradykinin antagonist, phenylalanine bradykinin, inhibited the bradykinin-induced increase in LH-stimulated ovulations. The addition of LH, but not of bradykinin, increased the levels of prostaglandin endoperoxide synthase in granulosa cells, but the levels of the enzyme in the residual ovarian tissue were negligible. In contrast, prostacyclin synthase was predominantly located in the residual ovarian tissue. This enzyme was not affected by LH or bradykinin. LH increased the tissue levels of prostaglandins, predominantly prostaglandin E2 (PGE2), at 7 h, whereas the stimulatory effect of bradykinin was smaller, with a preferential increase in prostacyclin (prostaglandin I2) levels. This study indicates a modulatory role of bradykinin, possibly involving prostacyclin late in the ovulatory process, in the rat.     

The effect of heterochromatin on synapsis of the sex chromosomes of Peromyscus (Rodentia, Cricetidae)

The pairing behavior of the sex chromosomes in male and female individuals representing seven species of Peromyscus was analyzed by electron microscopy of silver-stained zygotene and pachytene configurations. Six species possess submetacentric or metacentric X chromosomes with heterochromatic short arms. Sex-chromosome pairing in these species is initiated during early pachynema at an interstitial position on the X and Y axes. Homologous synapsis then progresses in a unidirectional fashion towards the telomeres of the X short arm and the corresponding arm of the heterochromatic Y chromosome. The distinctive pattern of synaptic initiation allowed a late-synapsing bivalent in fetal oocytes to be tentatively identified as that of the X chromosomes. In contrast to the other species, Peromyscus megalops possesses an acrocentric X chromosome and a very small Y chromosome. Sex-chromosome pairing in this species is initiated at the proximal telomeric region during late zygonema, and then proceeds interstitially towards the distal end of the Y chromosome. These observations suggest that the presence of X short-arm heterochromatin and corresponding Y heterochromatin interferes with late-zygotene alignment of the pairing initiation sites, thereby delaying XY synaptic initiation until early pachynema. The pairing initiation sites are conserved in the vicinity of the X and Y centromeres in Peromyscus, and consequently the addition of heterochromatin during sex-chromosome evolution essentially displaces these sites to an interstitial position.     

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro.

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradykinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5 microM) on: 1) prostaglandin biosynthesis (PGE2, PGF2 alpha and 6-keto-PGF1 alpha), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1 microgram/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE2, and PGF2 alpha, (p less than or equal to 0.01) but not on 6-keto-PGF1 alpha synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE2, PGF2 alpha and 6-keto-PGF1 alpha. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast to LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.     

Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis

Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.     

Multilocus genealogies reveal multiple cryptic species and biogeographical complexity in the California turret spider Antrodiaetus riversi (Mygalomorphae, Antrodiaetidae)

Antrodiaetus riversi (Araneae, Antrodiaetidae) is a dispersal-limited, habitat specialized mygalomorph spider species endemic to mesic woodlands of northern and central California. This species occupies a disjunct distribution, with populations in the Sierra Nevada and Coast Ranges, separated by the inhospitable Central Valley. Previous studies of morphological and allozyme variation have suggested that these populations may constitute cryptic species. We investigated the phylogeography of A. riversi using both nuclear and mitochondrial DNA sequences, collected for a comprehensive population sample. These data reveal the presence of at least five species in the A. riversi complex - these species are deeply diverged, and genealogically exclusive in both nuclear and mitochondrial genomes. Each of these species is characterized by extreme population subdivision and deep phylogeographical structuring, consistent with minimal gene flow across the dissected Californian landscape. Three species are restricted to the Coast Ranges, one to high altitudes of the central Sierran Nevada, and one species is found in both ranges. These species have allopatric distributions, although species parapatry is hypothesized to occur in several areas. Species diversification appears to have pulsed in the Late Miocene/Early Pliocene, a timing consistent with biogeographical reconstructions for many Californian taxa, and a time of turbulent geological activity in the region.     

Quantitative assessment of the structural bias in protein-protein interaction assays

With recent publications of several large-scale protein-protein interaction (PPI) studies, the realization of the full yeast interaction network is getting closer. Here, we have analysed several yeast protein interaction datasets to understand their strengths and weaknesses. In particular, we investigate the effect of experimental biases on some of the protein properties suggested to be enriched in highly connected proteins. Finally, we use support vector machines (SVM) to assess the contribution of these properties to protein interactivity. We find that protein abundance is the most important factor for detecting interactions in tandem affinity purifications (TAP), while it is of less importance for Yeast Two Hybrid (Y2H) screens. Consequently, sequence conservation and/or essentiality of hubs may be related to their high abundance. Further, proteins with disordered structure are over-represented in Y2H screens and in one, but not the other, large-scale TAP assay. Hence, disordered regions may be important both in transient interactions and interactions in complexes. Finally, a few domain families seem to be responsible for a large part of all interactions. Most importantly, we show that there are method-specific biases in PPI experiments. Thus, care should be taken before drawing strong conclusions based on a single dataset.     

Comparative Analyses of Reproductive Structures in Harvestmen (Opiliones) Reveal Multiple Transitions from Courtship to Precopulatory Antagonism

Explaining the rapid, species-specific diversification of reproductive structures and behaviors is a long-standing goal of evolutionary biology, with recent research tending to attribute reproductive phenotypes to the evolutionary mechanisms of female mate choice or intersexual conflict. Progress in understanding these and other possible mechanisms depends, in part, on reconstructing the direction, frequency and relative timing of phenotypic evolution of male and female structures in species-rich clades. Here we examine evolution of reproductive structures in the leiobunine harvestmen or “daddy long-legs” of eastern North America, a monophyletic group that includes species in which males court females using nuptial gifts and other species that are equipped for apparent precopulatory antagonism (i.e., males with long, hardened penes and females with sclerotized pregenital barriers). We used parsimony- and Bayesian likelihood-based analyses to reconstruct character evolution in categorical reproductive traits and found that losses of ancestral gift-bearing penile sacs are strongly associated with gains of female pregenital barriers. In most cases, both events occur on the same internal branch of the phylogeny. These coevolutionary changes occurred at least four times, resulting in clade-specific designs in the penis and pregenital barrier. The discovery of convergent origins and/or enhancements of apparent precopulatory antagonism among closely related species offers an unusual opportunity to investigate how major changes in reproductive morphology have occurred. We propose new hypotheses that attribute these enhancements to changes in ecology or life history that reduce the duration of breeding seasons, an association that is consistent with female choice, sexual conflict, and/or an alternative evolutionary mechanism.     

An assessment of the life time biosynthesis potential of the male boll weevil

The four monoterpene compounds [1, (+)-cis-2-isopropenyl-1-methycyclobutaneethanol; II, (Z)-3,3-dimethyl-Δ^1,β-cyclohexaneethanol; III, (Z)-3,3-dimethyl-Δ^1,α-cyclohexaneacetaldehyde; and IV, (E)-3,3-dimethyl-Δ^1,α-cyclohexaneacetaldehyde] which comprise the pheromone of the male boll weevil, Anthonomus grandis, were determined daily throughout the lifespan of the insect by GLC analysis of the whole body and the frass. The total content of the components in the male is never greater than 210 ng, the average content in the frass produced during 1 day is 1268 ng, and the lifetime production is at least 40,000 ng. The ratio of the four components in frass is 6 : 6 : 2 : 1/I : II : III : IV. None of the four components was found in females, but female frass contained traces in three instances. These data can be used to compare the potential attractancy of insects from various geographical areas, at different seasons, in different nutritional states, and after chemosterilization. Such differences in potential insect attractiveness could be crucial to effective large-scale control programmes.