Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.     

Genotype by environment interaction for growth of Eucalyptus globulus in Australia

This study assessed the genotype by environment (G × E) interaction for diameter growth in 15 Eucalyptus globulus progeny trials in Australia. Single-site analyses revealed significant subrace and family-within-subrace variance in all trials. Across-site subrace () and family () correlations were estimated by linear mixed model analyses of pairs of trials. Using a factor analytic structure for subrace and family random terms in a multi-environment mixed model analysis, best linear unbiased predictions of subrace effects were obtained for each trial. These were then averaged for each of four states (Victoria, Tasmania, South Australia and Western Australia) and across all sites. Statistically significant G × E interaction was detected, and weighted means across states for and were 0.73 and 0.76, respectively. Nevertheless, the three subraces from the Otway Ranges were both fast growing and relatively stable in their ranks over all sites. We evaluated the sensitivity of subraces to changing environmental conditions, on the basis of random coefficient models regressing subrace performance on selected trial climatic variables. The results suggested differential susceptibility of subraces to water, light and (to a less extent) temperature stresses during summer. Moreover, using multivariate techniques to visualize and interpret the across-site correlation structure for subrace effects, we could identify site clusters of reduced G × E interaction related to soil water availability and evaporative demand during summer. A revised site-type classification using these factors should allow a better capture of genetic gains from breeding and deployment.     

Genes invoked in the ovarian transition to menopause

Menopause and the associated declines in ovarian function are major health issues for women. Despite the widespread health impact of this process, the molecular mechanisms underlying the aging-specific decline in ovarian function are almost completely unknown. To provide the first gene–protein analysis of the ovarian transition to menopause, we have established and contrasted RNA gene expression profiles and protein localization and content patterns in healthy young and perimenopausal mouse ovaries. We report a clear distinction in specific mRNA and protein levels that are noted prior to molecular evidence of steroidogenic failure. In this model, ovarian reproductive aging displays similarities with chronic inflammation and increased sensitivity to environmental cues. Overall, our results indicate the presence of mouse climacteric genes that are likely to be major players in aging-dependent changes in ovarian function.     

Human cytomegalovirus UL84 protein contains two nuclear export signals and shuttles between the nucleus and the cytoplasm

Previous studies defined pUL84 of human cytomegalovirus as an essential regulatory protein with nuclear localization that was proposed to act during initiation of viral-DNA synthesis. Recently, we demonstrated that a complex domain of 282 amino acids within pUL84 functions as a nonconventional nuclear localization signal. Sequence inspection of this domain revealed the presence of motifs with homology to leucine-rich nuclear export signals. Here, we report the identification of two functional, autonomous nuclear export signals and show that pUL84 acts as a CRM-1-dependent nucleocytoplasmic shuttling protein. This suggests an unexpected cytoplasmic role for this essential viral regulatory protein.     

Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.     

Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions

Canonical Wnt/beta-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/beta-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/beta-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate beta-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/beta-catenin signaling.     

Potent and selective pyrazole-based inhibitors of B-Raf kinase

Herein we describe a novel pyrazole-based class of ATP competitive B-Raf inhibitors. These inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. A subset of these inhibitors has demonstrated the ability to inhibit downstream ERK phosphorylation in LOX tumors from mouse xenograft studies.     

Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets

We have developed a simplified, efficient approach for the 3D reconstruction and analysis of mammalian cells in toto by electron microscope tomography (ET), to provide quantitative information regarding ‘global’ cellular organization at 15–20 nm resolution. Two insulin-secreting beta cells—deemed ‘functionally equivalent’ by virtue of their location at the periphery of the same pancreatic islet—were reconstructed in their entirety in 3D after fast-freezing/freeze-substitution/plastic embedment in situ within a glucose-stimulated islet of Langerhans isolated intact from mouse pancreata. These cellular reconstructions have afforded several unique insights into fundamental structure–function relationships among key organelles involved in the biosynthesis and release of the crucial metabolic hormone, insulin, that could not be provided by other methods. The Golgi ribbon, mitochondria and insulin secretory granules in each cell were segmented for comparative analysis. We propose that relative differences between the two cells in terms of the number, dimensions and spatial distribution (and for mitochondria, also the extent of branching) of these organelles per cubic micron of cellular volume reflects differences in the two cells’ individual capacity (and/or readiness) to respond to secretagogue stimulation, reflected by an apparent inverse relationship between the number/size of insulin secretory granules versus the number/size of mitochondria and the Golgi ribbon. We discuss the advantages of this approach for quantitative cellular ET of mammalian cells, briefly discuss its application relevant to other complementary techniques, and summarize future strategies for overcoming some of its current limitations.