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Gram-negative bacteria secrete proteins using a type III secretion system (T3SS), which functions as a needle-like molecular machine. The many proteins involved in T3SS construction are tightly regulated due to its role in pathogenesis and motility. Here, starting with the 35 kb Salmonella pathogenicity island 1 (SPI-1), we eliminated internal regulation and simplified the genetics by removing or recoding genes, scrambling gene order and replacing all non-coding DNA with synthetic genetic parts. This process results in a 16 kb cluster that shares no sequence identity, regulation or organizational principles with SPI-1. Building this simplified system led to the discovery of essential roles for an internal start site (SpaO) and small RNA (InvR). Further, it can be controlled using synthetic regulatory circuits, including under SPI-1 repressing conditions. This work reveals an incredible post-transcriptional robustness in T3SS assembly and aids its control as a tool in biotechnology.

Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genetic memory can be implemented using enzymes that catalyze DNA inversions, where each orientation corresponds to a "bit". Here, we use two DNA invertases (FimE and HbiF) that reorient DNA irreversibly between two states with opposite directionality. First, we construct memory that is set by FimE and reset by HbiF. Next, we build a NOT gate where the input promoter drives FimE and in the absence of signal the reverse state is maintained by the constitutive expression of HbiF. The gate requires ∼3 h to turn on and off. The evolutionary stabilities of these circuits are measured by passaging cells while cycling function. The memory switch is stable over 400 h (17 days, 14 state changes); however, the gate breaks after 54 h (>2 days) due to continuous invertase expression. Genome sequencing reveals that the circuit remains intact, but the host strain evolves to reduce invertase expression. This work highlights the need to evaluate the evolutionary robustness and failure modes of circuit designs, especially as more complex multigate circuits are implemented.

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell-specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation., (© 2015 Au, Hermo, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body., (© 2015 The Authors.)

Genetic memory enables the recording of information in the DNA of living cells. Memory can record a transient environmental signal or cell state that is then recalled at a later time. Permanent memory is implemented using irreversible recombinases that invert the orientation of a unit of DNA, corresponding to the [0,1] state of a bit. To expand the memory capacity, we have applied bioinformatics to identify 34 phage integrases (and their cognate attB and attP recognition sites), from which we build 11 memory switches that are perfectly orthogonal to each other and the FimE and HbiF bacterial invertases. Using these switches, a memory array is constructed in Escherichia coli that can record 1.375 bytes of information. It is demonstrated that the recombinases can be layered and used to permanently record the transient state of a transcriptional logic gate.

The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.

Sir2 is a central regulator of yeast aging and its deficiency increases daughter cell inheritance of stress- and aging-induced misfolded proteins deposited in aggregates and inclusion bodies. Here, by quantifying traits predicted to affect aggregate inheritance in a passive manner, we found that a passive diffusion model cannot explain Sir2-dependent failures in mother-biased segregation of either the small aggregates formed by the misfolded Huntingtin, Htt103Q, disease protein or heat-induced Hsp104-associated aggregates. Instead, we found that the genetic interaction network of SIR2 comprises specific essential genes required for mother-biased segregation including those encoding components of the actin cytoskeleton, the actin-associated myosin V motor protein Myo2, and the actin organization protein calmodulin, Cmd1. Co-staining with Hsp104-GFP demonstrated that misfolded Htt103Q is sequestered into small aggregates, akin to stress foci formed upon heat stress, that fail to coalesce into inclusion bodies. Importantly, these Htt103Q foci, as well as the ATPase-defective Hsp104Y662A-associated structures previously shown to be stable stress foci, co-localized with Cmd1 and Myo2-enriched structures and super-resolution 3-D microscopy demonstrated that they are associated with actin cables. Moreover, we found that Hsp42 is required for formation of heat-induced Hsp104Y662A foci but not Htt103Q foci suggesting that the routes employed for foci formation are not identical. In addition to genes involved in actin-dependent processes, SIR2-interactors required for asymmetrical inheritance of Htt103Q and heat-induced aggregates encode essential sec genes involved in ER-to-Golgi trafficking/ER homeostasis.

Although the graphitic carbon contamination of synchrotron beamline optics has been an obvious problem for several decades, the basic mechanisms underlying the contamination process as well as the cleaning/remediation strategies are not understood and the corresponding cleaning procedures are still under development. In this study an analysis of remediation strategies all based on in situ low-pressure RF plasma cleaning approaches is reported, including a quantitative determination of the optimum process parameters and their influence on the chemistry as well as the morphology of optical test surfaces. It appears that optimum results are obtained for a specific pressure range as well as for specific combinations of the plasma feedstock gases, the latter depending on the chemical aspects of the optical surfaces to be cleaned.

Human ectopic pregnancy (EP) is a leading cause of pregnancy-related death, but the molecular basis underlying the onset of tubal EP is largely unknown. Female Dicer1 conditional knockout mice are infertile with dysfunctional Fallopian tube and have a different miRNA expression profile compared to wild-type mice, and we speculated that Dicer-mediated regulation of miRNA expression and specific miRNA-controlled targets might contribute to the onset of tubal EP. In the present study, we used microarray analysis and quantitative RT-PCR to examine the expression of miRNAs and core miRNA regulatory components in Fallopian tube tissues from women with EP. We found that the levels of DICER1, four miRNAs (let-7i, miR-149, miR-182, and miR-424), and estrogen receptor α distinguished the tubal implantation site from the non-implantation site. Computational algorithms and screening for interactions with the estrogen and progesterone receptor signaling pathways showed that the four miRNAs were predicted to target ten genes, including NEDD4, TAF15, and SPEN. Subsequent experiments showed differences in NEDD4 mRNA and protein levels between the implantation and non-implantation sites. Finally, we revealed that increases in smooth muscle cell NEDD4 and stromal cell TAF15, in parallel with a decrease in epithelial cell SPEN, were associated with tubal implantation. Our study suggests that changes in miRNA levels by the DICER-mediated miRNA-processing machinery result in aberrant expression of cell type-specific proteins that are potentially involved in the onset of tubal EP.

Heterogeneous nuclear ribonucleoproteins (hnRNPs), which are chromatin-associated RNA-binding proteins, participate in mRNA stability, transport, intracellular localization, and translation by acting as transacting factors. Several studies have shown that steroid hormones can regulate hnRNP expression. However, to date, the regulation of hnRNPs and their interactions with steroid hormone signaling in fallopian tubes and endometrium are not fully elucidated. In the present study, we determined whether hnRNP expression is regulated during the menstrual cycle and correlates with estrogen receptor (ER) and progesterone receptor (PR) levels in human fallopian tubes in vivo. Because of the limited availability of human tubal tissues for the research, we also explored the mechanisms of hnRNP regulation in human endometrium in vitro. Fallopian tissue was obtained from patients in the early, late, and postovulatory phases and the midsecretory phase and endometrial tissue from premenopausal and postmenopausal women undergoing hysterectomy. We measured expression of hnRNPs and assessed their intracellular localization and interactions with ERs and PRs. We also determined the effects of human chorionic gonadotropin, 17β-estradiol (E(2)), and progesterone (P(4)) on hnRNP expression. In fallopian tubes, mRNA and protein levels of hnRNP A1, AB, D, G, H, and U changed dynamically during ovulation and in the midsecretory phase. In coimmunolocation and coimmunoprecipitation experiments, hnRNPs interacted with each other and with ERs and PRs in fallopian tubes. After treatment with E(2) and/or P(4) to activate ERs and PRs, hnRNP A1, AB, D, G, and U proteins displayed overlapping but distinct patterns of regulation in the endometrium in vitro. Our findings expand the physiological repertoire of hnRNPs in human fallopian tubes and endometrium and suggest that steroid hormones regulate different hnRNPs directly by interacting with ERs and/or PRs or indirectly by binding other hnRNPs. Both actions may contribute to regulation of gene transcription.

A synthetic de novo designed heterodimeric coiled-coil was used to copurify two target fluorescent proteins, Venus and enhanced cyan fluorescent protein (ECFP). The coiled-coil consists of two 21-amino acid repetitive sequences, (EIAALEK)(3) and (KIAALKE)(3), named E3 and K3, respectively. These sequences were fused to the C-termini of ECFP or Venus followed by either a strep- or a his-tag, respectively, for affinity purification. Mixed lysates of Venus-K3 and ECFP-E3 were subjected to consecutive affinity purification and showed highly specific association between the coiled-coil pair by SDS-PAGE, gel filtration, isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET). The tagged proteins eluted as heterodimers at the concentrations tested. FRET analysis further showed that the coiled-coil pair was stable in buffers commonly used for protein purification, including those containing high salt concentration and detergent. This study shows that the E3/K3 pair is very well suited for the copurification of two target proteins expressed in vivo because of its high specificity: it forms exclusively heterodimers in solution, it does not interact with any cellular proteins and it is stable under different buffer conditions., (Copyright © 2012 The Protein Society.)

Objective: To apply a new statistical method (principle component analysis; PCA) to evaluate osseointegration., Materials and Methods: Two different commercially available implants were selected for the study. Twenty implants, 10 of each type, were placed in the rabbit tibiae (n = 10). The fluorochromes (FLCs) alizarin complexone and calcein green were administered after 20 days and 4 days before sacrifice for labeling. On the day of implantation and retrieval (6 weeks), implant stability was measured with a resonance frequency analyzer (RFA). The retrieved samples were ground sectioned for histomorphometric and FLC quantification. The collected data were analyzed by a PCA software program (Qlucore Omics Explorer, Lund, Sweden) to explore and determine the correlation between different study variables and to analyze the differences between different implants., Results: The RFA presented no significant differences at either time point. The bone-to-implant contact was significantly higher for the TiUnite (NobelBiocare, Gothenburg, Sweden); however, the bone area and FLC quantification showed higher values for the Osseotite (3i Implant Innovation, FL). Consistent with these results, the PCA indicated a strong correlation between TiUnite and high bone-to-implant contact values and between Osseotite and high bone area and FLC values. No correlation between RFA and the biological responses were found., Conclusion: The application of the PCA analysis may help interpret and correlate results obtained from numerous evaluations.

Context: Tissue-specific dicer1 knockout mice display severe, irreversible Fallopian tube damage and disrupted tubal transport. It is not known how Dicer1 affects human Fallopian tube function., Objective: The aim of the study was to investigate the regulation of tubal Dicer1 expression during ovulation and the midsecretory phase and to assess Dicer1-associated alterations in estrogen receptor (ER) subtype and progesterone receptor (PR) expression., Design: Fallopian tissue was obtained from patients at early (n = 4), late (n = 4), and postovulatory (n = 5) phases and the midsecretory phase (n = 4). Serum was obtained immediately before surgery (sterilization or hysterectomy) to confirm the phases. The localization and regulation of Dicer1, ER subtypes, and PR isoforms were determined by immunofluorescence, confocal microscopy, and quantitative RT-PCR., Results: Dicer1 protein was expressed most abundantly in Fallopian epithelial cells; mRNA and protein levels peaked in the late ovulatory phase. ER subtype and PR isoform mRNA levels were not related to ovulatory stages; however, ERβ1 and ERβ2 mRNA/protein levels were highest and PRA/B and PRB mRNA/protein levels were lowest in the midsecretory phase. Dicer1 mRNA expression correlated positively with ERα mRNA expression in the late ovulatory phase and negatively with ERβ2 mRNA expression in the midsecretory phase and PRB mRNA in the early ovulatory phase., Conclusion: Dicer1 expression is up-regulated in cell-specific fashion in human Fallopian tubes during ovulation. The stage-dependent expression of Dicer1 and its correlation with ERα, ERβ2, and PRB mRNA suggests that tubal Dicer1 helps regulate tubal expression of steroid hormone receptors in a cycle-dependent manner and may contribute to tubal transport in humans.

Objective: Previous findings demonstrate that enhanced expression of the forkhead transcription factor Foxc2 in adipose tissue leads to a lean and insulin-sensitive phenotype. These findings prompted us to further investigate the role of Foxc2 in the regulation of genes of fundamental importance for metabolism and mitochondrial function., Research Design and Methods: The effects of Foxc2 on expression of genes involved in mitochondriogenesis and mitochondrial function were assessed by quantitative real-time PCR. The potential of a direct transcriptional regulation of regulated genes was tested in promoter assays, and mitochondrial morphology was investigated by electron microscopy. Mitochondrial function was tested by measuring oxygen consumption and extracellular acidification rates as well as palmitate oxidation., Results: Enhanced expression of FOXC2 in adipocytes or in cells with no endogenous Foxc2 expression induces mitochondriogenesis and an elongated mitochondrial morphology. Together with increased aerobic metabolic capacity, increased palmitate oxidation, and upregulation of genes encoding respiratory complexes and of brown fat-related genes, Foxc2 also specifically induces mitochondrial fusion genes in adipocytes. Among tested forkhead genes, Foxc2 is unique in its ability to trans-activate the nuclear-encoded mitochondrial transcription factor A (mtTFA/Tfam) gene--a master regulator of mitochondrial biogenesis. In human adipose tissue the expression levels of mtTFA/Tfam and of fusion genes also correlate with that of Foxc2., Conclusions: We previously showed that a high-calorie diet and insulin induce Foxc2 in adipocytes; the current findings identify a previously unknown role for Foxc2 as an important metabo-regulator of mitochondrial morphology and metabolism.

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.

Little is known about the regulation and cellular distribution of androgen receptors (ARs) in female rodent brains at various stages of the oestrous cycle. This information is critical for further studies of androgen signalling in the regulation of brain function under physiological and pathophysiological conditions. In this report, we show that the distribution of AR immunoreactivity in the female rat brain is consistent with reported AR mRNA hybridisation signals in the male brain, except for the dentate gyrus of the hippocampus. Immunohistochemical and Western blot analyses performed herein revealed that the onset of region-specific changes in AR proteins was strongly correlated with circulating and ovarian levels of estradiol and testosterone across the oestrous cycle. During the metestrus and diestrus stages, however, the highest levels of AR expression were abolished by chronic dihydrotestosterone (DHT) treatment. This demonstrates that fluctuations in endogenous androgens are required for the regulation of AR expression in the female rat brain. Colocalisation studies revealed that: (1) anatomical variations in AR protein localisation existed between female and male brains, (2) AR immunoreactivity was both neuronal and non-neuronal, and (3) AR protein expression was lower in female rat brains at all stages of the oestrous cycle compared to age-matched males. Our results indicate the presence of regional sex differences in AR expression and changes in the proportion of AR between different subcellular compartments. Furthermore, DHT was found to down-regulate the level of AR in the subcellular compartment in females in a region-specific manner. As a whole, the present study provides the first step toward understanding the dynamics of AR expression and regulation in the brain during normal physiological conditions and for differences in neuronal androgen effects based on sex., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Adult female rats continuously exposed to androgens from prepuberty have reproductive and metabolic features of polycystic ovary syndrome (PCOS). We investigated whether such exposure adversely affects estrous cyclicity and the expression and distribution of gonadotropin-releasing hormone (GnRH), GnRH receptors, and corticotrophin-releasing hormone (CRH) in the hypothalamus and whether the effects are mediated by the androgen receptor (AR). We also assessed the effect of low-frequency electro-acupuncture (EA) on those variables. At 21 days of age, rats were randomly divided into three groups (control, PCOS, and PCOS EA; n = 12/group) and implanted subcutaneously with 90-day continuous-release pellets containing vehicle or 5alpha-dihydrostestosterone (DHT). From age 70 days, PCOS EA rats received 2-Hz EA (evoking muscle twitches) five times/week for 4-5 weeks. Hypothalamic protein expression was measured by immunohistochemistry and western blot. DHT-treated rats were acyclic, but controls had regular estrous cycles. In PCOS rats, hypothalamic medial preoptic AR protein expression and the number of AR- and GnRH-immunoreactive cells were increased, but CRH was not affected; however, GnRH receptor expression was decreased in both the pituitary and hypothalamus. Low-frequency EA restored estrous cyclicity within 1 week and reduced the elevated hypothalamic GnRH and AR expression levels. EA did not affect GnRH receptor or CRH expression. Interestingly, nuclear AR co-localized with GnRH in the hypothalamus. Thus, rats with DHT-induced PCOS have disrupted estrous cyclicity and an increased number of hypothalamic cells expressing GnRH, most likely mediated by AR activation. Repeated low-frequency EA normalized estrous cyclicity and restored GnRH and AR protein expression. These results may help explain the beneficial neuroendocrine effects of low-frequency EA in women with PCOS.

The action of interleukin-6 (IL-6) impacts female reproduction. Although IL-6 was recently shown to inhibit cilia activity in human fallopian tubes in vitro, the molecular mechanisms underlying IL-6 signaling to tubal function remain elusive. Here, we investigate the cellular localization, regulation, and possible function of two IL-6 receptors (IL-6R alpha and gp130) in mouse and human fallopian tubes in vivo. We show that IL-6R alpha is restricted to the cilia of epithelial cells in both mouse and human fallopian tubes. Exogenous 17beta-estradiol (E(2)), but not progesterone (P(4)), causes a time-dependent decrease in IL-6R alpha expression, which is blocked by the estrogen receptor (ER) antagonist ICI-182,780. Exposure of different ER-selective agonists propyl-(1H)-pyrazole-1,3,5-triyl-trisphenol or 2,3-bis-(4-hydroxyphenyl)-propionitrile demonstrated an ER subtype-specific regulation of IL-6R alpha in mouse fallopian tubes. In contrast to IL-6R alpha, gp130 was detected in tubal epithelial cells in mice but not in humans. In humans, gp130 was found in the muscle cells and was decreased in the periovulatory and luteal phases during the reproductive cycles, indicating a species-specific expression and regulation of gp130 in the fallopian tube. Expression of tubal IL-6R alpha and gp130 in IL-6 knockout mice was found to be normal; however, E(2) treatment increased IL-6R alpha, but not gp130, in IL-6 knockout mice when compared with wild-type mice. Furthermore, expression levels of IL-6R alpha, but not gp130, decreased in parallel with estrogenic accelerated oocyte-cumulus complex (OCC) transport in mouse fallopian tubes. Our findings open the possibility that cilia-specific IL-6R alpha may play a role in the regulation of OCC transport and suggest an estrogen-regulatory pathway of IL-6R alpha in the fallopian tube.

Clomiphene citrate (CC) therapy for disorders of anovulatory infertility has been linked to an increased frequency of tubal ectopic pregnancy. Although CC enhances apoptotic processes in the ovaries, villi, and decidual tissues, its effect on apoptosis in the fallopian tube is unknown. Here, we show that chronic treatment with CC induces tubal apoptosis, but not necrosis, through an intrinsic mitochondria-dependent signaling pathway in vivo. The apoptosis was specific to epithelial cells in the isthmus, and the damage was reversed with 17beta-estradiol (E2); however, pretreatment or concomitant treatment with E2 did not protect against tubal apoptosis induced by chronic treatment with CC. Chronic treatment activated estrogen receptors (ESRs), particularly cilia-localized ESR2A (formerly ERbeta2). In contrast to E2, acute treatment of superovulating rats with a high dose of CC or the ESR2-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN) significantly delayed the transport of oocyte-cumulus complexes through the fallopian tube. Our findings suggest that in response to chronic CC therapy, isthmus-specific apoptosis of epithelial cells and activation of cilia-ESR2A act in parallel to block gamete and embryo passage through the fallopian tube, eventually resulting in tubal ectopic pregnancy.

We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed.

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.

Estrogen receptors (ERs) are members of the nuclear receptor superfamily and are involved in regulation of fallopian tube functions (i.e., enhancement of protein secretion, formation of tubal fluid, and regulation of gamete transport). However, the ER subtype-mediated mechanisms underlying these processes have not been completely clarified. Recently, we identified ERbeta expression and localization in rat fallopian tubes, suggesting a potential biological function of ERbeta related to calcium-dependent ciliated beating. Here we provide for the first time insight into the less studied ERalpha isoforms, which mediate estrogen-dependent production and secretion of IGFs in vivo. First, Western blot studies revealed that three ERalpha isoforms were expressed in mouse fallopian tubes. Subsequent immunohistochemical analysis showed that ERalpha was detected in all cell types, whereas ERbeta was mainly localized in ciliated epithelial cells. Second, ERalpha isoform levels were dramatically downregulated in mouse fallopian tubes by treatment with E(2) or PPT, an ERalpha agonist, in a time-dependent manner. Third, the presence of ICI 182,780, an ER antagonist, blocked the E(2)- or PPT-induced downregulation of tubal ERalpha isoform expression in mice. However, alteration of ERalpha immunoreactivity following ICI 182,780 treatment was only detected in epithelial cells of the ampullary region. Fourth, changes in ERalpha isoform expression were found to be coupled to multiple E(2) effects on tubal growth, protein synthesis, and secretion in mouse fallopian tube tissues and fluid. In particular, E(2) exhibited positive regulation of IGF-I and IGF-II protein levels. Finally, using growth hormone receptor (GHR) gene-disrupted mice, we showed that regulation by E(2) of IGF production was independent of GH-induced GHR signaling in mouse fallopian tubes in vivo. These data, together with previous studies from our laboratory, suggest that the long-term effects of estrogen agonist promote IGF synthesis and secretion in mouse tubal epithelial cells and fallopian tube fluid via stimulation of ERalpha.

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.

Background: Non-palpable breast lesions are diagnosed frequently posing the problem of localization and removal. When such lesions are malignant, axillary node status must be determined. We report our experience using radio-guided occult lesion localization (ROLL) for locating and removing non-palpable breast lesions together with sentinel node biopsy (SNB) to assess axillary status. We call the technique SNOLL., Methods: From March 1997 to April 2004, 1046 consecutive patients presented suspicious non-palpable breast lesions and were programmed for conservative surgery and SNB. In 87 patients intraoperative histological examination revealed a benign lesion and SNB was not performed. The remaining 959 patients, with cytologically or histologically proven cancer, underwent SNOLL with immobile radiotracer injected under mammographic or ultrasound (US) guidance into the lesion, and subsequent injection of mobile tracer subdermally to localize the sentinel node (SN). Patients then underwent breast surgery and SNB., Results: Breast lesions were localized by ROLL in 99.6% of cases and were removed radically with negative margins in 91.9% of cases. Sentinel nodes were detected in all but one case. Intraoperative or definitive histological examination revealed 776 invasive/microinvasive carcinomas and 182 with in situ disease. Sentinel nodes were positive in 154 (19.8%) of 776 invasive/microinvasive cancers and in two with ductal intraepithelial neoplasia (1.1%)., Conclusions: In SNOLL the injection procedures are performed separately, but both lesion and SNs are removed together; axillary dissection is performed if the SN is positive, thus definitive treatment of malignant non-palpable lesions occurs in a single surgical session.

Several ERbeta isoforms have been identified in human and rodent tissues, but it is unclear whether each isoform has distinctly different cellular targeting characteristics and physiological functions. We have investigated the intracellular localization and regulatory patterns for ERbeta isoforms in rat fallopian tubes. Western blot analysis reveals that two ERbeta isoforms corresponding to ERbeta1 and ERbeta2 are expressed in rat fallopian tubes. However, ERbeta2 is the predominant form of ERbeta in this tissue. High-resolution confocal imaging and immunohistochemical analysis provide ample evidence that ERbeta expression is limited almost exclusively to the ciliated epithelial cells, in contrast to ERalpha, which is widely distributed. Furthermore, within the ciliated epithelial cells, ERbeta is colocalized with beta-tubulin IV at stem portion of the cilia. We show that ERbeta2 protein expression is tightly regulated by E(2) or DPN in a time-dependent manner without changes in ERbeta1 expression. These estrogenic effects are inhibited by an ER antagonist, ICI 182,780. In addition, significant alteration of ERbeta immunoreactivity is detected only histologically in the ampullary region. Since the cilia are considered an essential determinant of tubal transport, we further demonstrate that E(2)- or DPN-induced ERbeta2 activation is associated with alterations in tubal protein expression crucial for the regulation of calcium-dependent ciliary beating. Given the coordinated regulation and interaction of ER and progesterone receptor in the cilia, we hypothesize that tubal ERbeta2 may facilitate the estrogen-mediated transport process by processing protein-protein interaction under physiological and/or pathological conditions. We show for the first time that a previously unrecognized localization of ERbeta isoform in rat fallopian tubes can combine with estrogen to individually control the expression of ER beta-isoforms in normal target tissues.

Female mice lacking AR display alterations in ovarian and uterine function. However, the biology of AR in the fallopian tube is not fully understood. To gain an insight into potential roles of AR in this tissue, we demonstrated that eCG treatment increased AR expression in a time-dependent manner and subsequent treatment with hCG decreased AR expression in mouse fallopian tubes. This expression pattern was positively associated with 17beta-estradiol and testosterone levels in vivo. Immunohistochemical analysis of fallopian tube epithelial cells revealed that nuclear localization of AR increased in parallel with decreased AR in the cytoplasm following eCG treatment. Moreover, we found that treatment with flutamide upregulated AR expression in immature mice in association with a decrease in serum testosterone levels, whereas the same treatment resulted in downregulation of AR expression in gonadotropin-stimulated mice with concomitant decreases in serum 17beta-estradiol concentrations, suggesting that androgen differs from estrogen in the regulation of AR expression. Furthermore, we demonstrated that DES increased both AR protein expression and nuclear location over a 48-h time course. DHT had rapid effects, with induction of AR expression and translocation at 6 h after injection, but unlike DES it had prolonged efficacy. In addition, we provided direct in vivo evidence that nuclear protein interaction between AR and p21(Cip1), a previously reported AR-regulated gene, was enhanced by gonadotropin stimulation. To our knowledge, this study provides the first demonstration to illustrate that estrogen as a principal regulator may contribute to regulate and activate AR in the fallopian tubes in vivo.

We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.

Progesterone (P(4)) regulates many aspects of physiological functions via two nuclear P(4) receptors (PR), PRA and PRB, which are members of a structurally related nuclear hormone receptor superfamily that includes glucocorticoid receptors (GR). The regulation and cellular distribution of PR protein isoforms have been extensively studied in reproductive tissues, but this is not the case in the lung. In the present study, reverse transcriptase (RT)-PCR, Western blotting, and immunolocalization supported the presence of PRA in the lung of female mice, with PRA protein levels significantly increased between postnatal day 7 and 12, declined at postnatal day 26, and minimal in adults when compared to postnatal day 2. The peak was temporally related to postnatal lung maturation in rodents. Immunoreactivity for PR was detected in the alveolar and bronchial epithelia. We then extended this study to examine, for the first time, the regulation of PRA protein expression in female mouse lung in vivo. Neither the increase in endogenous P(4) nor treatment with exogenous P(4) regulated PRA protein expression in female mouse lung. However, treatment of mice with the GR/PR antagonist RU 486, but not Org 31710 (a specific PR antagonist), significantly increased PRA protein expression in parallel to a decrease in GR protein expression. In addition, treatment with the synthetic glucocorticoid dexamethasone led to a decrease in PRA protein expression independent of endogenous P(4) levels. Furthermore, immunoprecipitation followed by Western blot analysis revealed that, under in vivo conditions, PRA physically interacted with GR in mouse lung. Confocal laser microscopy revealed that PRA and GR co-localized in the nuclei of alveolar epithelia cells, whereas nuclear PR and cytoplasmic GR were detected in bronchial epithelium. Taken together, our observations suggest that PRA may be an important physiological factor involved in postnatal lung development and that the regulation of PRA protein expression is not dependent on P(4), but rather on functional glucocorticoid/GR signaling mediated by protein-protein interaction in the mouse lung.

Progesterone and its interaction with nuclear progesterone receptors (PR) PR-A and PR-B play a critical role in the regulation of female reproductive function in all mammals. However, our knowledge of the regulation and possible cellular function of PR protein isoforms in the fallopian tube and uterus in vivo is still very limited. In the present study, we revealed that equine chorionic gonadotropin (eCG) treatment resulted in a time-dependent increase in expression of both isoforms, reaching a maximal level at 48 h in the fallopian tube. Regulation of PR-A protein expression paralleled that of PR-B protein expression. However, in the uterus PR-B protein levels increased and peaked earlier than PR-A protein levels after eCG treatment. With prolonged exposure to eCG, PR-B protein levels decreased, whereas PR-A protein levels continued to increase. Furthermore, subsequent treatment with human (h)CG decreased the levels of PR protein isoforms in both tissues in parallel with increased endogenous serum progesterone levels. To further elucidate whether progesterone regulates PR protein isoforms, we demonstrated that a time-dependent treatment with progesterone (P(4)) decreased the expression of PR protein isoforms in both tissues, whereas decreases in p27, cyclin D(2), and proliferating cell nuclear antigen protein levels were observed only in the uterus. To define the potential PR-mediated effects on apoptosis, we demonstrated that the PR antagonist treatment increased the levels of PR protein isoforms, induced mitochondrial-associated apoptosis, and decreased in epidermal growth factor (EGF) and EGF receptor protein expression in both tissues. Interestingly, immunohistochemistry indicated that the induction of apoptosis by PR antagonists was predominant in the epithelium, whereas increase in PR protein expression was observed in stromal cells of both tissues. Taken together, these observations suggest that 1) the tissue-specific and hormonal regulation of PR isoform expression in mouse fallopian tube and uterus, where they are potentially involved in regulation of mitochondrial-mediated apoptosis depending on the cellular compartment; and 2) a possible interaction between functional PR protein and growth factor signaling may have a coordinated role for regulating apoptotic process in both tissues in vivo.

CD43 is a transmembrane molecule that contains a 123-aminoacids-long cytoplasmic tail and a highly O-glycosylated extracellular domain of mucin type. Endogenous CD43 expressed in COLO 205, K562 and Jurkat cells revealed a membrane-associated, 20 kDa CD43-specific cytoplasmic tail fragment (CD43-CTF) upon inhibition of gamma-secretase. This fragment was formed by an extracellular cleavage, as it was not accumulated after treating cells with 1,10-phenanthroline, a metalloprotease inhibitor. When CD43 was transfected into HEK-293 cells expressing dominant-negative PS1 (presenilin-1), the CD43-CTF was accumulated, but not in cells with wild-type PS1. Owing to its accumulation in the presence of a non-functional PS variant, it may thus be a novel gamma-secretase substrate. This CTF is formed by an extracellular cleavage close to the membrane, is a fragment that can be concluded to be a substrate for gamma-secretase. However, the intracellular gamma-secretase product has not been possible to detect, suggesting a quick processing of this product. During normal growth the CTF was not found without gamma-secretase inhibition, but when the cells (COLO 205) were very confluent the fragment could be detected. The intracellular domain of CD43 has previously been shown to contain a functional nuclear localization signal, and has been suggested to be involved in gene activation. From this and the present results, a novel way to explain how mucin-type molecules may transduce intracellular signals can be proposed.

The MUC1 tumor antigen is overexpressed on most breast tumors and metastases. It interacts with signaling proteins such as the ErbB kinases and beta-catenin, and is involved in mammary gland oncogenesis and tumor progression. Herein, we report a novel interaction between MUC1 and adenomatous polyposis coli (APC), a tumor suppressor involved in downregulating beta-catenin signaling. Initially identified in colorectal cancer, APC is also downregulated in breast tumors and presumably involved in mammary carcinogenesis. MUC1 and APC co-immunoprecipitate from the ZR-75-1 human breast carcinoma cell line and co-localize in mouse mammary glands and tumors. These studies also indicate that the association of MUC1 and APC may be increased by epidermal growth factor stimulation. Intriguingly, the co-immunoprecipitation of MUC1 and APC increases in human breast tumors and metastases as compared to adjacent normal tissues, indicating that this association may play a role in the formation and progression of breast tumors.

CD43 is a transmembrane molecule with a highly O-glycosylated extracellular domain of mucin type. It is a normal constituent of leukocytes and found in colon adenoma, but not in normal colon epithelia. Here it is shown that the cytoplasmic tail of CD43 contains a functional bipartite nuclear localization signal directing it to the nucleus. The intracellular domain of CD43 interacts with beta-catenin and causes an upregulation of the beta-catenin target genes c-MYC and CyclinD1. The present results suggest that CD43 can be involved in nuclear signaling and via beta-catenin interaction be involved in cell proliferation.

CD43 is an abundant transmembrane sialoglycoprotein in leukocyte-type cell lines, but it has also been suggested to be present in colon adenomas and colon carcinomas. We have now shown that CD43 is expressed in a variety of cell lines of different origins (CaSKI, A549, 293, MTSV1-7, MCF7, HT-1080, Jurkat, K562, COLO 205, HT-29, Caco-2, DLD-1 and SW480). The level of expression of CD43 mRNA was analyzed by reverse transcriptase-polymerase chain reaction and that of the protein by immunoprecipitation and Western blot, flow cytometry and confocal microscopy using two monoclonal anti-CD43 antibodies (L10 and 4D2). As all cell lines expressed CD43, it is suggested that CD43 has a more fundamental function than previously believed and thus cannot be considered only as a specific leukocyte marker., (Copyright 2002 S. Karger AG, Basel)

The mucins secreted from the colon carcinoma cell line COLO 205 have the MUC1 and CD43 (leukosialin) as core proteins, where both carry sialyl-Lewis a and MUC1 sialyl-Lewis x epitopes. The adhesion of E-selectin expressing CHO cells to the coated mucins was analyzed in a flow system revealing that the MUC1 mucin adhered better than the CD43 mucin. One reason could be their different glycosylation, a difference that was explored by analyzing the biosynthesis of MUC1 and CD43 in COLO 205 cells. Both the MUC1 and CD43 mucins became sialyl-Lewis a reactive, but after different times as revealed by pulse-chase studies. However, only MUC1 became sialyl-Lewis x reactive. These differences suggest that MUC1 and CD43 are synthesized in different compartments of the cell. It was also observed that the mucins from colon carcinoma patients had MUC1-type mucins that carried both sialyl-Lewis a and x epitopes and CD43-type sialyl-Lewis a mucins with only low levels of sialyl-Lewis x epitopes. One could hypothesize that colon carcinoma derived MUC1 is decorated with potent E-selectin epitopes, and that this could be one of several reasons for the involvement of MUC1 in cancer development.

CD43 is a leukocyte-associated sialoglycoprotein which is also expressed in human colon adenoma and carcinoma. To obtain monoclonal antibodies (MAbs) that would react with CD43 in a glycosylation-independent way, antibodies were raised against a peptide corresponding to a portion of the CD43 cytoplasmic domain. Hybridomas were screened on paraffin sections from CD43-positive colon tumours. The reactivity of the antibodies with CD43 was verified by Western blot analysis of lysate of CHO cells transfected with human CD43 cDNA and by immunoprecipitation of lysates from CD43+ cell lines. Epitope mapping of antibodies was done using overlapping heptameric peptides. A detailed characterisation of one of the novel antibodies (CD43-3A1) is presented. This antibody reacts with the CD43 protein regardless of its glycosylation in Western blot analysis, immunoprecipitation and immuno-histochemistry of paraffin sections. Immuno-histochemical analysis of paraffin sections from colon adenoma and carcinoma tissues as well as colon cancer cell lines revealed that CD43 was predominantly localised intracellularly, in contrast to leukocyte-type cells. The MAb reacted more efficiently with paraffin-embedded colon adenoma and carcinoma cells than previously characterised CD43-specific antibodies. This should facilitate the evaluation of a potential role of CD43 during cancer development.

As part of a population-based case-control study on diet and breast cancer in Spain, the role of dietary fat and vegetable oils in breast cancer etiology was examined. A validated, semi-quantitative food-frequency questionnaire was completed by 762 women, 18-75 years of age, with histologically confirmed, newly diagnosed breast cancer, and 988 randomly selected female controls. For each food item and nutrient, the study subjects were divided into quartiles according to intake levels, with the lowest quartile serving as the reference category. Adjustment for total energy intake and other potential confounders was made using multiple logistic regression for all women as well as separately for pre- and post-menopausal women. Neither total fat intake nor specific types of fat were significantly associated with breast cancer in pre- or post-menopausal women. However, higher consumption of olive oil (rich in monounsaturated fat) was significantly related to a lower risk of breast cancer [for highest vs. lowest quartile of consumption, odds ratio (OR) = 0.66; 95% CI, 0.46-0.97] with a significant dose-response trend. While these findings do not support a relation between total fat intake and breast cancer risk, they do provide evidence for an inverse association between olive oil (and suggest one between monounsaturated fat) and risk of breast cancer.