Phosphoserine-86-HSPB1 (pS86-HSPB1) is cytoplasmic and highly induced in rat myometrium at labour.

Uterine myocytes during pregnancy proceed through a series of adaptations and collectively transform into a powerfully contractile tissue by term. Previous work has indicated that members of the heat shock protein (HSP) B family of stress proteins are associated with the process of adaptation and transformation. Utilizing immunoblot analyses, widefield epifluorescence and total internal reflection (TIRF) microscopy, this study investigated the temporal and spatial detection of HSPB1 phosphorylated on serine-86 (pS86-HSPB1) in rat myometrium during pregnancy, the role of uterine distension in regulation of pS86-HSPB1, and the comparative localization with pS15-HSPB1 in rat myometrial tissue as well as in an immortalized human myometrial cell line. Immunoblot detection of pS86-HSPB1 was significantly elevated during late pregnancy and labour. In particular, pS86-HSPB1 was significantly increased at day (d)22 and d23 (labour) compared with all other timepoints assessed. Localization of pS86-HSPB1 in myometrium became prominent at d22 and d23 with cytoplasmic detection around myometrial cell nuclei. Furthermore, pS86-HSPB1 detection was found to be significantly elevated in the gravid rat uterine myometrium compared with the non-gravid tissue at d19 and d23. Both widefield epifluorescence and TIRF microscopy examination of human myometrial cells demonstrated that pS15-HSPB1 was prominently localized to focal adhesions, while pS82-HSPB1 (homologous to rodent pS86-HSPB1) was primarily located in the cell cytoplasm. Our data demonstrate that levels of phosphorylated HSPB1 increase just prior to and during labour, and that uterine distension is a stress-inducing signal for HSPB1 phosphorylation. The exact roles of these phosphorylated forms in myometrial cells remain to be determined.

Examination of FERMT1 expression in placental chorionic villi and its role in HTR8-SVneo cell invasion.

Transmembrane integrin receptors mediate cell-extracellular matrix as well as cell-cell adhesion. As placental trophoblast cells undergo differentiation they display changes in integrin expression or switching, but the mechanism(s) of integrin activation that supports this differentiation is still unknown. The Fermitin family of adapter proteins (FERMT 1-3) are integrin activators that mediate integrin-mediated signaling. In this study, we examined the spatiotemporal pattern of expression of FERMT1 in human chorionic villi throughout gestation and its role in HTR8-SVneo substrate adhesion and invasion. Placental villous tissue was obtained from patients undergoing elective terminations at weeks 8-14, as well as from term deliveries at weeks 37-40 and analyzed by immunofluorescence. Additionally, HTR8-SVneo trophoblast cells were transfected with FERMT1-specific siRNA or non-targeting siRNA (control) and used in cell-substrate adhesion as well as invasion assays. FERMT1 was primarily localized to membrane-associated regions at the base or around the periphery of the villous cytotrophoblast and proximal as well as distal cell column trophoblast. FERMT1 was also localized to endothelial cells of blood vessels in chorionic villi. siRNA-mediated depletion of FERMT1 in HTR8-SVneo cells did not markedly alter HTR8-SVneo cell-substrate adhesion but did significantly decrease invasion (P < 0.05) compared to control cells. These novel findings identify the presence of the integrin activator FERMT1 in trophoblast cells and that FERMT1 can regulate HTR8-SVneo cell invasion. FERMT1 may directly influence integrin activation and the subsequent integrin-mediated signaling and differentiation that underlies the acquisition of the invasive trophoblast phenotype in vivo.

Inducible heat shock protein A1A (HSPA1A) is markedly expressed in rat myometrium by labour and secreted via myometrial cell-derived extracellular vesicles.

The myometrium goes through physiological, cellular and molecular alterations during gestation that necessitate effective cellular proteostasis. Inducible heat shock protein A1A (HSPA1A) is a member of the 70-kDa heat shock protein A (HSPA) family, which acts as a chaperone to regulate proteostasis; however, HSPA1A also participates as a cytokine in inflammatory regulation, leading to its designation as a chaperokine. This study examined the spatiotemporal expression of HSPA1A protein in the rat myometrium throughout gestation and assessed whether it is secreted as cargo of myometrial cell-derived extracellular vesicles (EVs). Immunoblot analysis demonstrated that HSPA1A expression was markedly elevated during late pregnancy and labour and increased by uterine distension. Myometrial HSPA1A expression insitu increased in myocytes of longitudinal and circular muscle layers from Day 19 through to postpartum, specifically in the cytoplasm and nuclei of myocytes from both muscle layers, but frequently detectable just outside myocyte membranes. Scanning electron microscopy examination of samples isolated from hTERT-HM cell-conditioned culture medium, using EV isolation spin columns, confirmed the presence of EVs. EV lysates contained HSPA8, HSPA1A and the EV markers apoptosis-linked gene 2-interacting protein X (Alix), the tetraspanin cluster of differentiation 63 (CD63), tumour susceptibility gene 101 (TSG101) and HSP90, but not the endoplasmic reticulum protein calnexin. These results indicate that HSPA1A may act as a chaperokine in the myometrium during pregnancy.

The porcine trophoblast cell line PTr2 is susceptible to porcine reproductive and respiratory syndrome virus-2 infection.

INTRODUCTION: Porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) breaches the maternal-fetal interface (MFI) to infect porcine fetuses, yet the exact mechanism(s) of transmission is not understood. The objective of this study was to determine the susceptibility of porcine trophoblast cell line (PTr2) to PRRSV-2 infection to understand the potential role of the trophoblast in viral transmission to fetuses in vivo. METHODS: PTr2 cells were exposed in vitro to PRRSV-2 and then subjected to immunofluorescence analysis (IF), flow cytometry (FCM), real-time quantitative PCR (RT-qPCR), transmission electron microscopy (TEM) and immunogold electron microscopy (IEM) to assess viral infection. The effects of PRRSV-2 on PTr2 cell cycle progression and apoptosis, as well as the ability of PTr2 cells to produce infectious viral particles were also examined. RESULTS: PRRSV-2 was readily detected in PTr2 cells by IF, FCM, RT-qPCR, TEM and IEM techniques. RT-qPCR and FCM results of a time course of infection of PTr2 cells indicated PRRSV-2 load decreased over time after initial infection up to 72 h. PRRSV-2 infection altered PTr2 cell cycle with a selective increase of cells within the G2/M phase and also induced apoptosis. TEM and IEM demonstrated PRRSV-2 within and on the surface of PTr2 cells and PRRSV-2 virions released from PTr2 cells infected naïve MARC-145 cells inducing cytopathic effects. DISCUSSION: Trophoblast cells are susceptible to PRRSV-2 infection and release live virions capable of inducing cytopathic effects in naïve cells. This suggests a possible mechanism by which PRRSV-2 can breach the MFI resulting in fetal infection and death.

Spatiotemporal immunofluorescent evaluation of porcine reproductive and respiratory syndrome virus transmission across the maternal-fetal interface.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe reproductive failure characterized by high fetal morbidity and mortality leading to substantial economic losses to the swine industry. Evaluation of spatiotemporal transmission of PRRSV at the maternal-fetal interface (MFI) is critical for understanding fetal infection. Localization of PRRSV-2 strain NVSL 97-7895 at different regions of the MFI in 20 pregnant gilts at 2, 5, 8, 12 and 14 days post-inoculation (dpi) were analyzed by immunofluorescence (IF). Samples of MFI were collected from 15 inoculated and 5 control gilts and transplacental PRRSV transmission assessed in randomly selected fetuses from each litter. Localization of NVSL 97-7895 antigen immunoreactivity in the MFI was focused in three major areas: endometrial connective tissues (ENDO), the feto-maternal junction (FMJ) and fetal placenta (PLC). NVSL 97-7895 was detected at the FMJ by 2 dpi. At 2, 5 and 8 dpi, NVSL 97-7895 was localized within the ENDO and FMJ, whereas at 12 and 14 dpi, it was mainly localized in the PLC. Using a novel IF strategy for counting and size sorting NVSL 97-7895 viral antigen in situ, results of this study indicate that non-cell-associated mechanisms are involved in PRRSV transmission across the MFI.

Induction of expression and phosphorylation of heat shock protein B5 (CRYAB) in rat myometrium during pregnancy and labour.

During pregnancy the myometrium undergoes a programme of differentiation induced by endocrine, cellular, and biophysical inputs. Small heat shock proteins (HSPs) are a family of ten (B1-B10) small-molecular-weight proteins that not only act as chaperones, but also assist in processes such as cytoskeleton rearrangements and immune system activation. Thus, it was hypothesized that HSPB5 (CRYAB) would be highly expressed in the rat myometrium during the contractile and labour phases of myometrial differentiation when such processes are prominent. Immunoblot analysis revealed that myometrial CRYAB protein expression significantly increased from day (D) 15 to D23 (labour; P<0.05). In correlation with these findings, serine 59-phosphorylated (pSer59) CRYAB protein expression significantly increased from D15 to D23, and was also elevated 1-day post-partum (P<0.05). pSer59-CRYAB was detected in the cytoplasm of myocytes within both uterine muscle layers mid- to late-pregnancy. In unilaterally pregnant rats, pSer59-CRYAB protein expression was significantly elevated in the gravid uterine horns at both D19 and D23 of gestation compared with non-gravid horns. Co-immunolocalization experiments using the hTERT-human myometrial cell line and confocal microscopy demonstrated that pSer59-CRYAB co-localized with the focal adhesion protein FERMT2 at the ends of actin filaments as well as with the exosomal marker CD63. Overall, pSer59-CRYAB is highly expressed in myometrium during late pregnancy and labour and its expression appears to be regulated by uterine distension. CRYAB may be involved in the regulation of actin filament dynamics at focal adhesions and could be secreted by exosomes as a prelude to involvement in immune activation in the myometrium.

Developmental differences in the expression of FGF receptors between human and mouse embryos.

INTRODUCTION: Fibroblast growth factor (FGF) signaling is essential for early trophoblast expansion and maintenance in the mouse, but is not required for trophectoderm specification during blastocyst formation. This signaling pathway is stably activated to expand the trophoblast stem cell compartment in vivo, while in vitro, FGFs are used for the derivation of trophoblast stem (TS) cells from blastocysts and early post-implantation mouse embryos. However, the function of FGFs during human trophoblast development is not known. METHODS: We sought to derive TS cells from human blastocysts in a number of culture conditions, including in the presence of FGFs and stem cell factor (SCF). We also investigated the expression of FGF receptors (FGFRs) in blastocysts, and the expression of FGFR2 and activated ERK1/2 in first trimester human placentae. RESULTS: We found that SCF, but not FGF2/4, improved the quality of blastocyst outgrowths, but we were unable to establish stable human TS cell lines. We observed CDX2 expression in the trophectoderm of fully blastocysts, but rarely observed transcription of FGFRs. FGFR2 protein was not detected in human blastocysts, but was strongly expressed in mouse blastocysts. However, we found robust FGFR2 expression and activated ERK1/2 in the cytotrophoblast layer of early human placenta. DISCUSSION: Our data suggests that initiation of FGF-dependent trophoblast expansion may occur later in human development, and is unlikely to drive maintenance of a TS cell compartment during the peri-implantation period. These findings suggest that cytotrophoblast preparations from early placentae may be a potential source of FGF-dependent human TS cells.

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle.

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.

Spatiotemporal expression of alpha(1), alpha(3) and beta(1) integrin subunits is altered in rat myometrium during pregnancy and labour.

Integrins are transmembrane extracellular matrix (ECM) receptors composed of alpha- and beta-subunits. Integrins can cluster to form focal adhesions and, because there is significant ECM remodelling and focal adhesion turnover in the rat myometrium during late pregnancy, we hypothesised that the expression of alpha(1), alpha(3) and beta(1) integrin subunits in the rat myometrium would be altered at this time to accommodate these processes. Expression of alpha(1) and beta(1) integrin subunit mRNA was significantly increased on Days 6-23 of pregnancy compared with non-pregnant (NP) and postpartum (PP) time points (P < 0.05). In contrast, alpha(3) integrin subunit mRNA expression was significantly increased on Days 14, 21 and 22 compared with NP, Day 10, 1 day PP and 4 days PP (P < 0.05). A relative gene expression study revealed that, of the integrins studied, the expression of beta(1) integrin mRNA was highest in pregnant rat myometrium. The alpha(1), alpha(3) and beta(1) integrin subunit proteins became immunolocalised to myocyte membranes in situ by late pregnancy and labour in both myometrial muscle layers. Increased alpha(1), alpha(3) and beta(1) integrin gene expression during gestation and the specific detection of these subunits in myocyte membranes during late pregnancy and labour may contribute to the cell-ECM interactions required for the development of a mechanical syncytium.

Catabolite repression of SOS-dependent and SOS-independent spontaneous mutagenesis in stationary-phase Escherichia coli.

Previous work in our laboratory established that a spontaneous mutagenesis process operating in stationary-phase Escherichia coli cells undergoing selection is subject to regulation by the global regulatory mechanism known as catabolite repression (formerly also called glucose-repression). Here, we set out to determine the identity of this hitherto unknown catabolite-repressible spontaneous mutation generation mechanism(s). We used two different spontaneous mutation detection assays, reversion of a Lac(-) (lacI33OmegalacZ) frameshift marker and forward mutation to valine-resistance, and tested the effects of varying the nature of the carbon source(s) present in the selective plating medium on the mutability of bacterial cells carrying known defects in the recA, umuDC and dinB genes, three well-known SOS response genes, whose products are important for mutagenesis in E. coli. Consistent with the results of our previous Lac(-)-->Lac(+) assay using otherwise SOS-proficient bacterial cells, we found that the overall numbers of spontaneous Lac(+)E. coli revertants were highest when the selective medium contained lactose and lowest when it contained lactose plus the non-utilizable but strongly catabolite-repressing glucose analogue, methyl-alpha-d-glucopyranoside (alphaMG). In contrast, we found that the numbers of Lac(+) revertants appearing on the lactose and lactose+alphaMG selection plates were greatly diminished and not significantly different when the bacterial cells concerned carried either a DeltarecA or DeltadinB mutation. Furthermore, introducing the DeltadinB mutant allele into bacterial cells over-expressing the recA gene reduced the numbers of Lac(+) mutations to those being recovered with the DeltadinB cells. These results appear to suggest that (i) the DinB-dependent mutation generation pathway is alone responsible for spontaneous reversion of the lacI33OmegalacZ frameshift marker, and (ii) the varying numbers of Lac(+) colonies that we recover on the lactose and lactose+alphaMG plates provide a direct measure of the differential effects of these particular carbon compounds on the overall expression of the dinB gene. Interestingly, the yields of spontaneous Val mutations arising in wild-type, DeltarecA, DeltadinB and DeltaumuDC cells were found to be similar, but always tended to be highest when the medium contained only a non-repressing carbon source (glycerol) and lowest when it had been supplemented with a strong catabolite repressor such as glucose or alphaMG. Together, our results would seem to establish that stationary-phase E. coli cells exposed to strong selection pressures can accumulate spontaneous mutations via SOS-dependent and SOS-independent mutation generation pathways whose levels of expression are regulated by catabolite repression.

Morphological and electrical properties of human trophoblast choriocarcinoma, BeWo cells.

The syncytiotrophoblast of the human placenta arises from fusion of stem cells called cytotrophoblasts. The molecular mechanisms associated with cell fusion and syncytiation of cytotrophoblastic cells remain largely unknown. In the present study, we investigated the morphological and electrical properties of BeWo cells, a human choriocarcinoma-derived trophoblast cell model, with several features of the human cytotrophoblast. Cultured cells tended to cluster, but only fused into small, multinucleated syncytia in the presence of cAMP (72 h). The morphological features of both the actin and microtubular cytoskeletons indicated that within 72 h of constant exposure to cAMP, intracellular cortical actin cytoskeleton disappeared, which was the most prominent inducing factor of multi-nucleation. The presence of the cation channel protein, polycystin-2 (PC2), a TRP-type cation channel, associated with placental ion transport in term human syncytiotrophoblast, co-localised with acetylated tubulin in midbodies, but was found non-functional under any conditions. Different electrical phenotypes were observed among control BeWo cells, where only 26% (8 of 31 cells) displayed a voltage-dependent outwardly rectifying conductance. Most quiescent BeWo cells had, however, a low, slightly outwardly rectifying basal whole cell conductance. Acute exposure to intracellular cAMP (<15 min) increased the whole cell conductance by 122%, from 0.72 nS/cell to 1.60 nS/cell, and eliminated the voltage-regulated conductance. The encompassed evidence indicates that the early events in BeWo cell fusion and syncytiation occur by cAMP-associated changes in ionic conductance but not morphological changes associated to chronic exposure to the second messenger. This suggests a tight regulation, and important contribution of cation conductances in cytotrophoblastic cells prior to syncytiation.

The coordination number of silicon in silicon-oxygen compounds: the special case of 6-fold coordination in thaumasite.

In most silicon-oxygen compounds, the silicon atom is 4-fold coordinated. Exceptionally, silicon is found in 6-fold coordination, as in the mineral thaumasite, which also may be formed in the degradation, and sometimes subsequent weakening, of certain concretes. The connection between coordination number and chemical bonding in oxidic compounds generally is explored using the optical basicity concept to estimate the extent of negative charge provided by oxide(-II) donor atoms in the coordination sphere. A correlation is established between this charge and the heat of formation of silicate (per oxide(-II) atom), which indicates that reduction in charge decreases thermodynamic stability. Calculations for thaumasite show that 4-fold coordination would provide a very small charge, but that 6-fold provides a charge comparable with that for stable 4-fold coordinated silicates such as K2Si2O5.

Expression of small heat shock-related protein 20 (HSP20) in rat myometrium is markedly decreased during late pregnancy and labour.

The underlying mechanisms regulating uterine contractions during labour are still poorly understood. Heat shock protein 20 (HSP20) is known to be present at high levels in smooth muscle and implicated in muscle relaxation, but HSP20 expression in the myometrium is completely undetermined. Since HSP20 has been implicated in smooth muscle relaxation, we hypothesized that HSP20 would be highly expressed in the rat myometrium during early and mid-pregnancy when the myometrium is relatively quiescent. Northern blot analysis particularly demonstrated that HSP20 mRNA detection was significantly decreased from day (d) 22 of pregnancy to 1-day post-partum (PP) compared with d6 (P < 0.05). HSP20 mRNA detection was also significantly decreased from d22 to d23 of gestation compared with non-pregnant (NP) samples. Immunoblot analysis showed that detection of HSP20 was significantly decreased at d23 compared with d12 and d15 (P < 0.05). HSP20 detection also significantly decreased at PP compared with d15 (P < 0.05). Immunofluorescence analysis demonstrated that after d15, plasma membrane-associated localization of HSP20 decreased markedly in both circular and longitudinal muscle layers. In addition, HSP20 was detectable near cell membranes at much higher levels in the longitudinal muscle layer of progesterone-treated pregnant rats (delayed labour) at all gestational time points examined, compared with controls. Our results demonstrate that HSP20 mRNA and protein are highly expressed during early and mid-pregnancy and then the expression markedly decreases during late pregnancy and labour. The observed patterns of HSP20 expression are consistent with a potential role for HSP20 in facilitating myometrium quiescence during early and mid-pregnancy.

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line.

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.

Expression of alpha5 integrin (Itga5) is elevated in the rat myometrium during late pregnancy and labor: implications for development of a mechanical syncytium.

The underlying mechanisms controlling uterine contractions during labor are still poorly understood. Integrins are heterodimeric, transmembrane receptors composed of alpha and beta subunits that can be found in focal adhesions. Because these structures play an important role in the regulation of smooth muscle contractility and cell adhesion, we hypothesized that alpha5 integrin mRNA (Itga5) and protein (ITGA5) expression would be induced in the rat myometrium during late pregnancy and labor. Itga5 mRNA expression was significantly increased (P < 0.05) from Day 17 to labor, noticeably decreasing 1 day postpartum (PP). Immunoblot analysis illustrated a continual increase in ITGA5 levels during pregnancy, labor, and PP, with levels reaching significance at labor (P < 0.05). Analysis of ITGA5 expression by immunocytochemistry demonstrated that it is primarily localized to myometrial cell membranes in the longitudinal muscle layer of the myometrium from before pregnancy to Day 6, and in both the longitudinal and circular muscle layers from Day 15 to PP. Treatment of late-pregnant rats with progesterone blocked labor and resulted in sustained expression of Itga5 mRNA expression to Day 24. In addition, immunocytochemistry experiments showed ITGA5 was detectable at higher levels in cell membranes of both myometrial layers in progesterone-treated animals on Days 23 and 24, compared with vehicle controls. We propose that ITGA5, with its sole known partner, ITGB1, may be important in promoting cellular cohesion during late pregnancy. This process may aid the development of a mechanical syncytium for efficient force transduction during the sustained, coordinated, and powerful contractions of labor.

Small heat shock protein 27 (Hsp27) expression is highly induced in rat myometrium during late pregnancy and labour.

The underlying mechanisms that regulate uterine contractions during labour are still poorly understood. A candidate regulatory protein is heat shock protein 27 (Hsp27). It belongs to the small heat shock protein family and can regulate actin cytoskeleton dynamics, act as a chaperone, and may regulate contractile protein activation. As a result, we hypothesized that Hsp27 expression would be highly induced during late pregnancy and labour. Hsp27 mRNA expression was significantly elevated (P <0.05) on days 17 to 22 of gestation. In addition, immunoblot analysis demonstrated that detection of total Hsp27 increased (P <0.05) between day 21 and 1 day post-partum (PP) inclusive. Since phosphorylation of Hsp27 has been reported to be a prerequisite for smooth muscle contraction, we examined the temporal and spatial expression of Ser-15 phosphorylated Hsp27. Immunoblot analysis showed that the detection of Ser-15 phosphorylated Hsp27 significantly increased (P <0.05) between days 19 and 23 (active labour) inclusive, in parallel with detection of total Hsp27. Immunocytochemical analysis of Ser-15 phosphorylated Hsp27 expression in situ demonstrated that phosphorylated Hsp27 in circular muscle became detectable in peri-nuclear and membrane regions on days 19 to 22, but was primarily restricted to the cytoplasm on days 23 to PP. In contrast, phosphorylated Hsp27 in longitudinal muscle was primarily detected in myocyte membranes on days 15 to 22, and then also became detectable in the cytoplasm of myocytes on days 23 and PP. Our results demonstrate that Hsp27 expression is highly upregulated during late pregnancy and labour and suggest that Hsp27 is a potential candidate contraction-associated protein.

Radiation dose-dependent increases in inflammatory response markers in A-bomb survivors.

PURPOSE: The well-documented increases in malignant tumours in the A-bomb survivors have recently been supplemented by reports that non-cancer diseases, including cardiovascular disease, may also have increased in incidence with increasing radiation dose. Given that low-level inflammatory responses are widely accepted as a significant risk factor for such diseases, we undertook a detailed investigation of the long-term effects of ionizing radiation on the levels of the inflammatory markers C-reactive protein (CRP) and interleukin 6 (IL-6) in A-bomb survivors. MATERIALS AND METHODS: Blood samples were taken from 453 participants in a long-term epidemiological cohort of A-bomb survivors. Plasma levels of CRP and IL-6 were measured using standard antibody-mediated procedures. Relationships between CRP or IL-6 levels and radiation dose were then investigated by multivariate regression analysis. Blood lymphocytes from each individual were used for immunophenotyping by flow cytometry with murine monoclonal antibodies to CD3, CD4 and CD8. RESULTS: CRP levels were significantly increased by about 31% Gy(-1) of estimated A-bomb radiation (p=0.0001). Higher CRP levels also correlated with age, male gender, body mass index and a history of myocardial infarction. After adjustments for these factors, CRP levels still appeared to have increased significantly with increasing radiation dose (about 28% increase at 1Gy, p=0.0002). IL-6 levels also appeared to have increased with radiation dose by 9.3% at 1Gy (p=0.0003) and after multiple adjustments by 9.8% at 1Gy (p=0.0007). The elevated CRP and IL-6 levels were associated with decreases in the percentages of CD4(+) helper T-cells in peripheral blood lymphocyte populations. CONCLUSIONS: Our results appear to indicate that exposure to A-bomb radiation has caused significant increases in inflammatory activity that are still demonstrable in the blood of A-bomb survivors and which may lead to increased risks of cardiovascular disease and other non-cancer diseases.