Although Bid is considered to be a cell apoptotic mediator, current studies suggest that it has a possible role in cell survival for mouse embryonic fibroblasts (MEFs) in response to low doses of anti-(±)-5- methylchrysene-l,2-diol-3,4-epoxide (<0.25µM) (5-MCDE). We found that the exposure of MEFs to 0.25 µM 5-MCDE resulted in a slight apoptotic induction, while this apoptotic response was substantially increased in the Bid knockout MEFs (Bid−/−), suggesting that there is a Bid-mediated anti-apoptotic function in this response. This notion was further supported by the findings that re-constitution expression of Bid into Bid−/− cells could inhibit the increased apoptosis. Further studies show that the antiapoptotic function of Bid was associated with its mediation of COX-2 expression. This conclusion was based the reduction of COX-2 expression in Bid−/− cells, the restoration of low sensitivity to 5-MCDE-induced apoptosis by the introduction of Bid into Bid−/− cells, and increased sensitivity of WT MEFs to 5-MCDE-induced apoptosis by the knockdown of COX-2 expression. Furthermore, we found that Bid mediated COX-2 expression through the IKKβ/NFκB pathway because the deficiency of Bid in Bid−/− MEFs resulted in the blockade of IKK/NFκB activation and knockout of IKKβ caused abrogation of COX-2 expression induced by 5-MCDE. Collectively, our results demonstrate that Bid is critical for COX-2 induction through the IKKβ/NFκB pathway, which mediates its anti-apoptotic function, in cell response to low doses of 5-MCDE exposure.
Bid; COX-2; 5-MCDE; NFκB; apoptosis
Cyclooxygenase-2 (COX-2) is a critical enzyme implicated in chronic inflammation-associated cancer development. Our studies have shown that the exposure of Beas-2B cells, a human bronchial epithelial cell line, to lung carcinogenic nickel compounds results in increased COX-2 expression. However, the signaling pathways leading to nickel-induced COX-2 expression are not well understood. In the current study, we found that the exposure of Beas-2B cells to nickel compounds resulted in the activation of both nuclear factor of activated T cell (NFAT) and nuclear factor-κB (NF-κB). The expression of COX-2 induced upon nickel exposure was inhibited by either a NFAT pharmacological inhibitor or the knockdown of NFAT3 by specific siRNA. We further found that the activation of NFAT and NF-κB was dependent on each other. Since our previous studies have shown that NF-κB activation is critical for nickel-induced COX-2 expression in Beas-2B cells exposed to nickel compounds under same experimental condition, we anticipate that there might be a cross-talk between the activation of NFAT and NF-κB for the COX-2 induction due to nickel exposure in Beas-2B cells. Furthermore, we showed that the scavenging of reactive oxygen species (ROS) by introduction of mitochondrial catalase inhibited the activation of both NFAT and NF-κB, and the induction of COX-2 due to nickel exposure. Taken together, our results defining the evidence showing a key role of the cross-talk between NFAT and NF-κB pathways in regulating nickel-induced COX-2 expression, further provide insight into the understanding of the molecular mechanisms linking nickel exposure to its lung carcinogenic effects.
Beas-2B cells; COX-2; nickel; NFAT; NF-κB; ROS
Metal and its oxide nanoparticles show ideal pharmacological activity, especially in anti-tumor therapy. Our previous study demonstrated that cuprous oxide nanoparticles (CONPs) selectively induce apoptosis of tumor cells in vitro. To explore the anti-tumor properties of CONPs in vivo, we used the particles to treat mouse subcutaneous melanoma and metastatic lung tumors, based on B16-F10 mouse melanoma cells, by intratumoral and systemic injections, respectively. The results showed that CONPs significantly reduced the growth of melanoma, inhibited the metastasis of B16-F10 cells and increased the survival rate of tumor-bearing mice. Importantly, the results also indicated that CONPs were rapidly cleared from the organs and that these particles exhibited little systemic toxicity. Furthermore, we observed that CONPs targeted the mitochondria, which resulted in the release of cytochrome C from the mitochondria and the activation of caspase-3 and caspase-9 after the CONPs entered the cells. In conclusion, CONPs can induce the apoptosis of cancer cells through a mitochondrion-mediated apoptosis pathway, which raises the possibility that CONPs could be used to cure melanoma and other cancers.
cuprous oxide nanoparticles; melanoma; apoptosis; mitochondrion-mediated apoptosis; anti-tumor nanomedicine
The corpus luteum (CL) is a transient endocrine organ that is essential for maintenance of pregnancy in both ruminants and primates. The cellular and endocrine mechanisms that regulate the CL in these species have commonalities and some distinct and intriguing differences. Both species have similar cellular content with large luteal cells derived from the granulosa cells of the follicle, small luteal cells from follicular thecal cells, and large numbers of capillary endothelial cells that form the vasculature that has an essential role in optimal CL function. Intriguingly, the large luteal cells in ruminants grow larger than in primates and acquire a capacity for high constitutive progesterone (P4) production that is independent of stimulation from LH. In contrast, the primate CL and the granulosa lutein cells from primates continue to require stimulation by LH/CG throughout the luteal phase. Although the preovulatory follicle of women and cows had similar size and steroidogenic output (10 to 20 mg/h), the bovine CL had about ten-fold greater P4 output compared to the human CL (17.4 vs. 1.4 mg/h), possibly due to the development of high constitutive P4 output by the bovine large luteal cells. The continued dependence of the primate CL on LH/CG/cAMP also seems to underlie luteolysis, as there seems to be a requirement for greater luteotropic support in the older primate CL than is provided by the endogenous LH pulses. Conversely, regression of the ruminant CL is initiated by PGF from the nonpregnant uterus. Consequently, the short luteal phase in ruminants is primarily due to premature secretion of PGF by the nonpregnant uterus and early CL regression, whereas CL insufficiency in primates is related to inadequate luteotropic support and premature CL regression. Thus, the key functions of the CL, pregnancy maintenance and CL regression in the absence of pregnancy, are produced by common cellular and enzymatic pathways regulated by very distinct luteotropic and luteolytic mechanisms in the CL of primates and ruminants.
corpus luteum; primate; progesterone; ruminant
picardial adipose tissue is associated with coronary artery disease, however the causal relationship between perivascular adipose tissue and local atherogenesis is unclear.
Methods and Results
Apolipoprotein E deficient (ApoE−/−) mice underwent transplantation of visceral or subcutaneous adipose tissue immediately adjacent to the right common carotid artery. Carotid arteries with fat transplants were analyzed for atherosclerosis by surface oil-red-O staining and cross-sectional analysis. Vascular function of the carotid arteries was assessed using pressure myography. Visceral fat transplants were also performed to ApoE−/− mice with neutralization of P-selectin glycoprotein ligand-1 (Psgl-1). Atherosclerosis surface area and lesion thickness were greater in mice receiving the perivascular visceral fat compared to the subcutaneous fat. Mice with visceral fat transplants also displayed more complicated atherosclerotic lesions with evidence of atherothrombosis. Serum Mcp-1 was higher in mice receiving visceral fat transplants compared to subcutaneous transplants. Visceral fat transplantation also caused impaired endothelial-dependent relaxation of the carotid artery. Psgl-1 deficiency or neutralization of Psgl-1 with an anti-Psgl-1 antibody was protective against perivascular visceral adipose tissue-induced atherosclerosis and was associated with reduced Mcp-1 levels.
Perivascular visceral fat leads to endothelial dysfunction and accelerated atherosclerosis. This proatherogenic effect of perivascular adipose tissue is blocked by neutralization of Psgl-1.
atherosclerosis; adipocyte; cytokine; visceral obesity
The aim of the study was to describe emtricitabine pharmacokinetics during pregnancy and postpartum.
The International Maternal Pediatric and Adolescent AIDS Clinical Trials (IMPAACT), formerly Pediatric AIDS Clinical Trials Group (PACTG), study P1026s is a prospective pharmacokinetic study of HIV-infected pregnant women taking antiretrovirals for clinical indications, including a cohort taking emtricitabine 200 mg once daily. Intensive steady-state 24-hour emtricitabine pharmacokinetic profiles were performed during the third trimester and 6–12 weeks postpartum, and on maternal and umbilical cord blood samples collected at delivery. Emtricitabine was measured by liquid chromatography–mass spectrometry with a quantification limit of 0.0118 mg/L. The target emtricitabine area under the concentration versus time curve, from time 0 to 24 hours post dose (AUC0-24), was ≥7 mg h/L (≤30% reduction from the typical AUC of 10 mg h/L in nonpregnant historical controls). Third-trimester and postpartum pharmacokinetics were compared within subjects.
Twenty-six women had pharmacokinetics assessed during the third trimester (median 35 weeks of gestation) and 22 postpartum (median 8 weeks postpartum). Mean [90% confidence interval (CI)] emtricitabine pharmacokinetic parameters during the third trimester vs. postpartum were, respectively: AUC: 8.0 (7.1–8.9) vs. 9.7 (8.6–10.9) mg h/L (P = 0.072); apparent clearance (CL/F): 25.0 (22.6–28.3) vs. 20.6 (18.4–23.2) L/h (P = 0.025); 24 hour post dose concentration (C24): 0.058 (0.037–0.063) vs. 0.085 (0.070–0.010) mg/L (P = 0.006). The mean cord:maternal ratio was 1.2 (90% CI 1.0–1.5). The viral load was <400 HIV-1 RNA copies/mL in 24 of 26 women in the third trimester, in 24 of 26 at delivery, and in 15 of 19 postpartum. Within-subject comparisons demonstrated significantly higher CL/F and significantly lower C24 during pregnancy; however, the C24 was well above the inhibitory concentration 50%, or drug concentration that suppresses viral replication by half (IC50) in all subjects.
While we found higher emtricitabine CL/F and lower C24 and AUC during pregnancy compared with postpartum, these changes were not sufficiently large to warrant dose adjustment during pregnancy. Umbilical cord blood concentrations were similar to maternal concentrations.
emtricitabine; HIV; pregnancy; pharmacokinetics
A biomimetic replacement for tooth enamel is urgently needed because dental caries is the most prevalent infectious disease to affect man. Here, design specifications for an enamel replacement material inspired by Nature are deployed for testing in an animal model. Using genetic engineering we created a simplified enamel protein matrix precursor where only one, rather than dozens of amelogenin isoforms, contributed to enamel formation. Enamel function and architecture were unaltered, but the balance between the competing materials properties of hardness and toughness was modulated. While the other amelogenin isoforms make a modest contribution to optimal biomechanical design, the enamel made with only one amelogenin isoform served as a functional substitute. Where enamel has been lost to caries or trauma a suitable biomimetic replacement material could be fabricated using only one amelogenin isoform, thereby simplifying the protein matrix parameters by one order of magnitude.
Biomimetic material; Biomineralization; ECM (extracellular matrix); Fracture toughness: Genetic engineering; Mechanical properties; Molecular biology
Background and Objectives
Studies have demonstrated that some cutaneous biophysical properties vary with age, gender and body sites. However, the characteristics of the skin friction coefficient in different genders and age groups have not yet been well established. In the present study, we assess the skin friction coefficient in a larger Chinese population.
A total of 633 subjects (300 males and 333 females) aged 0.15–79 years were enrolled. A Frictiometer® FR 770 and Corneometer® CM 825 (C&K MPA 5) were used to measure the skin friction coefficient and stratum corneum hydration, respectively, on the dorsal surface of the hand, the forehead and the canthus.
In the females, the maximum skin friction coefficients on both the canthus and the dorsal hand skin were observed around the age of 40 years. In the males, the skin friction coefficient on the dorsal hand skin gradually increased from 0 to 40 years of age, and changed little afterward. Skin friction coefficients on some body sites were higher in females than in age-matched males in some age groups. On the canthus and the dorsal hand skin of females, a positive correlation was found between skin friction coefficient and stratum corneum hydration (p < 0.001 and p < 0.0001, respectively). In contrast, in males, the skin friction coefficient was positively correlated with stratum corneum hydration on the forehead and the dorsal hand skin (p < 0.05 and p < 0.0001, respectively).
The skin friction coefficient varies with age, gender and body site, and positively correlates with stratum corneum hydration on some body sites.
Age; Gender; Stratum corneum hydration; Skin friction coefficient
Fenofibrate has been widely used for the treatment of dyslipidaemia with a long history. Species differences of its metabolism were reported, but its metabolites in rodent have not been fully investigated.Urine and plasma samples were collected before and after oral dosages of fenofibrate in Sprague–Dawley rats. Urine samples were subjected to ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) analysis, and projection to latent structures discriminant analysis was used for the identification of metabolites.New metabolites in urine and plasma were also studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The metabolism pathway was studied in rat hepatocytes. Synthesized and purchased authentic compounds were used for metabolite identification by LC-MS/MS.Five ever-reported metabolites were identified and another four new ones were found. Among these new metabolites, fenofibric acid taurine and reduced fenofibric acid taurine indicate new phase II conjugation pathway of fenofibrate.
Metabolomics; metabolites; metabolism pathway; fenofibrate; rat
Wnt/β-catenin signaling plays a pivotal role in modulating cellular proliferation, differentiation, tissue organization and embryonic development. Earlier, we found that the endocytic adaptor disabled-2 (Dab2) could attenuate Wnt/β-catenin signaling by stabilizing Axin and preventing its translocation to the membrane. Recently, protein phosphatase 1 (PP1) has been shown to interact with, and dephosphorylate Axin, leading to its destabilization. Here, we show that Dab2 functions upstream of PP1 to block the interaction between Axin and PP1, inhibiting Axin dephosphorylation and thereby stabilizing its expression, ultimately leading to inhibition of Wnt/β-catenin. We show that Dab2 acts as a competitive inhibitor of PP1 by binding to the same C-terminal domain of Axin. Both PP1 and Axin bind to the N-terminus of Dab2 and a Dab2 truncation mutant consisting of the N-terminal phosphotyrosine binding domain blocks PP1–Axin interactions and inhibits Wnt signaling. We confirm the inhibitory effect of Dab2 on Wnt/β-catenin signaling in zebrafish embryos, showing that its ectopic expression phenocopies Axin overexpression resulting in altered dorsoventral patterning. We conclude that Dab2 stabilizes Axin and attenuates Wnt/β-catenin signaling by preventing PP1 from binding Axin.
Disabled-2 (Dab2); Axin–protein phosphatase 1 (PP1); Wnt–β-catenin signaling
The ErbB3 binding protein (Ebp1) is a transcriptional corepressor that inhibits the activity of proliferation-associated genes and the growth of human breast cancer cell lines. Treatment of breast cancer cells with the ErbB3 ligand heregulin (HRG) results in increased phosphorylation of Ebp1 and transcriptional repression. The p21-activated serine/threonine kinase 1 (PAK1), which plays an important role in breast cancer progression and resistance to the anti-oestrogen tamoxifen, is also activated by HRG. We therefore examined the ability of PAK1 to phosphorylate and regulate the function of Ebp1. We found that PAK1 phosphorylated Ebp1 in vitro and mapped the phosphorylation site to threonine 261. Both HRG treatment and expression of a constitutively activated PAK1 in MCF-7 breast cancer cells enhanced threonine phosphorylation of Ebp1. In MCF-7 cells, ectopically expressed Ebp1 bound endogenous PAK1 and this association was enhanced by treatment with HRG. Mutation of the PAK1 phosphorylation site to glutamic acid, mimicking a phosphorylated state, completely abrogated the ability of Ebp1 to repress transcription, inhibit growth of breast cancer cell lines and contribute to tamoxifen sensitivity. These studies demonstrate for the first time that Ebp1 is a substrate of PAK1 and the importance of the PAK1 phosphorylation site for the functional activity of Ebp1 in breast cancer cells.
Ebp1; PA2G4; phosphorylation; PAK1; breast cancer
Serodiagnosis of human immunodeficiency virus (HIV) infection in the United States has traditionally relied on a sequential two-test algorithm: an initial screen with an enzyme immunoassay (EIA) and reflex testing of EIA-reactive specimens with a more specific supplemental test such as Western blotting or immunofluorescence. The supplemental tests are tedious, subjective, and expensive. In addition, there have been major improvements in the performance and accuracy of the EIA tests as well as the introduction of rapid serologic tests (RT) and HIV nucleic acid amplification tests (NAAT). Related to these improvements is the possibility that alternative algorithms using combinations of currently approved HIV tests may function as well as if not better than the current algorithm, with more flexibility, improved accuracy, and lower cost. To this end, we evaluated the performance of 12 currently licensed tests and 1 in-house HIV test (6 EIA, 4 RT, and 3 NAAT) on panels of plasma samples from HIV-infected (n = 621 HIV type 1 [HIV-1] and 34 HIV-2) and uninfected (n = 513) people and of sequential specimens from people early in seroconversion (183 specimens from 15 patients). Test combinations were analyzed in two dual-test (sensitivity-optimized and specificity-optimized) algorithms and in a three-test (tie-breaking) algorithm, and performance was compared to the conventional algorithm. The results indicate that alternative algorithm strategies with currently licensed tests compare favorably with the conventional algorithm in detecting and confirming established HIV infection. Furthermore, there was a lower frequency of discordant or indeterminate results that require follow-up testing, and there was improved detection of early infection.
To analyse the neointimal coverage of sirolimus-eluting stent (SES) and bare-metal stent (BMS) visualised in vivo by optical coherence tomography (OCT).
OCT images were obtained in 26 coronary vessels of 24 patients at 5–93 months after SES or BMS deployment. The short-term BMS group (BMS1) consisted of eight BMS in seven patients at 5–10 months of follow-up, the long-term BMS group (BMS2) consisted of six BMS in six patients at 23–93 months of follow-up, and the SES group (SES) consisted of 13 SES in 10 patients at 6–12 months of follow-up. The strut apposition, strut coverage and mean maximal and minimal neointimal thicknesses (NIT) for both BMS groups and SES were compared.
OCT images were acquired successfully. Significant differences between completely apposed and malapposed stent struts (p<0.0001) and between covered and uncovered stent struts (p<0.0001) were found among the three groups. The mean maximal and minimal NIT in the SES group were all significantly less than those of the BMS1 or BMS2 group, the minimal NIT in the BMS1 group was significantly less than that of the BMS2 but the mean maximal NIT was no significant difference between the BMS1 and BMS2 groups. In an open bifurcation artery, 19 struts visualised by OCT had no discernible coverage or were surrounded by either thrombus or a thick tissue layer.
OCT imaging can clearly visualise stent apposition and neointimal coverage of stent struts. Incomplete strut apposition and lack of strut coverage occurred with a significantly higher frequency in SES than in BMS. These findings may explain the occurrence of late thrombosis in SES.
The Nef from a highly virulent strain of simian immunodeficiency virus (SIV), SIVpbj14, and a Nef from the traditional strain SIVmac239 bearing the mutation from RQ to YE (YE-Nef) both induce an acute lethal disease in monkeys. The YE mutation and its surrounding sequence resemble the immunoreceptor tyrosine-based activation motif (ITAM), which is present in the cytoplasmic tail of T- and B-cell antigen receptors and mediates signaling during lymphocyte activation. We show here that the ITAM from YE-Nef performs the same function. First, not only does YE-Nef increase the activity of the transcription factor NFAT, which is one of the downstream targets of T-cell activation, but the ITAM from the YE-Nef by itself also activates NFAT. Second, the ITAM from YE-Nef is phosphorylated on tyrosine residues by Lck and associates with ZAP-70, a T-cell-specific tyrosine kinase. The phosphorylation of both conserved tyrosine residues on the ITAM is required for the recruitment of ZAP-70. Finally, Lck is required for the activation of NFAT by YE-Nef. These results demonstrate that YE-Nef contains a functional ITAM and elucidate the molecular mechanisms underlying the pathogenesis of SIVpbj14.
Results from an animal model of vaginal candidiasis suggest that Candida-specific cell-mediated immunity in the systemic circulation does not mediate protection against vaginitis. The present study used cellular depletion analysis to examine the circulation of immune effector function between the vagina and the periphery. Results showed that anti-Thy-1.2 antibodies given intravenously to mice depleted Thy-1+ T lymphocytes in the systemic compartment but not in the vaginal mucosa, while the same antibodies injected intravaginally significantly reduced Thy-1+ T cells in both the vaginal and systemic compartments. These results support a lack or low level of circulation of immune effector function from the periphery to the vaginal mucosa.
Previously, model systems were developed in our laboratory to study murine immune responses to the 23-valent pneumococcal polysaccharide vaccine Pnu-Imune, both in vivo and in vitro (M. Garg and B. Subbarao, Infect. Immun. 60:2329-2336, 1992; M. Garg, A. M. Kaplan, and S. Bondada, J. Immunol. 152: 1589-1596, 1994). Using these systems, we found that aged mice did not respond to the vaccine in vivo or in vitro. Cell separation studies showed that the unresponsiveness of the aged spleen cells to the vaccine was not due to an intrinsic B-cell defect or to T-cell-mediated immunosuppression but resulted from an accessory cell deficiency. Irradiated spleen cells from young mice enabled the old mouse spleen cells to respond to the vaccine. Interestingly, irradiated spleen cells from old mice also restored the vaccine responsiveness in old mice but were required in greater numbers than the young mouse spleen cells to induce similar levels of response. The accessory cell was an adherent cell that could be removed by passage through Sephadex G-10 and thus may be a macrophage. Accessory function could also be provided by the cytokine interleukin-1 (IL-1), IL-4, or IL-5 but not IL-2 or IL-6. Thus, one reason for the deficient immune response to pneumococcal vaccine in aged mice is a quantitative defect in adherent accessory cells.
Growth of cells in vitro in the presence of fatty acids can alter the membrane composition and hence fluidity and permeability. Exposure of both doxorubicin (2780AD) and cisplatin (2780CP) resistant human ovarian cell lines to non-toxic concentrations of polyunsaturated fatty acids (gamma-linolenic acid and eicosapentaenoic acid) either before or during exposure to the cytotoxic drug did not modulate drug sensitivity. However, the fatty acids were toxic in their own right. Whilst the ovarian cell lines 2780AD and 2780CP showed a small degree of cross resistance to both fatty acids the doxorubicin resistant breast cell line MCF7/Adr was slightly more sensitive than MCF7. When the interactions between the polyunsaturated fatty acids and cytotoxic drugs were analysed by the isobologram method the toxicities were shown to be additive. The combination of polyunsaturated fatty acids and cytotoxic drugs may have clinical potential provided that the normal tissue toxicities of the two treatments are not additive.
Four purine and two pyrimidine 2',3'-dideoxynucleosides were studied for their ability to inhibit duck hepadnavirus replication. The purine 2',3'-dideoxynucleosides were more potent antiviral agents than the pyrimidine 2',3'-dideoxynucleosides. The concentration for 50% inhibition of viral replication (IC50) was determined for each of the effective agents. Two drugs with low IC50s, 2,6-diaminopurine 2',3'-dideoxyriboside and 2',3'-dideoxyadenosine, were chosen for in vivo studies. Animals received 10 mg/kg intramuscularly twice daily. Rapid clearance of hepadnavirus DNA from the sera of the animals was seen as a result of treatment with 2,6-diaminopurine 2',3'-dideoxyriboside; however, treatment with 2',3'-dideoxyadenosine did not clear the virus.