Cloning, identification, and functional characterization of novel prophenoloxidases (ShproPO) from the freshwater crab Sinopotamon henanense in response to cadmium exposure and Aeromonas hydrophila infection.

Prophenoloxidase (proPO) is essential in the prophenoloxidase-activating system (proPO-AS) which is important for defense against foreign infection in crustaceans. However, most studies have focused on expression in the presence of a single pathogenic bacterium, and very few have addressed the presence of environmental contaminants simultaneously, such as cadmium (Cd) and Aeromonas hydrophila. Our study aimed to investigate the function of proPO in the freshwater crab Sinopotamon henanense and the changes in its expression by Cd and infection of A. hydrophila. A novel proPO from the hemocytes of S. henanense (ShproPO) was found in this research, the full-length cDNA of ShproPO was 2620 bp of encoding a protein of 678 amino acids containing three typical hemocyanin domains. The ShproPO protein could be found in both the granular (GHc) and the semi-granular hemocytes (SGHc). The ShproPO mRNA was found to be abundantly expressed in hemocytes and could be influenced by A. hydrophila infection. These results indicate that ShproPO could be involved in the antibacterial process. Further research found that low concentrations of Cd could promote its expression after infection with A. hydrophila. Therefore, it was hypothesized that Cd disrupted the response of crabs to A. hydrophila infection. Subsequently, PO enzyme activity was found to be significantly reduced through in vivo RNA interference with ShproPO, and the results suggested that ShproPO is likely to be a key enzyme in the melanization response. Finally, ShproPO was found to significantly enhance the phagocytosis of A. hydrophila-infected hemocytes by in vitro recombination, confirming that ShproPO is involved in hemocyte-mediated melanization and phagocytosis. Our findings reveal completely new insight into the immunotoxicity of Cd and the immune function of ShproPO in S. henanense.

Enhancing expression of the pseudorabies virus glycoprotein E in yeast and its application in an indirect sandwich ELISA.

AIMS: The purpose of this study was to produce a recombinant pseudorabies virus (PRV) glycoprotein E (gE) protein with the correct antigenicity for use as a low-cost diagnostic antigen. METHODS AND RESULTS: The gene fragment encoding the amino-terminal immunodominant region of PRV gE (codons 31-270) (gEN31-270) was codon optimized and expressed constitutively and secreted using a Pichia pastoris expression system. Yeast-expressed gEN31-270 (ygEN31-270) was harvested from the culture supernatant, and ygEN31-270 was shown to exhibit N-linked glycosylation. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) was developed using ygEN31-270 as a coating antigen, and the results showed that the assay had high sensitivity and specificity, as well as almost perfect concordance with a commercial gE ELISA kit. CONCLUSIONS: The immunodominant region (amino acids 31-270) of gE was expressed successfully in P. pastoris using a codon optimization strategy. ygEN31-270 was secreted and N-glycosylated. The ygEN31-270-based indirect sandwich ELISA showed high sensitivity and specificity to detect gE-specific antibodies in swine serum samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ygEN31-270-based indirect sandwich ELISA may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost.

Two-Dimensional Nanoparticle Cluster Formation in Supercritical Fluid CO2.

Supercritical fluid carbon dioxide (sc-CO2) is capable of depositing nanoparticles in small structures of silicon substrates because of its gas-like penetration, liquid-like solvation abilities, and near-zero surface tension. In nanometer-sized shallow wells on silicon surface, formation of two-dimensional (2D) monolayer metal nanoparticle (NP) clusters can be achieved using the sc-CO2 deposition method. Nanoparticles tend to fill nanostructured holes first, and then, if sufficient nanoparticles are available, they will continue to cover the flat areas nearby, unless defects or other surface imperfections are available. In addition, SEM images of two-dimensional gold (Au) nanoparticle clusters formed on a flat silicon surface with two to a dozen or more of the nanoparticles are provided to illustrate the patterns of nanoparticle cluster formation in sc-CO2.

Impact of lower concentrations of phytoestrogens on the effects of estradiol in breast cancer cells.

OBJECTIVE: To explore the impact of lower concentrations of phytoestrogens on 17ß-estradiol (E2) in the growth of MCF-7 breast cells. METHODS: MCF-7 cells were treated with E2 (10(-8) mol/l), one of three phytoestrogens (genistein, resveratrol, and quercetin) (10(-7)  to 10(-5) mol/l), and with a combination of both E2 and one of these phytoestrogens for 48 h. These cells were then extracted for MTT and TUNEL assay. Western blot was utilized to evaluate the proteins involved in the proliferative and apoptotic pathways, as well as the differential effects on ERα and ß. RESULTS: MCF-7 cell proliferations were induced by both E2 alone and E2 plus one of the three phytoestrogens (at concentrations ≥ 10(-6) mol/l). Apoptotic cells were significantly increased in the phytoestrogen-treated MCF-7 cells and, conversely, suppressed in the cells treated with both E2 and phytoestrogens. Proliferating cell nuclear antigen, PI3K and p-Akt were increased in the cultures with E2 and substantially more in the cultures with E2 plus a phytoestrogen. The combination of E2 and phytoestrogen significantly inhibited the increase in FADD, cytochrome C, truncated Bid, caspase-9, caspase-3 and ERß that was induced by phytoestrogens in the MCF-7. ERα expression was significantly induced by E2 regardless of the presence of these phytoestrogens. CONCLUSIONS: This study demonstrates that, in the presence of E2, genistein, resveratrol, and quercetin may stimulate breast cancer cells, even at low physiological concentrations. Therefore, the conflicting results regarding the effects of phytoestrogens on breast cells may be attributed to the endogenous estrogen present in breast tissue.

Phytoestrogens induce apoptosis through a mitochondria/caspase pathway in human breast cancer cells.

OBJECTIVE: To explore the effect and pathway of phytoestrogens on the growth of breast cancer cell line MCF-7. METHODS: MCF-7 cells (human estrogen receptor-positive and progesterone receptor-positive breast cancer cells) were cultured in serum-free medium for 24 h and then treated with genistein, resveratrol, and quercetin (10(-10)-10(-4) mol/l). After further incubation for 24, 48, 72, and 92 h, the cells were harvested and extracted for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. According to the above results, the proteins involving proliferative and apoptotic pathways were evaluated by Western blot analysis. RESULTS: Genistein, resveratrol, and quercetin significantly inhibited cellular proliferation in a dose- and time-dependent manner. Significantly elevated caspase-3 activity was noted after treatment with genistein (10(-9)-10(-7) mol/l), as well as with resveratrol and quercetin (10(-9)-10(-5) mol/l). Significant reduction of PI3K and AKT protein and significant increase of Fas ligand, Fas-associated protein with death domain, cytochrome C, truncated Bid, caspase-9, and caspase-3 were all noted after genistein, resveratrol, and quercetin treatment. 17ß-Estradiol induced completely opposite results. Estrogen receptor (ER) α expression was significantly increased with 17ß-estradiol, whereas ERß expression was significantly elevated in the cultures with genistein, resveratrol, and quercetin. CONCLUSIONS: These data demonstrate that genistein, resveratrol, and quercetin have antiproliferative effects on breast cancer cells. Their induction of apoptosis involves the activation of both the intrinsic and extrinsic apoptotic pathways, which may be related to the differential affinity to ERα and ERß. Whether phytoestrogens have similar effects on normal breast cells remains to be examined.

Lower concentrations of phthalates induce proliferation in human breast cancer cells.

OBJECTIVE: To explore the effect and pathway of phthalates on the growth of MCF-7 breast cancer cells. METHODS: MCF-7 cells were treated with benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP) (10(-10)-10(-4) mol/l). After incubation for 24, 48, 72, and 92 h, the cells were harvested and extracted for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The proteins involving proliferative and apoptotic pathways were evaluated by Western blot analysis. RESULTS: MTT assay revealed cell toxicity at more than 10(-5) mol/l for DEHP and at 10(-4) mol/l for both BBP and DBP in MCF-7 cells. Cell proliferation was significantly increased at 10(-8)-10(-5) mol/l of BBP and DBP, and at 10(-8)-10(-6) mol/l of DEHP treatment. Proliferating cell nuclear antigen (PCNA) was substantially increased in cultures with DEHP (10(-8)-10(-6) mol/l), BBP (10(-8)-10(-5) mol/l), and DBP (10(-7)-10(-5) mol/l). Obvious increases in PI3K, p-AKT, and PCNA were noted in cultures with 17ß-estradiol, BBP, DBP, and DEHP. Estrogen receptor α expression was also notably increased in treatment with estradiol, BBP, DBP, and DEHP. CONCLUSIONS: The present study demonstrates that, even at a very low concentration, BBP, DBP, and DEHP were not only still capable of inducing a proliferative effect through the PI3K/AKT signaling pathway but also displaying estrogenic activity. Therefore, the current reference doses for phthalates defined by governments should be further evaluated.

Phytoestrogens induce differential effects on both normal and malignant human breast cells in vitro.

OBJECTIVE: To explore the effect and pathway of phytoestrogens in vitro on the growth of both normal and malignant breast cells. METHODS: Normal breast MCF-10A cells and breast cancer MCF-7 cells were incubated with 10 (-10)-10(-4)  mol/l genistein, resveratrol, and quercetin (plasma concentrations in human: 10 nmol/l-10 µmol/l) for 48 h and were then extracted for a cell proliferation assay (MTT), and for a cell death assay (TUNEL) assay. The proteins involved in the proliferative and apoptotic pathways were evaluated by Western blot analysis. Additionally, a comparison with 17ß-estradiol as well as an evaluation of the differential effects on estrogen receptors (ER) α and ß were performed. RESULTS: MCF-7 cell proliferation was significantly inhibited at the concentrations greater than 10(-4) mol/l for all three phytoestrogens and from 10 (-5) mol/l for resveratrol and quercetin. MCF-10A cell proliferation was significantly increased at the concentrations from 10 (-8) to 10 (-5) mol/l for genistein and resveratrol and only at 10 (-5) mol/l for quercetin. Apoptotic cells were significantly increased by these phytoestrogens in the MCF-7 cells. At a concentration of 10 (-7)  mol/l of these phytoestrogens, a significant reduction of PI3K and Akt and an increase of Fas ligand, Fas-associated protein with death domain, cytochrome C, truncated Bid, caspase-9, and caspase-3 were noted in the MCF-7; PI3K and Akt were significantly increased in the MCF-10A. ERß expression was significantly elevated in MCF-10A and MCF-7 with these phytoestrogens. The effects of estradiol on normal and malignant breast cells were completely opposite to those of phytoestrogens. CONCLUSIONS: This study demonstrates that phytoestrogens have antiproliferative effects on breast cancer cells via an ER-dependent mechanism, even at low concentrations, but are also capable of maintaining the survival of normal breast cell via ER-independent or other mechanisms.

Probable Blastomyces dermatitidis infection in a young rat.

A 21-week-old male untreated control SHR/NCrlNarl rat was found dead during an experiment. Grossly, pulmonary lesions were characterized by multifocal to coalescing firm gray-white nodules randomly scattered on the surface. Microscopically, bronchopneumonia was found with pyogranulomas containing neutrophils, macrophages, and numerous thick-walled yeast cells. Yeast cells, 5 to 25 µm in diameter, with no branching of hyphae were observed by staining with hematoxylin and eosin, Diff-Quik, and periodic acid-Schiff. Furthermore, polymerase chain reaction (PCR) using panfungal and nested PCR primers were used for detection of Blastomyces dermatitidis DNA in the lung tissue. After sequencing and matching with DNA sequences in the GenBank, the sample showed a similarity of 94.6% and 97% to Ajellomyces dermatitidis (B. dermatitidis), respectively. On the basis of these results, probable pulmonary blastomycosis was diagnosed. The origin of the infection in the colony rat is undetermined.

Supercritical fluid deposition of uniform PbS nanoparticle films for energy-transfer studies.

Using supercritical fluid CO(2) (Sc-CO(2)) as a medium, PbS nanoparticles can be uniformly deposited on surfaces of various substrates. Sc-CO(2) deposition of PbS nanoparticles on carbon-coated copper grids, into small holes in silicon, and formation of uniform PbS nanoparticle films on glass are described. Fluorescence spectra of PbS nanoparticles obtained from the films prepared by the Sc-CO(2) method indicate effective energy transfer between PbS nanoparticles of different sizes.

Free-standing arrays of isolated TiO2 nanotubes through supercritical fluid drying.

A common complication in fabricating arrays of TiO(2) nanotubes is that they agglomerate into tightly packed bundles during the inevitable solvent evaporation step. This problem is particularly acute for template-fabricated TiO(2) nanotubes, as the geometric tunability of this technique enables relatively large inter-pore spacings or, from another perspective, more space for lateral displacement. Our work showed that agglomeration results from the surface tension forces that are present as the ambient solvent is evaporated from the nanotube film. Herein, we report a processing and fabrication approach that utilizes supercritical fluid drying (CO(2)) to prepare arrays of template-fabricated TiO(2) nanotubes that are free-standing and spatially isolated. This approach could be beneficial to many emerging technologies, such as solid-state dye-sensitized solar cells and vertically-oriented carbon nanotube electrodes.

Effect of antidiabetic agents added to metformin on glycaemic control, hypoglycaemia and weight change in patients with type 2 diabetes: a network meta-analysis.

AIM: Most guidelines recommend metformin as first-line therapy in patients with type 2 diabetes. However, the choice of a second-line drug lacks consistent consensus. We aimed to assess available information of antidiabetic drugs added to metformin on the change in glycated haemoglobin A1c (A1C), risk of hypoglycaemia and change in body weight. METHODS: PubMed and Cochrane Central Register of Controlled Trials were searched for randomized controlled trials (RCTs) written in English through December 2011. We analysed direct and indirect comparisons of different treatments using Bayesian network meta-analysis. RESULTS: Thirty-nine RCTs involving 17 860 individuals were included. Glucagon-like peptide-1 (GLP-1) analogues resulted in greater decrease in A1C compared with sulfonylureas, glinides, thiazolidinediones, α-glucosidase inhibitors and DPP-4 inhibitors [-0.20% (95% CI -0.34 to -0.04%), -0.31% (95% CI -0.61 to -0.02%), -0.20% (95% CI -0.38 to -0.00), -0.36% (95% CI -0.64 to -0.07%), -0.32% (95% CI -0.47 to -0.17%), respectively] and was comparable with basal insulin and biphasic insulin. A1C decrease was greater for sulfonylureas compared with DPP-4 inhibitors [-0.12% (-0.23 to -0.03%)], and for biphasic insulin compared with glinides (-0.36%; 95% CI -0.82 to -0.11%). Compared with placebo, the risk of hypoglycaemia was increased in the sulfonylureas, glinides, basal insulin and biphasic insulin. Weight increase was seen with sulfonylureas, glinides, thiazolidinediones, basal insulin and biphasic insulin, and weight loss was seen with α-glucosidase inhibitors and GLP-1 analogues. CONCLUSIONS: Biphasic insulin, GLP-1 analogues and basal insulin were ranked the top three drugs in terms of A1C reduction. GLP-1 analogues did not increase the risk of hypoglycaemia and resulted in a significant decrease in body weight. Most oral antidiabetic drugs had similar effects on A1C, but some agents had a lower risk of hypoglycaemia and body weight gain.

Impact of interleukin-8 gene polymorphisms and environmental factors on oral cancer susceptibility in Taiwan.

OBJECTIVES: Interleukin-8 (IL-8), which is an angiogenic chemokine with a high expression level in tumor tissues, plays important roles in developing many human malignancies including oral squamous cell carcinoma (OSCC). This study was designed to examine the association of IL-8 gene polymorphisms with the susceptibility and clinicopathological characteristics of OSCC. METHODS: A total of 270 patients with OSCC and 350 healthy control subjects were recruited. Four single nucleotide polymorphisms (SNPs) of IL-8 genes were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping analysis. RESULTS: Results showed that four IL-8 SNPs (-251 T/A, +781 C/T, +1633 C/T, and +2767 A/T) were not associated with oral cancer susceptibility as well as clinicopathological parameters. But among 345 smokers, IL-8 polymorphisms carriers with betel quid chewing were found to have a 17.41- to 23.14-fold risk to have oral cancer compared to IL-8 wild-type carriers without betel quid chewing. Among 262 betel quid chewers, IL-8 polymorphisms carriers with smoking have a 10.54- to 20.44-fold risk to have oral cancer compared to those who carried wild type without smoking. CONCLUSIONS: Our results suggest that the combination of IL-8 gene polymorphisms and environmental carcinogens might be highly related to the risk of oral cancer.

Role of lipocalin 2 and its complex with matrix metalloproteinase-9 in oral cancer.

OBJECTIVES: Recent evidence demonstrated that lipocalin (LCN)2 is induced in many types of human cancer, while the detection of its complex with matrix metalloproteinase (MMP)-9 is correlated with the cancer disease status. We attempted to evaluate plasma expressions of LCN2, MMP-9, and their complex (LCN2/MMP-9) during the diagnostic work-up of patients with oral squamous cell carcinoma (OSCC) and investigated their correlations with disease progression. METHODS: In total, 195 patients with OSCC and 81 healthy controls were recruited. Expression levels of LCN2, MMP-9, and LCN2/MMP-9 were determined with immunoenzymatic assays. RESULTS: Patients with OSCC exhibited significantly higher levels of LCN2, MMP-9, and LCN2/MMP-9 compared with healthy controls (LCN2: P < 0.001; MMP-9: P < 0.001; LCN2/MMP-9: P < 0.01). Plasma levels of LCN2, MMP-9, and LCN2/MMP-9 in patients with OSCC were significantly correlated with each other and were associated with more-advanced clinical stages (P < 0.05) and/or a larger tumor size (P < 0.05), but were not associated with positive lymph-node metastasis or distal metastasis. CONCLUSION: Our results suggest that plasma levels of LCN2 and the LCN2/MMP-9 complex may be useful in non-invasively monitoring OSCC progression, while supporting their potential role as biomarkers of oral cancer disease status.

Role of mitochondrial DNA variants and copy number in diabetic atherogenesis.

Hyperglycemia-induced reactive oxygen species production can cause diabetes and its complications, including atherosclerosis. The role of mitochondrial DNA variants and mitochondrial copy number in the pathogenesis of diabetic atherogenesis is not well understood. We examined 36 diabetic patients who had undergone amputation for diabetic foot and seven non-diabetic patients who had undergone amputation after traumatic injury. Mitochondrial DNA was extracted and used for sequencing. Single nucleotide polymorphisms (SNPs) relative to the Cambridge reference sequence were analyzed. Mitochondrial DNA copy number was quantified by real-time PCR. Twenty-one novel variants were detected in 29 diabetic patients with arterial stenosis; six of the variants were heteroplasmic, and most occurred in highly evolutionarily conserved residues. These variants were more prevalent in patients with arterial stenosis than in those without stenosis. The novel variants included four in complex I (ND1: C3477A/C, A3523A/G; ND5: C13028A/C, C13060A/C), one in complex IV (COX1: T6090A/T), and one in rRNA (12srRNA: G857G/T). Compared with non-diabetic patients, the diabetic patients had significantly less mitochondrial DNA. Furthermore, among diabetic patients with arterial stenosis, there was a significant positive correlation between mitochondrial DNA copy number and the number of total SNPs. In conclusion, we identified six novel heteroplasmic mitochondrial DNA variants among diabetic patients with arterial stenosis, and we found that diabetic atherogenesis is associated with decreased amounts of mitochondrial DNA.

Cadmium-induced ultrastructural changes and apoptosis in the gill of freshwater mussel Anodonta woodiana.

This study investigated the acute toxicity of cadmium (Cd) to the freshwater mussel Anodonta woodiana. The freshwater mussels were exposed to five concentrations of Cd (0 mg/L, 8.43 mg/L, 16.86 mg/L, 33.72 mg/L, and 67.45 mg/L) for up to 96 h. The 24-h, 48-h, 72-h, and 96-h LC50 values for Cd were estimated as 562.3 mg/L, 331.1 mg/L, 182.0 mg/L, and 134.9 mg/L, respectively. Caspase-3, caspase-8, caspase-9, and Ca-ATPase activities; protein and H2O2 levels; DNA fragmentation; and ultrastructure of the gill were also investigated. The activities of caspase-3 and caspase-9 in mussels were increased by Cd in a dose-dependent manner, where higher doses of Cd (33.72 mg/L and 67.45 mg/L) significantly increased the enzyme activities compared to the controls (P < 0.05). The caspase-8 activity was significantly depressed by a low dose of Cd (8.43 mg/L) but was clearly induced by higher doses of Cd (16.86 mg/L, 33.72 mg/L, and 67.45 mg/L) (P < 0.05). The Ca-ATPase activity and H2O2 levels were elevated and reached maximum values under the medium dose of Cd (16.86 mg/L). However, protein levels were decreased by Cd in an inverse dose-dependent manner. In the gills of the mussels, Cd treatment induced DNA fragmentation as demonstrated by DNA ladders observed via agarose gel electrophoresis. Moreover, ultrastructural alterations in gill cells of mussels treated with Cd (16.86 mg/L and 67.45 mg/L) for 96 h were observed by electronic microscopy. The ultrastructure abnormalities were characterized by the following features: (1) a disordered arrangement and breaking off of microvilli of epithelial cells; (2) chromatin condensed near the nuclear membrane and the appearances of extremely irregular nuclei, some with a fingerlike shape and an unclear, swollen, invaginated, or ruptured nuclear membrane and apoptotic bodies; (3) swollen and vacuolating mitochondria, some with disintegrated or missing cristae; (4) a disintegrated rough endoplasmic reticulum containing different sizes of vesicles; and (5) shrinking and deformation of Golgi bodies with decreased vesicle numbers. Our results demonstrated that Cd could activate caspase-3, caspase-8, caspase-9, and Ca-ATPase; cause ultrastructural changes; and produce DNA fragmentation in the mussels investigated. Based on the information obtained through this study, it is reasonable to conclude that Cd can induce apoptosis in the gills of the mussels, eventually leading to tissue damage.

Transgenic expression of murine chemokine decoy receptor D6 by islets reveals the role of inflammatory CC chemokines in the development of autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: Autoimmune diabetes results from a progressive destruction of insulin-producing beta cells in the pancreatic islets by chemokine-attracted lymphocytes. Because islet cells in NOD mice produce chemokines during the development of autoimmune diabetes, we investigated the role of inflammatory CC chemokines in disease progression in these mice. METHODS: We generated a transgenic NOD mouse model that overproduces the inflammatory CC chemokine decoy receptor D6 in pancreatic islets. RESULTS: The frequency of diabetes and insulitis scores of transgenic mice were decreased significantly, compared with non-transgenic control littermates. Transgenic expression of D6 (also known as Ccbp2) did not affect systemic lymphocyte development or alter: (1) the T cell subsets such as T helper (Th)1, Th2 and T regulatory cells; or (2) antigen-presenting cells such as dendritic cells or macrophages. The percentages and numbers of T and B lymphocytes were decreased significantly in the pancreas. Activation status, autoantigen-specific proliferation and diabetogenicity of lymphocytes were also markedly reduced. CONCLUSIONS/INTERPRETATION: Inflammatory CC chemokines play a critical role in the development of autoimmune diabetes. Transgenic expression of D6 in pancreatic islets of NOD mice reduced this pathogenic process by suppressing activation of autoreactive lymphocytes and by reducing migration of lymphocytes to the pancreas.

Effects of different progestogens on human breast tumor cell growth.

OBJECTIVE: To examine the effect of progestogens currently used in hormone therapy on growth of human breast tumor cells. METHODS: MCF-7 cells were incubated in 10 nmol/l progestogens, including progesterone (P4), medroxyprogesterone acetate (MPA), norethisterone acetate (NETA), and cyproterone acetate (CPA), with or without 17ß-estradiol (E(2), 1 nmol/l and 10 nmol/l), as well as E(2) alone. Cell proliferation, apoptosis, and the expression of caspase-3 and proliferating cell nuclear antigen (PCNA) were evaluated. RESULTS: The ratios of apoptosis : proliferation significantly increased in cultures with progestogens alone, 1 nmol/l E(2) plus progestogens (except P4), and 10 nmol/l E(2) plus NETA. Caspase-3 significantly diminished in cultures with E(2) alone; this was completely reversed when progestogens were added. MPA alone or with 1 nmol/l E(2) and 10 nmol/l E(2) plus NETA significantly increased caspase-3. Using progestogens alone, except P4, significantly decreased PCNA expression. CONCLUSIONS: The results demonstrate that various progestogens have different effects on growth of breast tumor cells, especially with the combined usage of E(2). Progestogen-induced apoptosis may be partly inhibited with the presence of E(2), but less severe with lower E(2) concentrations. Therefore, the choice of progestogens, as well as the doses of E(2) and/or progestogen, may influence breast cancer risk.

Pt, Rh and Pt-Rh nanoparticles on modified single-walled carbon nanotubes for hydrogenation of benzene at room temperature.

Nanometer-sized Pt, Rh, and bimetallic Pt-Rh particles can be deposited on surface of phenylacetic acid functionalized single-walled carbon nanotubes (SWCNTs) by a microemulsion method. The SWCNT-supported metallic nanoparticles show much greater catalytic activities compared with commercially available carbon-supported Pt and Rh catalysts for hydrogenation of neat benzene under mild experimental conditions. The bimetallic Pt-Rh nanoparticle catalyst synthesized by this method shows an enhanced activity relative to individual SWCNT-supported Pt and Rh nanoparticle catalysts. The SWCNT-supported metal nanoparticle catalysts can be recycled and reused at least five times without losing their activity. The hydrogenation reactions performed under our experimental conditions would not affect the pi-pi stacking holding phenylacetic acid on SWCNT surface.

T cell-independent interleukin 15Ralpha signals are required for bystander proliferation.

Cytokine driven or "bystander" proliferation of T cells occurs in vivo independently of major histocompatibility complex-T cell receptor interactions. This process may be important for supporting T cell homeostasis and facilitating T cell responses to microbial antigens, and may involve the cytokine interleukin (IL)-15. In this study, we find that IL-15Ralpha-deficient (IL-15Ralpha(-/-)) mice fail to undergo poly I:C or IL-15 driven bystander proliferation of CD8(+) T cells. Surprisingly, IL-15Ralpha(-/-) CD8(+) T cells proliferate in response to poly I:C when adoptively transferred into normal mice, and normal CD8(+) T cells fail to proliferate in IL-15Ralpha(-/-) mice. Normal mice reconstituted with IL-15Ralpha(-/-) bone marrow cells also fail to exhibit bystander responses. Thus, CD8(+) T cell independent IL-15Ralpha signals from radiation sensitive hematopoietic cells are likely required for bystander responses. Moreover, normal CD8(+) T cells proliferate in IL-15Ralpha(-/-) mice after treatment with IL-15. Therefore, IL-15Ralpha signals may mediate a positive feedback loop involving the further physiological production of IL-15. These findings provide new insights into how IL-15Ralpha supports memory phenotype CD8(+) T cell proliferation, and suggest novel mechanisms by which memory CD8(+) T cells are maintained in vivo.

Inspiratory muscle dysfunction in patients with severe obstructive sleep apnoea.

Repetitive inspiratory effort against an obstructed airway and intermittent hypoxia may be deleterious to the inspiratory muscles in patients with obstructive sleep apnoea (OSA). We investigated muscular dysfunction by comparing the strength, endurance and fatigability of inspiratory muscles and knee extensors in patients with newly diagnosed severe OSA compared with matched controls. The measurements included strength and endurance tests of both muscles, and a fatigue trial with simultaneous surface electromyography of the diaphragm and the vastus lateralis during voluntary contractions and in response to magnetic stimulation. To our knowledge, this is the first investigation to assess peripheral muscle performance in severe OSA patients versus controls. Patients in the OSA group exhibited significantly lower strength and endurance in both muscles than the control group. The fatigue index decreased significantly exclusively in the inspiratory muscles of OSA patients. Magnetic stimulation-evoked compound muscle action potential latencies increased and the amplitudes decreased significantly in the diaphragm, but not in the vastus lateralis after a fatigue test in the OSA group. In conclusion, a significantly lower functional performance was shown for both inspiratory muscles and knee extensors in the OSA group. However, higher fatigability was only seen in the inspiratory muscles of patients with severe OSA.