Effects of dietary physical or nutritional factors on morphology of rumen papillae and transcriptome changes in lactating dairy cows based on three different forage-based diets.

BACKGROUND: Rumen epithelial tissue plays an important role in nutrient absorption and rumen health. However, whether forage quality and particle size impact the rumen epithelial morphology is unclear. The current study was conducted to elucidate the effects of forage quality and forage particle size on rumen epithelial morphology and to identify potential underlying molecular mechanisms by analyzing the transcriptome of the rumen epithelium (RE). To achieve these objectives, 18 mid-lactation dairy cows were allocated to three groups (6 cows per group), and were fed with one of three different forage-based diets, alfalfa hay (AH), corn stover (CS), and rice straw (RS) for 14 weeks, respectively. Ruminal volatile fatty acids (VFAs) and epithelial thickness were determined, and RNA-sequencing was conducted to identify the transcriptomic changes of rumen epithelial under different forage-based diets. RESULTS: The RS diet exhibited greater particle size but low quality, the AH diet was high nutritional value but small particle size, and CS diet was low quality and small particle size. The ruminal total VFA concentration was greater in AH compared with those in CS or RS. The width of the rumen papillae was greater in RS-fed cows than in cows fed AH or CS. In total, 31, 40, and 28 differentially expressed (DE, fold change > 2, FDR < 0.05) genes were identified via pair-wise comparisons including AH vs. CS, AH vs. RS, and RS vs. CS, respectively. Functional classification analysis of DE genes revealed dynamic changes in ion binding (such as DSG1) between AH and CS, proliferation and apoptotic processes (such as BAG3, HLA-DQA1, and UGT2B17) and complement activation (such as C7) between AH or RS and CS. The expression of HLA-DQA1 was down-regulated in RS compared with AH and CS, and the expression of UGT2B17 was down-regulated in RS compared with CS, with positive (R = 0.94) and negative (R = -0.96) correlation with the width of rumen epithelial papillae (P < 0.05), respectively. CONCLUSION: Our results suggest that both nutrients (VFAs) and particle sizes can alter expression of genes involved in cell proliferation/apoptosis process and complement complex. Our results suggest that particle size may be more important in regulating rumen epithelial morphology when animals are fed with low-quality forage diets and the identified DE genes may affect the RE nutrient absorption or morphology of RE. Our findings provide insights into the effects of the dietary particle size in the future management of dairy cow feeding, that when cows were fed with low-quality forage (such as rice straw), smaller particle size may be beneficial for nutrients absorption and milk production.

Newly Emergent Highly Pathogenic H5N9 Subtype Avian Influenza A Virus.

UNLABELLED: The novel H7N9 avian influenza virus (AIV) was demonstrated to cause severe human respiratory infections in China. Here, we examined poultry specimens from live bird markets linked to human H7N9 infection in Hangzhou, China. Metagenomic sequencing revealed mixed subtypes (H5, H7, H9, N1, N2, and N9). Subsequently, AIV subtypes H5N9, H7N9, and H9N2 were isolated. Evolutionary analysis showed that the hemagglutinin gene of the novel H5N9 virus originated from A/Muscovy duck/Vietnam/LBM227/2012 (H5N1), which belongs to clade 2.3.2.1. The neuraminidase gene of the novel H5N9 virus originated from human-infective A/Hangzhou/1/2013 (H7N9). The six internal genes were similar to those of other H5N1, H7N9, and H9N2 virus strains. The virus harbored the PQRERRRKR/GL motif characteristic of highly pathogenic AIVs at the HA cleavage site. Receptor-binding experiments demonstrated that the virus binds α-2,3 sialic acid but not α-2,6 sialic acid. Identically, pathogenicity experiments also showed that the virus caused low mortality rates in mice. This newly isolated H5N9 virus is a highly pathogenic reassortant virus originating from H5N1, H7N9, and H9N2 subtypes. Live bird markets represent a potential transmission risk to public health and the poultry industry. IMPORTANCE: This investigation confirms that the novel H5N9 subtype avian influenza A virus is a reassortant strain originating from H5N1, H7N9, and H9N2 subtypes and is totally different from the H5N9 viruses reported before. The novel H5N9 virus acquired a highly pathogenic H5 gene and an N9 gene from human-infecting subtype H7N9 but caused low mortality rates in mice. Whether this novel H5N9 virus will cause human infections from its avian host and become a pandemic subtype is not known yet. It is therefore imperative to assess the risk of emergence of this novel reassortant virus with potential transmissibility to public health.

Enhanced immune response to foot-and-mouth disease vaccine by oral administration of ginseng stem-leaf saponins.

Vaccination is an important approach to the control of foot-and-mouth disease (FMD). This study evaluated the effect of oral administration of ginseng stem-leaf saponins (GSLS) on the immune response to FMD vaccine and the gut mucosal immunity in mice. In experiment 1, mice were orally administered GSLS or not treated as a control. The animals were then immunized twice with FMD vaccine. Blood was sampled weekly within five weeks after the boost immunization for measurement of serum IgG and the isotypes. In experiment 2, mice were orally administrated GSLS or not treated as a control. After that, splenocytes were prepared from sacrificed mice for lymphocyte proliferation assay and intestinal tissues were sampled for immunohistochemistry and histological examination. The results showed that oral administration of GSLS significantly enhanced serum IgG and the isotype responses to FMD vaccine as well as the number of intestinal intraepithelial lymphocytes (IELs) and immunoglobulin A (IgA)+ cells. Therefore, GSLS may be a potent oral adjuvant and deserve further study to improve vaccination in susceptible animals.

Modeling time-dependent transcription effects of HER2 oncogene and discovery of a role for E2F2 in breast cancer cell-matrix adhesion.

MOTIVATION: Oncogenes are known drivers of cancer phenotypes and targets of molecular therapies; however, the complex and diverse signaling mechanisms regulated by oncogenes and potential routes to targeted therapy resistance remain to be fully understood. To this end, we present an approach to infer regulatory mechanisms downstream of the HER2 driver oncogene in SUM-225 metastatic breast cancer cells from dynamic gene expression patterns using a succession of analytical techniques, including a novel MP grammars method to mathematically model putative regulatory interactions among sets of clustered genes. RESULTS: Our method highlighted regulatory interactions previously identified in the cell line and a novel finding that the HER2 oncogene, as opposed to the proto-oncogene, upregulates expression of the E2F2 transcription factor. By targeted gene knockdown we show the significance of this, demonstrating that cancer cell-matrix adhesion and outgrowth were markedly inhibited when E2F2 levels were reduced. Thus, validating in this context that upregulation of E2F2 represents a key intermediate event in a HER2 oncogene-directed gene expression-based signaling circuit. This work demonstrates how predictive modeling of longitudinal gene expression data combined with multiple systems-level analyses can be used to accurately predict downstream signaling pathways. Here, our integrated method was applied to reveal insights as to how the HER2 oncogene drives a specific cancer cell phenotype, but it is adaptable to investigate other oncogenes and model systems. AVAILABILITY AND IMPLEMENTATION: Accessibility of various tools is listed in methods; the Log-Gain Stoichiometric Stepwise algorithm is accessible at http://www.cbmc.it/software/Software.php.

Identification and functional analysis of the novel ORF4 protein encoded by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. To date, viral proteins Cap, Rep, Rep', and ORF3, encoded by the PCV2 genome, have been described. Here, transcription and translation of a novel viral gene within the PCV2 genome (designated ORF4) was determined and functionally analyzed in vitro and in vivo. Northern blot analysis indicated that the RNA transcribed from the ORF4 gene is about 180 bp in length and overlaps ORF3 in the same direction. Site-directed mutagenesis confirmed that the viral ORF4 protein is not essential for virus replication in PK-15 cells and in mice infected with an ORF4-deficient PCV2 (PCV2Δ). PCV2Δ triggered higher activity levels of caspase-3 and -8 than wild-type PCV2 (wPCV2) in PK-15 cells. The antigenic epitopes of two mouse monoclonal antibodies (MAbs) raised against the viral ORF4 protein were mapped to the same 19KSSASPR25 peptide. Expression of ORF4 was confirmed using the specific MAbs in wPCV2-infected PK-15 cells and mice. Mice infected with PCV2Δ had a higher serum viral load (genomic copies) and more severe lymphoid tissue damage in the spleen than those infected with wPCV2. Meanwhile, flow-cytometric analysis indicated that the PCV2Δ infection caused a significant decrease of CD4(+) and CD8(+) T lymphocytes. Our results demonstrate that ORF4 is a newly discovered viral protein that is not essential for PCV2 replication but plays a role in suppressing caspase activity and regulating CD4(+) and CD8(+) T lymphocytes during PCV2 infection.

Detection of mcr-1-harbouring Escherichia coli by quantum dot labelling of synthetic small peptides mimicking lipopolysaccharide receptors.

Overuse of antibiotics and the emergence of multidrug-resistant bacteria has made colistin the last line of defence against complex infections. In previous studies, MCR-1-mediated colistin resistance was mainly detected through PCR or antimicrobial susceptibility testing. However, intuitive detection methods for phenotype are rarely reported. In this study, two small peptide antibodies were constructed for immunofluorescence detection of mcr-1-harbouring Escherichia coli: one was a small peptide labelled with a quantum dot antibody; and the other was a small peptide labelled with a fluorescein isothiocyanate (FITC) antibody. Whether using FITC or quantum dots, colistin-resistant bacteria in the sample could be qualitatively detected. The assembled antibodies achieved the desired goals in terms of sensitivity, specificity, precision and repeatability. The non-specific problem of sandwich antigen recognition of lipid A binding to small peptides in modified lipopolysaccharide (LPS) was resolved, and this relatively developed immunofluorescence technique standardised the detection process. Together, in addition to PCR, both fluorescent antibodies can be used for immunofluorescent detection of mcr-1-harbouring E. coli.

Integrated expression profiles of mRNA and miRNA in a gerbil model of fatty liver fibrosis treated with exenatide.

BACKGROUND: The morbidity of nonalcoholic fatty liver disease (NAFLD) has increased consistently in recent years. Exenatide could reverse liver fibrosis and lower the occurrence of fatty liver. The aim of the study was to identify and characterize mRNA and miRNA expression to elucidate the mechanism of exenatide in the gerbil model. METHODS: Gerbils were fed a high-fat diet for 8 weeks to induce a fibrosis model; then, the gerbil models were treated with exenatide for 4 weeks. The total RNA extracted from the liver tissue samples was used to prepare the library and sequence on a HiSeq 2000. Bioinformatic methods were employed to analyze the sequence data to identify the mRNAs and miRNAs and to acquire the miRNA-mRNA regulatory network. RESULTS: By RNA-seq, 2344 differentially expressed genes (DEGs) and 72 miRNAs were found in the model group. Compared with the model group, 591 DEGs and 19 miRNAs were found in the quercetin group, whereas 876 DEGs and 18 miRNAs were found in the treatment group. The miRNA-mRNA regulatory network was constructed in a gerbil model. Immunohistochemistry and RNA sequencing confirmed that the therapeutic effect of exenatide may be derived from extrahepatic signal transduction. The key differential genes are CYP3A, CYP4A11, ACAA1, ACSM, PHX1, MAO, FMO, UGT, ACOX2, ABAT, PIK3C and PLCG1. The key miRNAs are miR-15a, miR-27b, miR-532-3P, miR-627, miR-3596, miR-142-3P, Let-7e-5p, miR-214-5, miR-101-3p, miR-378d. New miRNAs, such as novel_127, novel_143, novel_15, novel_204 are associated with liver fibrosis, while novel_127, novel_15, and novel_54 are associated with reverse treated with exenatide. CONCLUSIONS: Our research represents the first description of mRNA/miRNA profiles in a gerbil model of fatty liver fibrosis treated with exenatide, which may provide insights into the pathogenesis or treatment of the metabolic syndrome.

Gene expression profiles in primary pancreatic tumors and metastatic lesions of Ela-c-myc transgenic mice.

BACKGROUND: Pancreatic carcinoma usually is a fatal disease with no cure, mainly due to its invasion and metastasis prior to diagnosis. We analyzed the gene expression profiles of paired primary pancreatic tumors and metastatic lesions from Ela-c-myc transgenic mice in order to identify genes that may be involved in the pancreatic cancer progression. Differentially expressed selected genes were verified by semi-quantitative and quantitative RT-PCR. To further evaluate the relevance of some of the selected differentially expressed genes, we investigated their expression pattern in human pancreatic cancer cell lines with high and low metastatic potentials. RESULTS: Data indicate that genes involved in posttranscriptional regulation were a major functional category of upregulated genes in both primary pancreatic tumors (PT) and liver metastatic lesions (LM) compared to normal pancreas (NP). In particular, differential expression for splicing factors, RNA binding/pre-mRNA processing factors and spliceosome related genes were observed, indicating that RNA processing and editing related events may play critical roles in pancreatic tumor development and progression. High expression of insulin growth factor binding protein-1 (Igfbp1) and Serine proteinase inhibitor A1 (Serpina1), and low levels or absence of Wt1 gene expression were exclusive to liver metastatic lesion samples. CONCLUSION: We identified Igfbp1, Serpina1 and Wt1 genes that are likely to be clinically useful biomarkers for prognostic or therapeutic purposes in metastatic pancreatic cancer, particularly in pancreatic cancer where c-Myc is overexpressed.

c-Myc-induced chemosensitization is mediated by suppression of cyclin D1 expression and nuclear factor-kappa B activity in pancreatic cancer cells.

PURPOSE: Pancreatic cancer is a highly aggressive disease that remains refractory to various chemotherapeutic agents. Because the proto-oncogene c-myc can modulate apoptosis in response to cytotoxic insults and is commonly overexpressed in pancreatic cancer, we investigated the value of c-myc as a potential modulator of cellular response to various chemotherapeutic agents. EXPERIMENTAL DESIGN: Stable overexpression or small interfering RNA (siRNA)-mediated knockdown of c-myc and restoration of cyclin D1 were done in the Ela-myc pancreatic tumor cell line. Cell viability after cisplatin treatment of c-myc-overexpressing, control, and siRNA-transfected cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and drug-induced apoptosis was measured by DNA fragmentation, sub-G(1), and poly(ADP-ribose) polymerase cleavage analyses. Protein expression profile after cisplatin treatment was determined by Western blotting and DNA binding activity of nuclear factor-kappaB was examined by electrophoretic mobility shift assay. RESULTS: Ectopic overexpression of c-myc in murine and human pancreatic cancer cell lines, Ela-myc and L3.6pl, respectively, resulted in increased sensitivity to cisplatin and other chemotherapeutic drugs. Increased sensitivity to cisplatin in c-myc-overexpressing cells was due, in part, to the marked increase in cisplatin-induced apoptosis. Conversely, down-regulation of c-myc expression in stable c-myc-overexpressing cells by c-myc siRNA resulted in decreased sensitivity to cisplatin-induced cell death. These results indicate an important role of c-myc in chemosensitivity of pancreatic cancer cells. The c-myc-induced cisplatin sensitivity correlated with inhibition of nuclear factor kappaB activity, which was partially restored by ectopic cyclin D1 overexpression. CONCLUSIONS: Our results suggest that the c-myc-dependent sensitization to chemotherapy-induced apoptosis involves suppression of cyclin D1 expression and nuclear factor kappaB activity.

[Correlation between porcine CAST gene polymorphism with muscle fiber histological traits and carcass characteristics].

Three polymorphisms were identified in the sixth intron of the CAST gene by PCR-RFLP using enzymes Msp I, Hinf I and Rsa I in 45 Jinpi F2 pigs. However, only three genotypes AACCEE, BBDDFF, and ABCDEF were detected in those pigs and the genotype frequencies were 0.1778, 0.2222 and 0.6000, respectively. Longissimus dorsi muscles were moved for paraffin serial sections and stained with hematoxylin-eosin or myosin heavy chain (MHC) by immunohistochemistry respectively. Fiber cross-sectional area, fiber density, fiber diameter, the rate of MHC type I fiber and the carcass characteristics were recorded. Correlation analysis showed that the skeletal muscle fiber area and the eye muscle area of the BBDDFF individual were significantly higher than those of the ABCDEF individual (P < 0.05).

Characterization of pancreatic lesions from MT-tgf alpha, Ela-myc and MT-tgf alpha/Ela-myc single and double transgenic mice.

In order to identify good animal models for investigating therapeutic and preventive strategies for pancreatic cancer, we analyzed pancreatic lesions from several transgenic models and made a series of novel findings. Female MT-tgf alpha mice of the MT100 line developed pancreatic proliferation, acinar-ductal metaplasia, multilocular cystic neoplasms, ductal adenocarcinomas and prominent fibrosis, while the lesions in males were less severe. MT-tgf alpha-ES transgenic lines of both sexes developed slowly progressing lesions that were similar to what was seen in MT100 males. In both MT100 and MT-tgf alpha-ES lines, TGF alpha transgene was expressed mainly in proliferating ductal cells. Ela-myc transgenic mice with a mixed C57BL/6, SJL and FVB genetic background developed pancreatic tumors at 2-7 months of age, and half of the tumors were ductal adenocarcinomas, similar to what was reported originally by Sandgren et al 1. However, in 20% of the mice, the tumors metastasized to the liver. MT100/Ela-myc and MT-tgf alpha-ES/Ela-myc double transgenic mice developed not only acinar carcinomas and mixed carcinomas as previously reported but also various ductal-originated lesions, including multilocular cystic neoplasms and ductal adenocarcinomas. The double transgenic tumors were more malignant and metastasized to the liver at a higher frequency (33%) compared with the Ela-myc tumors. Sequencing of the coding region of p16ink4, k-ras and Rb cDNA in small numbers of pancreatic tumors did not identify mutations. The short latency for tumor development, the variety of tumor morphology and the liver metastases seen in Ela-myc and MT-tgf alpha/Ela-myc mice make these animals good models for investigating new therapeutic and preventive strategies for pancreatic cancer.

Live Streptococcus suis type 5 strain XS045 provides cross-protection against infection by strains of types 2 and 9.

Streptococcus suis is one of the common pathogens causing diseases in pigs and covers 35 serotypes with the type 2 strains being more pathogenic and zoonotic. Existing inactivated or subunit vaccines, in clinical use or under trial, could not provide cross protection against other serotypes. We identified a natural low-virulence S. suis type 5 strain XS045 as a live vaccine candidate because it is highly adhesive to the cultured HEp-2 cells, but with no apparent pathogenicity in mice and piglets. We further demonstrate that subcutaneous administration of the live XS045 strain to mice induced high antibody responses and was able to provide cross protection against challenges by a type 2 strain HA9801 (100% protection) and a type 9 strain JX13 (85% protection). Induction of high-titer antibodies with opsonizing activity as well as their cross-reactivity to surface proteins of the types 2 and 9 strains and anti-adhesion effect could be the mechanisms of cross protection. This is the first report that a live vaccine candidate S. suis type 5 strain could induce cross-protection against strains of types 2 and 9. This candidate strain is to be further examined for safety in pigs of different ages and breeds as well as for its protection against other serotypes or other strains of the type 2, a serotype of particular importance from public health concern.

Mouse extrahepatic hepatoma detected on MicroPET using copper (II)-64 chloride uptake mediated by endogenous mouse copper transporter 1.

PURPOSE: This study was conducted to assess the positron-emitting copper (II)-64 chloride ((64)CuCl(2)) as a probe for imaging mouse extrahepatic hepatoma expressing mouse copper transporter 1 (mCtr1) with positron emission tomography (PET). PROCEDURES: Following the intravenous administration of (64)CuCl(2), athymic mice bearing extrahepatic hepatoma grafts were subjected to whole-body static PET imaging with a Concorde microPET R4 tomograph. Upon completion of the imaging study, immunohistochemistry (IHC) study of mCtr1 was performed with postmortem tissues. RESULTS: The mouse extrahepatic hepatoma grafts were well visualized on static microPET images. Quantitative analysis demonstrated that the tracer concentration in the hepatoma was significantly higher than those in the soft tissue of the right shoulder opposite to the tumor site and the brain (p < 0.001). mCtr1 immunoreactivity in the hepatoma graft was approximately 70% of that in liver, whereas (64)CuCl(2) concentration in the graft was approximately 11% of the liver concentration. CONCLUSIONS: The extrahepatic mouse hepatoma grafts may be visualized by Cu-64 PET, taking advantage of the (64)CuCl(2) uptake mediated by the functional endogenous mCtr1.

Reduced 64Cu uptake and tumor growth inhibition by knockdown of human copper transporter 1 in xenograft mouse model of prostate cancer.

UNLABELLED: Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased (64)Cu radioactivity were visualized previously by PET using (64)CuCl2 as a radiotracer ((64)CuCl2 PET). This study aimed to determine whether the increased tumor (64)Cu radioactivity was due to increased cellular uptake of (64)Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic (64)CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed. METHODS: A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1-shRNA) was constructed for RNA interference-mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular (64)Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of (64)Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper. RESULTS: RNA interference-mediated knockdown of hCtr1 was associated with the reduced cellular uptake of (64)Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer (64)CuCl2, the (64)Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU-145) was significantly lower than the (64)Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 %ID/g in Lenti-SCR-shRNA-DU-145, P < 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm(3) for Lenti-hCtr1-shRNA-PC-3 or 39 ± 22 mm(3) for Lenti-hCtr1-shRNA-DU-145) were significantly smaller than those without knockdown of hCtr1 (536 ± 191 mm(3) for Lenti- SCR-shRNA-PC-3 or 208 ± 104 mm(3) for Lenti-SCR-shRNA-DU-145, P < 0.01). CONCLUSION: Overall, data indicated that hCtr1 is a promising theranostic target, which can be further developed for metabolic imaging of prostate cancer using (64)CuCl2 PET/CT and personalized cancer therapy targeting copper metabolism.

Primary genetic investigation of a hyperlipidemia model: molecular characteristics and variants of the apolipoprotein E gene in Mongolian gerbil.

The objective of this work was to establish a novel Mongolian gerbil (Meriones unguiculatus) hyperlipidemia model and to investigate its susceptibility genetic basis. Two rodent (gerbil and rat) hyperlipidemia models were induced by feeding a high fat/high-cholesterol (HF/HC) diet. There were significant increases of serum total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in gerbils within a 4-week modeling period. About 10-30% of >8-month-old individuals developed hyperlipidemia spontaneously. The apolipoprotein E (ApoE) gene was cloned by merging a sequence of rapid amplification of cDNA ends (RACE) and nested polymerase chain reaction products. The results revealed an open reading frame of 948 bp, encoding a protein of 298 amino acids. The gene without a 5'-UTR region in the first intron was highly homologous to human Apo-A-I and rat Apo-A-IV. The distribution of expression of the ApoE gene in liver, brain, heart, lung, kidney, and adrenal gland was detected by an ABC immunohistochemical procedure. Three single nucleotide polymorphisms (SNPs; C97T, G781T, and A1774T) were first found using PCR-single-strand conformation polymorphism (PCR-SSCP) in a closed population containing 444 animals. Correlation analysis confirmed that new SNPs, age, and gender were associated significantly (P < 0.05) with hyperlipidemia.

The Predominant Proteins that React to the MC-20 Estrogen Receptor Alpha Antibody Differ in Molecular Weight between the Mammary Gland and Uterus in the Mouse and Rat.

There are many estrogen receptor α (ERα) antibodies available but few of them target a rodent ERα. Using the MC-20 antibody raised against the C-terminus of mouse ERα, we show in this communication that in the mammary gland of female mice and rats, the wild type (wt) ERα was detected on immunoblots as a dominant protein only during lactation, and the protein was lactating specific as it migrated slightly faster than the 67-kD wt ERα in the uterus, likely due to a different phosphorylation status. In contrast, in the nulliparous, pregnant, involuting and involuted mammary glands, the dominant protein recognized by MC-20 was about 61-kD, which is dubbed herein as "MC-20 reactive protein" or MC20RP in abbreviation as its identity is unknown. Our results showed that it was not derived from proteolysis or de-phosphorylation of the 67-kD ERα and was unlikely to be translated from an ERα mRNA variant. Ovariectomy decreased the lactating specific wt ERα but increased the 61-kD MC20RP in the mammary tumors from MMTV-c-myc transgenic mice but these two proteins in the uterus were unaffected. The 61-kD MC20RP was decreased in the mammary tumors, compared with proliferating mammary glands, in estrogen-treated ACI rats. These results suggest that while the lactating specific wt ERα alone or together with the MC20RP may sustain lactation, the MC20RP may support proliferation of the mammary gland and some mammary tumors.

Potent anticancer activity of pyrrolidine dithiocarbamate-copper complex against cisplatin-resistant neuroblastoma cells.

Effective drugs are urgently needed for the treatment of advanced neuroblastoma refractory to conventional chemotherapy. Pyrrolidine dithiocarbamate (PDTC) is a copper-binding ligand, which showed cytotoxicity on many human tumor cells after binding with copper ions. In this study, we synthesized a copper-PDTC complex, which was characterized as a Cu(PDTC)2 complex, with elemental analyses (Fourier transform infrared, electrospray ionization mass spectra, and ultraviolet-visible spectroscopy). The Cu(PDTC)2 complex suppressed the proliferation of BE(2)C cells, a human neuroblastoma cell line, with an IC50 of 8.0 micromol/l, which was more potent than cisplatin (IC50 of 80 micromol/l). Treatment of BE(2)C cells with the Cu(PDTC)2 complex caused the S-phase arrest of cell cycle progression, cellular apoptosis, and necrosis, and increased the expression of p53 protein. The Cu(PDTC)2 complex holds potential as a new drug for the treatment of refractory neuroblastoma in children.

Regulation of intracellular calcium release and PP1alpha in a mechanism for 4-hydroxytamoxifen-induced cytotoxicity.

Treatment with tamoxifen, or its metabolite 4-hydroxytamoxifen (4OHT), has cytostatic and cytotoxic effects on breast cancer cells in vivo and in culture. Although the effectiveness of 4OHT as an anti-breast cancer agent is due to its action as an estrogen receptor-alpha (ERalpha) antagonist, evidences show that 4OHT is also cytotoxic for ERalpha-negative breast cancer cells and can be effective therapy against tumors that lack estrogen receptors. These findings underscore 4OHT signaling complexities and belie the most basic understandings of 4OHT action and resistance. Here, we have investigated the effects of 4OHT on Ca2+ homeostasis and cell death in breast cancer cells in culture. Measurement of Ca2+ signaling in breast cancer cells showed that 4OHT treatment altered Ca2+ homeostasis and was cytotoxic for both an ERalpha+ and an ERalpha- cell line, MCF-7 and MDA-MB-231, respectively. Further investigation lead us to the novel discovery that 4OHT-induced increase of ATP-dependent Ca2+ release from the endoplasmic reticulum correlated with 4OHT-induced upregulation of protein phosphatase 1alpha (PP1alpha) and the inositol 1,4,5-trisphosphate receptor (IP3R). Blocking 4OHT-induced PP1alpha upregulation by siRNA strategy reduced the effects of 4OHT on both Ca2+ signaling and cytotoxicity. Results from these investigations strongly suggest a role for PP1alpha upregulation in a mechanism for 4OHT-induced changes to Ca2+ signaling that ultimately contribute to the cytotoxic effects of 4OHT.