The Golgi Localization of GOLPH2 (GP73/GOLM1) Is Determined by the Transmembrane and Cytoplamic Sequences

Golgi phosphoprotein 2 (GOLPH2) is a resident Golgi type-II membrane protein upregulated in liver disease. Given that GOLPH2 traffics through endosomes and can be secreted into the circulation, it is a promising serum marker for liver diseases. The structure of GOLPH2 and the functions of its different protein domains are not known. In the current study, we investigated the structural determinants for Golgi localization using a panel of GOLPH2 truncation mutants. The Golgi localization of GOLPH2 was not affected by the deletion of the C-terminal part of the protein. A truncated mutant containing the N-terminal portion (the cytoplasmic tail and transmembrane domain (TMD)) localized to the Golgi. Sequential deletion analysis of the N-terminal indicated that the TMD with a positively charged residue in the cytoplasmic N-terminal tail were sufficient to support Golgi localization. We also showed that both endogenous and secreted GOLPH2 exist as a disulfide-bonded dimer, and the coiled-coil domain was sufficient for dimerization. This structural knowledge is important for the understanding the pathogenic role of GOLPH2 in liver diseases, and the development of GOLPH2-based hepatocellular cancer diagnostic methods

Selection of suitable reference genes for quantitative real-time polymerase chain reaction in human meningiomas and arachnoidea

<p>Abstract</p> <p>Findings</p> <p>At first 32 housekeeping genes were analyzed in six randomly chosen meningiomas, brain and dura mater using geNorm, NormFinder, Bestkeeper-1 software and the comparative ΔCt method. Reference genes were ranked according to an integration tool for analyzing reference genes expression based on those four algorithms. Eight highest ranked reference genes (CASC3, EIF2B1, IPO8, MRPL19, PGK1, POP4, PPIA, and RPL37A) plus GAPDH and ACTB were then analyzed in 35 meningiomas, arachnoidea, dura mater and normal brain. NormFinder and Bestkeeper-1 identified RPL37A as the most stable expressed gene in meningiomas and their normal control tissue. NormFinder also determined the best combination of genes: RPL37A and EIF2B1. Commonly used reference genes GAPDH and ACTB were considered least stable genes. The critical influence of reference genes on qPCR data analysis is shown for VEGFA transcription patterns.</p> <p>Background</p> <p>In meningiomas quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is most frequently used for accurate determination of gene expression using various reference genes. Although meningiomas are a heterogeneous group of tissue, no data have been reported to validate reference genes for meningiomas and their control tissues.</p> <p>Conclusions</p> <p>RPL37A is the optimal single reference gene for normalization of gene expression in meningiomas and their control tissues, although the use of the combination of RPL37A and EIF2B1 would provide more stable results.</p

Radiosensitization of mammary carcinoma cells by telomere homolog oligonucleotide pretreatment

Introduction: Ionizing radiation (IR) is a widely used approach to cancer therapy, ranking second only to surgery in rate of utilization. Responses of cancer patients to radiotherapy depend in part on the intrinsic radiosensitivity of the tumor cells. Thus, promoting tumor cell sensitivity to IR could significantly enhance the treatment outcome and quality of life for patients. Methods: Mammary tumor cells were treated by a 16-base phosphodiester-linked oligonucleotide homologous to the telomere G-rich sequence TTAGGG (T-oligo: GGTTAGGTGTAGGTTT) or a control-oligo (the partial complement, TAACCCTAACCCTAAC) followed by IR. The inhibition of tumor cell growth in vitro was assessed by cell counting and clonogenic cell survival assay. The tumorigenesis of tumor cells after various treatments was measured by tumor growth in mice. The mechanism underlying the radiosensitization by T-oligo was explored by immunofluorescent determination of phosphorylated histone H2AX ($\gamma$H2AX) foci, $\beta$-galactosidase staining, comet and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assays. The efficacy of the combined treatment was assessed in a spontaneous murine mammary tumor model. Results: Pretreatment of tumor cells with T-oligo for 24 hours in vitro enhanced both senescence and apoptosis of irradiated tumor cells and reduced clonogenic potential. Radiosensitization by T-oligo was associated with increased formation and/or delayed resolution of $\gamma$H2AX DNA damage foci and fragmented DNA. T-oligo also caused radiosensitization in two in vivo mammary tumor models. Indeed, combined T-oligo and IR-treatment in vivo led to a substantial reduction in tumor growth. Of further significance, treatment with T-oligo and IR led to synergistic inhibition of the growth of spontaneous mammary carcinomas. Despite these profound antitumor properties, T-oligo and IR caused no detectable side effects under our experimental conditions. Conclusions: Pretreatment with T-oligo sensitizes mammary tumor cells to radiation in both in vitro and in vivo settings with minimal or no normal tissue side effects

Perilipin Overexpression in White Adipose Tissue Induces a Brown Fat-Like Phenotype

Background: Perilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model. Methodology and Principal Findings: When compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size. Conclusions: These findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders

Variation in life history traits and transcriptome associated with adaptation to diet shifts in the ladybird Cryptolaemus montrouzieri

Background: Despite the broad diet range of many predatory ladybirds, the mechanisms involved in their adaptation to diet shifts are not completely understood. Here, we explored how a primarily coccidophagous ladybird Cryptolaemus montrouzieri adapts to feeding on aphids. Results: Based on the lower survival rate, longer developmental time, and lower adult body weight and reproduction rate of the predator, the aphid Megoura japonica proved being less suitable to support C. montrouzieri as compared with the citrus mealybug Planococcus citri. The results indicated up-regulation of genes related to ribosome and translation in fourth instars, which may be related to their suboptimal development. Also, several genes related to biochemical transport and metabolism, and detoxification were up-regulated as a result of adaptation to the changes in nutritional and non-nutritional (toxic) components of the prey. Conclusion: Our results indicated that C. montrouzieri succeeded in feeding on aphids by regulation of genes related to development, digestion and detoxification. Thus, we argue that these candidate genes are valuable for further studies of the functional evolution of ladybirds led by diet shifts

Phyllanthus spp. Induces Selective Growth Inhibition of PC-3 and MeWo Human Cancer Cells through Modulation of Cell Cycle and Induction of Apoptosis

BACKGROUND: Phyllanthus is a traditional medicinal plant that has been used in the treatment of many diseases including hepatitis and diabetes. The main aim of the present work was to investigate the potential cytotoxic effects of aqueous and methanolic extracts of four Phyllanthus species (P.amarus, P.niruri, P.urinaria and P.watsonii) against skin melanoma and prostate cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Phyllanthus plant appears to possess cytotoxic properties with half-maximal inhibitory concentration (IC(50)) values of 150-300 µg/ml for aqueous extract and 50-150 µg/ml for methanolic extract that were determined using the MTS reduction assay. In comparison, the plant extracts did not show any significant cytotoxicity on normal human skin (CCD-1127Sk) and prostate (RWPE-1) cells. The extracts appeared to act by causing the formation of a clear "ladder" fragmentation of apoptotic DNA on agarose gel, displayed TUNEL-positive cells with an elevation of caspase-3 and -7 activities. The Lactate Dehydrogenase (LDH) level was lower than 15% in Phyllanthus treated-cancer cells. These indicate that Phyllanthus extracts have the ability to induce apoptosis with minimal necrotic effects. Furthermore, cell cycle analysis revealed that Phyllanthus induced a Go/G1-phase arrest on PC-3 cells and a S-phase arrest on MeWo cells and these were accompanied by accumulation of cells in the Sub-G1 (apoptosis) phase. The cytotoxic properties may be due to the presence of polyphenol compounds such as ellagitannins, gallotannins, flavonoids and phenolic acids found both in the water and methanol extract of the plants. CONCLUSIONS/SIGNIFICANCE: Phyllanthus plant exerts its growth inhibition effect in a selective manner towards cancer cells through the modulation of cell cycle and induction of apoptosis via caspases activation in melanoma and prostate cancer cells. Hence, Phyllanthus may be sourced for the development of a potent apoptosis-inducing anticancer agent

Interaction of Virstatin with Human Serum Albumin: Spectroscopic Analysis and Molecular Modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ∼pH 7.2, B form ∼pH 9.0 and F form ∼pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants Ka for N and B isomers were found to be 6.09×105 M−1 and 4.47×105 M−1, respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1∶1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling

Regulation of Inflammatory Gene Expression in PBMCs by Immunostimulatory Botanicals

Many hundreds of botanicals are used in complementary and alternative medicine for therapeutic use as antimicrobials and immune stimulators. While there exists many centuries of anecdotal evidence and few clinical studies on the activity and efficacy of these botanicals, limited scientific evidence exists on the ability of these botanicals to modulate the immune and inflammatory responses. Using botanogenomics (or herbogenomics), this study provides novel insight into inflammatory genes which are induced in peripheral blood mononuclear cells following treatment with immunomodulatory botanical extracts. These results may suggest putative genes involved in the physiological responses thought to occur following administration of these botanical extracts. Using extracts from immunostimulatory herbs (Astragalus membranaceus, Sambucus cerulea, Andrographis paniculata) and an immunosuppressive herb (Urtica dioica), the data presented supports previous cytokine studies on these herbs as well as identifying additional genes which may be involved in immune cell activation and migration and various inflammatory responses, including wound healing, angiogenesis, and blood pressure modulation. Additionally, we report the presence of lipopolysaccharide in medicinally prepared extracts of these herbs which is theorized to be a natural and active component of the immunostimulatory herbal extracts. The data presented provides a more extensive picture on how these herbs may be mediating their biological effects on the immune and inflammatory responses

Synthetic Double-Stranded RNAs Are Adjuvants for the Induction of T Helper 1 and Humoral Immune Responses to Human Papillomavirus in Rhesus Macaques

Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C12U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C12U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC—but not CpG-C given at the same dose—also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell–activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell–attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates