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Sample records for early protein expression

  1. Matrix Gla Protein expression pattern in the early avian embryo.

    PubMed

    Correia, Elizabeth; Conceição, Natércia; Cancela, M Leonor; Belo, José A

    2016-01-01

    MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

  2. Nuclear genes encoding plastid proteins expressed early in chloroplast development

    SciTech Connect

    Mullet, J.E.

    1991-01-01

    The overall objective of this grant was to characterize events which occur early in chloroplast biogenesis and to isolate nuclear genes encoding plastid proteins which are expressed during this developmental phase. In addition, the possible requirement of plastid transcription for the expression of the nuclear genes such as rbcS and cab was to be tested. The impetus for this research came from studies of chloroplast biogenesis in barley. We found that plastid DNA copy number was relatively high (120 copies/plastid vs 200 at maximal accumulation) in the meristematic region of the leaf base whereas plastid transcription activity was low in this plastid population. Later in chloroplast development transcription activity increased at least 5 fold per plastid or per template indicating that activation of plastid transcription occurred after most of the build up in plastid DNA per plastid. This suggested that activation of plastid DNA synthesis occurred early in chloroplast development and that nuclear genes involved in this activity must be regulated differently from genes such rbcS or cab which are expressed later in development. 3 refs., 7 figs.

  3. Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos.

    PubMed

    Bang, Jae-Il; Lee, Hyo-Sang; Deb, Gautam Kumar; Ha, A-Na; Kwon, Young-Sang; Cho, Seong-Keun; Kim, Byeong-Woo; Cho, Kyu-Woan; Kong, Il-Keun

    2013-01-15

    It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term.

  4. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  5. Expression and imaging of fluorescent proteins in the C. elegans gonad and early embryo.

    PubMed

    Green, Rebecca A; Audhya, Anjon; Pozniakovsky, Andrei; Dammermann, Alexander; Pemble, Hayley; Monen, Joost; Portier, Nathan; Hyman, Anthony; Desai, Arshad; Oegema, Karen

    2008-01-01

    The Caenorhabditis elegans gonad and early embryo have recently emerged as an attractive metazoan model system for studying cell and developmental biology. The success of this system is attributable to the stereotypical architecture and reproducible cell divisions of the gonad/early embryo, coupled with penetrant RNAi-mediated protein depletion. These features have facilitated the development of visual assays with high spatiotemporal resolution to monitor specific subcellular processes. Assay development has relied heavily on the emergence of methods to circumvent germline silencing to allow the expression of transgenes encoding fluorescent fusion proteins. In this chapter, we discuss methods for the expression and imaging of fluorescent proteins in the C. elegans germline, including the design of transgenes for optimal expression, the generation of transgenic worm lines by ballistic bombardment, the construction of multimarker lines by mating, and methods for live imaging of the gonad and early embryo.

  6. Early expression of surfactant proteins D in Fusarium solani infected rat cornea.

    PubMed

    Che, Cheng-Ye; Li, Xiao-Jing; Jia, Wen-Yan; Li, Na; Xu, Qiang; Lin, Jing; Wang, Qing; Jiang, Nan; Hu, Li-Ting; Zhao, Gui-Qiu

    2012-01-01

    To investigate the early expression of surfactant proteins D(SP-D) in Fusarium solani infected rat cornea. Wistar rats were divided into group A, B and C randomly. The right eyes were chosen as the experiment one. Group A was control group. Group B was not inoculated with Fusarium solani. Group C was taken as fusarium solani keratitis model. Five rats in group B and C were executed randomly at 6, 12, 24, 48 and 96 hours respectively after the experimental model being established. The expression of SP-D was assessed through immunohistochemistry and reverse transcription polymerase chain reaction(RT-PCR). RT-PCR detected that the SP-D mRNA expression was low in the corneal of normal rats and group B. The expression of fungal infected cornea increased gradually and reached the peak at 24 hours in group C. The synchronous expression of group B and C were in significant difference (P<0.01). Immunohistochemisty discovered the protein of SP-D expression was increased gradually from 12 hours and reached the peak at 48 hours in group C. The synchronous expression of group B and C were also in significant difference (P<0.01). There exists SP-D in rat corneal tissue and the expression is significantly increased at the early period of fusarium solani infected cornea. SP-D may play a role in the early innate immunity response of the corneal resistance to Fusarium solani infection.

  7. Redox Protein Expression Predicts Radiotherapeutic Response in Early-Stage Invasive Breast Cancer Patients

    SciTech Connect

    Woolston, Caroline M.; Al-Attar, Ahmad; Storr, Sarah J.; Ellis, Ian O.; Morgan, David A.L.; Martin, Stewart G.

    2011-04-01

    Purpose: Early-stage invasive breast cancer patients have commonly undergone breast-conserving surgery and radiotherapy. In a large majority of these patients, the treatment is effective; however, a proportion will develop local recurrence. Deregulated redox systems provide cancer cells protection from increased oxidative stress, such as that induced by ionizing radiation. Therefore, the expression of redox proteins was examined in tumor specimens from this defined cohort to determine whether such expression could predict response. Methods and Materials: The nuclear and cytoplasmic expression of nine redox proteins (glutathione, glutathione reductase, glutaredoxin, glutathione peroxidase 1, 3, and 4, and glutathione S-transferase-{theta}, -{pi}, and -{alpha}) was assessed using conventional immunohistochemistry on a tissue microarray of 224 tumors. Results: A high cytoplasmic expression of glutathione S-transferase-{theta} significantly correlated with a greater risk of local recurrence (p = .008) and, when combined with a low nuclear expression (p = .009), became an independent predictive factor (p = .002) for local recurrence. High cytoplasmic expression of glutathione S-transferase-{theta} also correlated with a worse overall survival (p = .009). Low nuclear and cytoplasmic expression of glutathione peroxidase 3 (p = .002) correlated with a greater risk of local recurrence and was an independent predictive factor (p = .005). These proteins did not correlate with tumor grade, suggesting their function might be specific to the regulation of oxidative stress rather than alterations of tumor phenotype. Only nuclear (p = .005) and cytoplasmic (p = .001) expression of glutathione peroxidase 4 correlated with the tumor grade. Conclusions: Our results support the use of redox protein expression, namely glutathione S-transferase-{theta} and glutathione peroxidase 3, to predict the response to radiotherapy in early-stage breast cancer patients. If incorporated into

  8. Synovial membrane protein expression differs between juvenile idiopathic arthritis subtypes in early disease

    PubMed Central

    2014-01-01

    Introduction Juvenile idiopathic arthritis (JIA) is the most common rheumatological disease of childhood with a prevalence of around 1 in 1,000. Without appropriate treatment it can have devastating consequences including permanent disability from joint destruction and growth deformities. Disease aetiology remains unknown. Investigation of disease pathology at the level of the synovial membrane is required if we want to begin to understand the disease at the molecular and biochemical level. The synovial membrane proteome from early disease-stage, treatment naive JIA patients was compared between polyarticular and oligoarticular subgroups. Methods Protein was extracted from 15 newly diagnosed, treatment naive JIA synovial membrane biopsies and separated by two dimensional fluorescent difference in-gel electrophoresis. Proteins displaying a two-fold or greater change in expression levels between the two subgroups were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry with expression further verified by Western blotting and immunohistochemistry. Results Analysis of variance analysis (P ≤ 0.05) revealed 25 protein spots with a two-fold or greater difference in expression levels between polyarticular and oligoarticular patients. Hierarchical cluster analysis with Pearson ranked correlation revealed two distinctive clusters of proteins. Some of the proteins that were differentially expressed included: integrin alpha 2b (P = 0.04); fibrinogen D fragment (P = 0.005); collagen type VI (P = 0.03); fibrinogen gamma chain (P = 0.05) and peroxiredoxin 2 (P = 0.02). The identified proteins are involved in a number of different processes including platelet activation and the coagulation system. Conclusions The data indicate distinct synovial membrane proteome profiles between JIA subgroups at an early stage in the disease process. The identified proteins also provide insight into differentially perturbed pathways

  9. Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression.

    PubMed Central

    Colberg-Poley, A M; Santomenna, L D; Harlow, P P; Benfield, P A; Tenney, D J

    1992-01-01

    We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or IE1 and in permissive human diploid fibroblasts (HFF) by IE1 or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37x1. The synergistic transactivation of hsp70 expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of IE1 or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression. Images PMID:1370097

  10. Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

    PubMed

    Morange, M; Diu, A; Bensaude, O; Babinet, C

    1984-04-01

    In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.

  11. Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

    PubMed Central

    Morange, M; Diu, A; Bensaude, O; Babinet, C

    1984-01-01

    In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells. Images PMID:6546970

  12. The Etl-1 gene encodes a nuclear protein differentially expressed during early mouse development.

    PubMed

    Schoor, M; Schuster-Gossler, K; Gossler, A

    1993-07-01

    Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae. Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development. ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis. Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm. In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development. After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1. During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1. Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription. In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage.

  13. Early changes in costameric and mitochondrial protein expression with unloading are muscle specific.

    PubMed

    Flück, Martin; Li, Ruowei; Valdivieso, Paola; Linnehan, Richard M; Castells, Josiane; Tesch, Per; Gustafsson, Thomas

    2014-01-01

    We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

  14. Expression of Mismatch Repair Proteins in Early and Advanced Gastric Cancer in Poland

    PubMed Central

    Karpińska-Kaczmarczyk, Katarzyna; Lewandowska, Magdalena; Ławniczak, Małgorzata; Białek, Andrzej; Urasińska, Elżbieta

    2016-01-01

    Background Mutations in DNA of mismatch repair (MMR) genes result in failure to repair errors that occur during DNA replication in microsatellites, resulting in accumulation of frameshift mutations in these genes and leading to DNA mismatch replication errors and microsatellite instability. Gastric cancers (GCs) with high MSI (MSI-H) are a well-defined subset of carcinomas showing distinctive clinicopathological features. In this study we investigated the rate of MSI and the correlation between MSI status and clinicopathological features of GC. Material/Methods The study included 107 patients with GCs: 61 with advanced gastric cancers (AGC) and 46 with early gastric cancer (EGC). MSI deficiency in GCs was assessed by the immunohistochemical analysis of expression of MMR proteins – MLH1, MSH2, MSH6, and PMS2 – using formalin-fixed and paraffin-embedded tissue. Results A total of 6 (5.6%) MSI-H were observed. The loss of MMR proteins expression was associated with the intestinal type of GC in Lauren classification, and tubular and papillary architecture in WHO classification. There was no statistically significant association between negative MMR expression and other selected clinical parameters: age, sex, tumor location, depth of invasion (EGC and AGC), lymph nodes status, presence of the ulceration, and lymphocytic infiltrate. Conclusions In the present era of personalized medicine, the histological type of GC and MMR proteins status in cancer cells are very important for the proper surveillance of patients with familial GC and sporadic GCs, as well as for selecting the proper follow-up and treatment. Larger collaborative studies are needed to verify the features of MSI-H GCs in Poland. PMID:27527654

  15. NUCLEAR EGFR PROTEIN EXPRESSION PREDICTS POOR SURVIVAL IN EARLY STAGE NON-SMALL CELL LUNG CANCER

    PubMed Central

    Traynor, Anne M.; Weigel, Tracey L.; Oettel, Kurt R.; Yang, David T.; Zhang, Chong; Kim, KyungMann; Salgia, Ravi; Iida, Mari; Brand, Toni M.; Hoang, Tien; Campbell, Toby C.; Hernan, Hilary R.; Wheeler, Deric L.

    2013-01-01

    Introduction Nuclear EGFR (nEGFR) has been identified in various human tumor tissues, including cancers of the breast, ovary, oropharynx, and esophagus, and has predicted poor patient outcomes. We sought to determine if protein expression of nEGFR is prognostic in early stage non-small cell lung cancer (NSCLC). Methods Resected stage I and II NSCLC specimens were evaluated for nEGFR protein expression using immunohistochemistry (IHC). Cases with at least one replicate core containing ≥5% of tumor cells demonstrating strong dot-like nucleolar EGFR expression were scored as nEGFR positive. Results Twenty-three (26.1% of the population) of 88 resected specimens stained positively for nEGFR. Nuclear EGFR protein expression was associated with higher disease stage (45.5% of stage II vs. 14.5% of stage I; p=0.023), histology (41.7% in squamous cell carcinoma vs. 17.1% in adenocarcinoma; p=0.028), shorter progression-free survival (PFS) (median PFS 8.7 months [95% CI 5.1–10.7 mo] for nEGFR positive vs. 14.5 months [95% CI 9.5–17.4 mo] for nEGFR negative; hazard ratio (HR) of 1.89 [95% CI 1.15–3.10]; p=0.011), and shorter overall survival (OS) (median OS 14.1 months [95% CI 10.3–22.7 mo] for nEGFR positive vs. 23.4 months [95% CI 20.1–29.4 mo] for nEGFR negative; HR of 1.83 [95% CI 1.12–2.99]; p=0.014). Conclusions Expression of nEGFR protein was associated with higher stage and squamous cell histology, and predicted shorter PFS and OS, in this patient cohort. Nuclear EGFR serves as a useful independent prognostic variable and as a potential therapeutic target in NSCLC. PMID:23628526

  16. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

    ERIC Educational Resources Information Center

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  17. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

    ERIC Educational Resources Information Center

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  18. Bone morphogenetic protein 1 is expressed in porcine ovarian follicles and promotes oocyte maturation and early embryonic development

    PubMed Central

    LEI, Xiaocan; CUI, Kuiqing; CAI, Xiaoyan; REN, Yanping; LIU, Qingyou; SHI, Deshun

    2016-01-01

    In the present study, we tried to determine whether bone morphogenetic protein 1 (BMP1) plays a role in ovarian follicular development and early embryo development. We systematically investigated the expression and influence of BMP1 during porcine follicle and early embryonic development. Immunohistochemistry demonstrated that the BMP1 protein is expressed in granular cells and oocytes during follicular development, from primary to pre-ovulatory follicles, including atretic follicles and the corpus luteum. The mRNA expression of BMP1 significantly increased as the porcine follicles grew. Immunofluorescence analysis indicated that BMP1 was expressed in cumulus-oocyte complexes (COCs), oocytes and porcine embryos during early in vitro culture. qPCR and western blot analysis showed that the expression of BMP1 was significantly up-regulated in mature porcine oocytes and COCs compared to immature oocytes and COCs. BMP1 is expressed in early porcine embryos, and its expression reaches a peak at the 8-cell stage. To determine the effect of BMP1 on the maturation of oocytes and the development of early embryos, various concentrations of BMP1 recombinant protein or antibody were added to the in vitro culture media, respectively. BMP1 significantly affected the porcine oocyte maturation rate, the cleavage rate and the blastocyst development rate of embryos cultured in vitro in a positive way, as well as the blastocyst cell number. In conclusion, BMP1 is expressed throughout porcine ovarian follicle development and early embryogenesis, and it promotes oocyte maturation and the developmental ability of embryos during early in vitro culture. PMID:27890905

  19. Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

    PubMed Central

    Jurata, L W; Kenny, D A; Gill, G N

    1996-01-01

    LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876198

  20. Differentially expressed proteins on postoperative 3 days healing in rabbit Achilles tendon rupture model after early kinesitherapy.

    PubMed

    Jialili, Ainuer; Jielile, Jiasharete; Abudoureyimu, Shajidan; Sabirhazi, Gulnur; Redati, Darebai; Bai, Jing-Ping; Bin, Liang; Duisabai, Sailike; Aishan, Jiangaguli; Kasimu, Haxiaobieke

    2011-04-01

    Surgical repair of Achilles tendon (AT) rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n equal to 16) received postoperative cast immobilization; Group B (early motion group, n equal to 16) received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C). The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF) and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI) protein database retrieval and then for bioinformatics analysis. A mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1, pro-alpha-1 type 1 collagen

  1. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  2. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  3. Expression of collapsin response mediator protein 1 in placenta of normal gestation and link to early-onset preeclampsia.

    PubMed

    Qiao, Chong; Wang, Chunhui; Jin, Feng; Zheng, Dongying; Liu, Caixia

    2015-04-01

    A human isoform of Collapsin Response Mediator Protein (CRMP) family proteins, CRMP-1, has been identified as a novel invasion suppressor. The aim of this study was to determine CRMP-1 expression pattern in placentas during normal pregnancy and elucidate the clinical significance of CRMP-1 expression in the placentas of women with early-onset preeclamptic pregnancies. We recruited 66 normal healthy pregnant Chinese women and 60 Chinese patients with preeclampsia [early-onset prereclampsia(ePE), n = 30 and late-onset preeclampsia(lPE) n = 30]. Gestational age-matched normal healthy pregnant women were used as controls of early-onset and late-onset preeclampsia, which were 23-33 + 6 weeks, n = 18 and control B: 34-40 weeks, n = 20). Quantitative RT-PCR, Western blot analysis and immunohistochemistry were used to analyze the expressions of CRMP-1 in placentas. Expression of CRMP-1 was detected in syncytio- and cytotrophoblasts of all groups using immunohistochemistry. CRMP-1 was most abundantly expressed in syncytiotrophoblasts, moderately in cytotrophoblasts and the intermediate trophoblasts especially in the first trimester. The placental expression of CRMP-1 is particularly striking in the first trimester and decreases throughout pregnancy. There is a significant increase in CRMP-1 expression in the placenta of ePE but not of lPE, as compared to gestational-matched controls. The aberrant upregulation of CRMP-1 expression may link to the mechanism of developing ePE. © The Author(s) 2014.

  4. Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene.

    PubMed Central

    Hayek, T; Masucci-Magoulas, L; Jiang, X; Walsh, A; Rubin, E; Breslow, J L; Tall, A R

    1995-01-01

    The human cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins. The activity of CETP results in a reduction in HDL cholesterol levels, but CETP may also promote reverse cholesterol transport. Thus, the net impact of CETP expression on atherogenesis is uncertain. The influence of hypertriglyceridemia and CETP on the development of atherosclerotic lesions in the proximal aorta was assessed by feeding transgenic mice a high cholesterol diet for 16 wk. 13 out of 14 (93%) hypertriglyceridemic human apo CIII (HuCIII) transgenic (Tg) mice developed atherosclerotic lesions, compared to 18 out of 29 (62%) controls. In HuCIII/CETPTg, human apo AI/CIIITg and HuAI/CIII/CETPTg mice, 7 of 13 (54%), 5 of 10 (50%), and 5 of 13 (38%), respectively, developed lesions in the proximal aorta (P < .05 compared to HuCIIITg). The average number of aortic lesions per mouse in HuCIIITg and controls was 3.4 +/- 0.8 and 2.7 +/- 0.6, respectively in HuCIII/CETPTg, HuAI/CIIIg, and HuAI/CIII/CETPTg mice the number of lesions was significantly lower than in HuCIIITg and control mice: 0.9 +/- 0.4, 1.5 +/- 0.5, and 0.9 +/- 0.4, respectively. There were parallel reductions in mean lesion area. In a separate study, we found an increased susceptibility to dietary atherosclerosis in nonhypertriglyceridemic CETP transgenic mice compared to controls. We conclude that CETP expression inhibits the development of early atherosclerotic lesions but only in hypertriglyceridemic mice. PMID:7560101

  5. Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood.

    PubMed

    Clinton, Sarah M; Glover, Matthew E; Maltare, Astha; Laszczyk, Ann M; Mehi, Stephen J; Simmons, Rebecca K; King, Gwendalyn D

    2013-08-21

    Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development.

  6. Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy.

    PubMed

    Yang, Yong-Jun; Liu, Wei-Min; Zhou, Jia-Xi; Cao, Yu-Jing; Li, Jing; Peng, Sha; Wang, Li; Yuan, Jiang-Gang; Duan, En-Kui

    2006-01-11

    Calcyclin-binding protein (Siah-1-Interacting Protein, CacyBP/SIP), is a calcium signaling protein involved in the degradation of beta-catenin, however, little is known about its role in reproductive biology. The present study was to character its temporospatial expression pattern and regulation in mouse uterus and to investigate whether it plays a role in the regulation of normal endometrial events. While prominently expressed in both luminal and glandular epithelia, CacyBP underwent dynamic changes during early pregnancy. CacyBP expression was observed weakly from days 1-4. An intense accumulation in luminal and glandular epithelia as well as decidua surrounding the embryo at later stages (days 5-7) was observed. Most notably, CacyBP accumulation in trophoblast was pronounced at day 7. Using ovariectomized and pseudopregnant mice, we found that progesterone (P(4)) and 17beta-estradiol (E(2)) led to increased expression of CacyBP gene and this could be abolished by Ru486 and tamoxifen, respectively. Antisense oligonucleotides (ODNs) against CacyBP significantly inhibited cultured endometrial stromal cells' (ESCs) apoptosis induced by UV irradiation. Injection of antisense ODNs into mouse uterine horn severely impaired the number of implanted blastocysts. Taken together, our results suggested that CacyBP expression was positively regulated by P(4) and E(2). CacyBP may be involved in the regulation of endometrial cell apoptosis during early pregnancy and play an important role in mouse endometrial events such as pregrancy establishment.

  7. Expression of FK506-binding protein 52 (FKBP52) in chorionic villi with early recurrent spontaneous abortion.

    PubMed

    Chen, Hong-Yan; Li, Ou-Yang; Pang, Li-Hong; Xu, Hong; Fan, Xiao-Jing; Liang, Hai-Feng; Chen, Xian-Feng; Qing, Jun-Zhen; Huang, Ru-Dian; Deng, Bi-Ye

    2015-07-01

    To investigate the mRNA and protein expression of FK506-binding protein 52 (FKBP52) in the chorionic villi of patients with recurrent spontaneous abortion (RSA) and normal women during early pregnancy. Fresh chorionic villus tissues were collected from 60 subjects. A total of 30 patients with a history of RSA were enrolled into the RSA group and 30 normal pregnant women were enrolled into the control group. The FKBP52 mRNA expression levels in chorionic villi of the RSA patients and healthy controls were measured via semiquantitative RT-PCR. The protein distribution and expression levels of FKBP52 in chorionic villi were analyzed through immunohistochemistry (IHC). The correlation between FKBP52 expression and RSA was analyzed. We demonstrated that FKBP52 mRNA is expressed in chorionic villi samples of normal pregnancy and RSA. RSA patients exhibited significantly lower FKBP52 gene expression levels compared with those in normal pregnancies (p < 0.05). FKBP52 immunoreactivity in chorionic villi was mainly observed in trophoblast cell cytoplasm. The FKBP52 protein expression levels in the chorionic villi of RSA patients was significantly lower than in normal women during pregnancy (p < 0.05). FKBP52 protein levels were decreased in the chorionic villi of RSA patients, which indicate that the decrease in FKBP52 may be associated with RSA. The low FKBP52 mRNA expression level, which is consistent with the IHC result, may affect embryonic development and even lead to abortion. FKBP52 may be involved in the pathogenesis of RSA and new therapies that increase the FKBP52 expression may help treat RSA.

  8. Temporal and spatial expression patterns of biomineralization proteins during early development in the stony coral Pocillopora damicornis.

    PubMed

    Mass, Tali; Putnam, Hollie M; Drake, Jeana L; Zelzion, Ehud; Gates, Ruth D; Bhattacharya, Debashish; Falkowski, Paul G

    2016-04-27

    Reef-building corals begin as non-calcifying larvae that, upon settling, rapidly begin to accrete skeleton and a protein-rich skeletal organic matrix that attach them to the reef. Here, we characterized the temporal and spatial expression pattern of a suite of biomineralization genes during three stages of larval development in the reef-building coral Pocillopora damicornis: stage I, newly released; stage II, oral-aborally compressed and stage III, settled and calcifying spat. Transcriptome analysis revealed 3882 differentially expressed genes that clustered into four distinctly different patterns of expression change across the three developmental stages. Immunolocalization analysis further reveals the spatial arrangement of coral acid-rich proteins (CARPs) in the overall architecture of the emerging skeleton. These results provide the first analysis of the timing of the biomineralization 'toolkit' in the early life history of a stony coral. © 2016 The Author(s).

  9. Temporal and spatial expression patterns of biomineralization proteins during early development in the stony coral Pocillopora damicornis

    PubMed Central

    Putnam, Hollie M.; Drake, Jeana L.; Zelzion, Ehud; Gates, Ruth D.; Bhattacharya, Debashish; Falkowski, Paul G.

    2016-01-01

    Reef-building corals begin as non-calcifying larvae that, upon settling, rapidly begin to accrete skeleton and a protein-rich skeletal organic matrix that attach them to the reef. Here, we characterized the temporal and spatial expression pattern of a suite of biomineralization genes during three stages of larval development in the reef-building coral Pocillopora damicornis: stage I, newly released; stage II, oral-aborally compressed and stage III, settled and calcifying spat. Transcriptome analysis revealed 3882 differentially expressed genes that clustered into four distinctly different patterns of expression change across the three developmental stages. Immunolocalization analysis further reveals the spatial arrangement of coral acid-rich proteins (CARPs) in the overall architecture of the emerging skeleton. These results provide the first analysis of the timing of the biomineralization ‘toolkit’ in the early life history of a stony coral. PMID:27122561

  10. Expression of regulatory proteins in choroid plexus changes in early stages of Alzheimer disease.

    PubMed

    Krzyzanowska, Agnieszka; García-Consuegra, Inés; Pascual, Consuelo; Antequera, Desiree; Ferrer, Isidro; Carro, Eva

    2015-04-01

    Recent studies indicate that the choroid plexus has important physiologic and pathologic roles in Alzheimer disease (AD). To obtain additional insight on choroid plexus function, we performed a proteomic analysis of choroid plexus samples from patients with AD stages I to II (n = 16), III to IV (n = 16), and V to VI (n = 11) and 7 age-matched control subjects. We used 2-dimensional differential gel electrophoresis coupled with mass spectrometry to generate a complete picture of changes in choroid plexus protein expression occurring in AD patients. We identified 6 proteins: 14-3-3 β/α, 14-3-3 ε, moesin, proteasome activator complex subunit 1, annexin V, and aldehyde dehydrogenase, which were significantly regulated in AD patient samples (p < 0.05, >1.5-fold variation in expression vs control samples). These proteins are implicated in major physiologic functions including mitochondrial dysfunction and apoptosis regulation. These findings contribute additional significance to the emerging importance of molecular and functional changes of choroid plexus function in the pathophysiology of AD.

  11. Human cytomegalovirus immediate early gene expression in the osteosarcoma line U2OS is repressed by the cell protein ATRX.

    PubMed

    McFarlane, Steven; Preston, Chris M

    2011-04-01

    The control of human cytomegalovirus (HCMV) immediate early (IE) gene expression in infected human fibroblasts was compared with that in the U2OS human osteosarcoma cells. Viral IE expression was stimulated by the virion protein pp71 and repressed by the cell protein hDaxx in fibroblasts, as expected from published data. Neither of these events occurred in infected U2OS cells, suggesting that this cell line lacks one or more factors that repress HCMV IE expression. The chromatin remodeling factor ATRX is absent from U2OS cells, therefore the effect of introducing this protein by electroporation of plasmid DNA was investigated. Provision of ATRX inhibited HCMV IE expression, and the presence of the HCMV-specified virion phosphoprotein pp71 overcame the repression. The experiments demonstrate that ATRX can act as a cellular intrinsic antiviral defense in U2OS cells by blocking gene expression from incoming HCMV genomes. In contrast, ATRX did not affect the replication of herpes simplex virus type 1, showing that there are differences in the way U2OS cells respond to the presence of the herpesviral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Early auditory enrichment with music enhances auditory discrimination learning and alters NR2B protein expression in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Cai, Rui; Zhang, Jiping; Sun, Xinde

    2009-01-03

    Previous studies have shown that the functional development of auditory system is substantially influenced by the structure of environmental acoustic inputs in early life. In our present study, we investigated the effects of early auditory enrichment with music on rat auditory discrimination learning. We found that early auditory enrichment with music from postnatal day (PND) 14 enhanced learning ability in auditory signal-detection task and in sound duration-discrimination task. In parallel, a significant increase was noted in NMDA receptor subunit NR2B protein expression in the auditory cortex. Furthermore, we found that auditory enrichment with music starting from PND 28 or 56 did not influence NR2B expression in the auditory cortex. No difference was found in the NR2B expression in the inferior colliculus (IC) between music-exposed and normal rats, regardless of when the auditory enrichment with music was initiated. Our findings suggest that early auditory enrichment with music influences NMDA-mediated neural plasticity, which results in enhanced auditory discrimination learning.

  13. Early changes in protein expression of barley following inoculation with erysiphe graminis f. sp. hordei

    SciTech Connect

    Simons, S.P.; Somerville, S.C. )

    1989-04-01

    Erysiphe graminis f. sp. hordei is an obligate pathogen of barley causing the powdery mildew disease. Resistance to this disease is the product of a highly specific interaction between barley lines with specific resistance alleles and pathogen races carrying complementary avirulence alleles. Using congenic barley lines which differ at the M1-a disease reaction locus, we hope to define the early molecular events of this interaction. Accordingly, resistant and susceptible barley seedlings were labelled with {sup 35}S-methionine and examined by two-dimensional electrophoresis at two hour intervals following inoculation. Infection related changes were observed with both isolines during the four to twelve hour time period. Additional differences existed constitutively between the barley lines. These differences have been quantified. Further characterization of these proteins will yield useful markers for events preceding or coinciding with cytological responses any may lead to identification and cloning of the M1-a gene.

  14. Loss of tumour-specific ATM protein expression is an independent prognostic factor in early resected NSCLC

    PubMed Central

    Petersen, Lars F.; Klimowicz, Alexander C.; Otsuka, Shannon; Elegbede, Anifat A.; Petrillo, Stephanie K.; Williamson, Tyler; Williamson, Chris T.; Konno, Mie; Lees-Miller, Susan P.; Hao, Desiree; Morris, Don; Magliocco, Anthony M.; Bebb, D. Gwyn

    2017-01-01

    Ataxia-telangiectasia mutated (ATM) is critical in maintaining genomic integrity. In response to DNA double-strand breaks, ATM phosphorylates downstream proteins involved in cell-cycle checkpoint arrest, DNA repair, and apoptosis. Here we investigate the frequency, and influence of ATM deficiency on outcome, in early-resected non-small cell lung cancer (NSCLC). Tissue microarrays, containing 165 formalin-fixed, paraffin-embedded resected NSCLC tumours from patients diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, between 2003 and 2006, were analyzed for ATM expression using quantitative fluorescence immunohistochemistry. Both malignant cell-specific ATM expression and the ratio of ATM expression within malignant tumour cells compared to that in the surrounding tumour stroma, defined as the ATM expression index (ATM-EI), were measured and correlated with clinical outcome. ATM loss was identified in 21.8% of patients, and was unaffected by clinical pathological variables. Patients with low ATM-EI tumours had worse survival outcomes compared to those with high ATM-EI (p < 0.01). This effect was pronounced in stage II/III patients, even after adjusting for other clinical co-variates (p < 0.001). Additionally, we provide evidence that ATM-deficient patients may derive greater benefit from guideline-recommended adjuvant chemotherapy following surgical resection. Taken together, these results indicate that ATM loss seems to be an early event in NSCLC carcinogenesis and is an independent prognostic factor associated with worse survival in stage II/III patients. PMID:28418844

  15. Loss of tumour-specific ATM protein expression is an independent prognostic factor in early resected NSCLC.

    PubMed

    Petersen, Lars F; Klimowicz, Alexander C; Otsuka, Shannon; Elegbede, Anifat A; Petrillo, Stephanie K; Williamson, Tyler; Williamson, Chris T; Konno, Mie; Lees-Miller, Susan P; Hao, Desiree; Morris, Don; Magliocco, Anthony M; Bebb, D Gwyn

    2017-06-13

    Ataxia-telangiectasia mutated (ATM) is critical in maintaining genomic integrity. In response to DNA double-strand breaks, ATM phosphorylates downstream proteins involved in cell-cycle checkpoint arrest, DNA repair, and apoptosis. Here we investigate the frequency, and influence of ATM deficiency on outcome, in early-resected non-small cell lung cancer (NSCLC). Tissue microarrays, containing 165 formalin-fixed, paraffin-embedded resected NSCLC tumours from patients diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, between 2003 and 2006, were analyzed for ATM expression using quantitative fluorescence immunohistochemistry. Both malignant cell-specific ATM expression and the ratio of ATM expression within malignant tumour cells compared to that in the surrounding tumour stroma, defined as the ATM expression index (ATM-EI), were measured and correlated with clinical outcome. ATM loss was identified in 21.8% of patients, and was unaffected by clinical pathological variables. Patients with low ATM-EI tumours had worse survival outcomes compared to those with high ATM-EI (p < 0.01). This effect was pronounced in stage II/III patients, even after adjusting for other clinical co-variates (p < 0.001). Additionally, we provide evidence that ATM-deficient patients may derive greater benefit from guideline-recommended adjuvant chemotherapy following surgical resection. Taken together, these results indicate that ATM loss seems to be an early event in NSCLC carcinogenesis and is an independent prognostic factor associated with worse survival in stage II/III patients.

  16. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery.

    PubMed

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-07-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood-brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.

  17. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery

    PubMed Central

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-01-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood–brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke. PMID:23571276

  18. Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

    PubMed

    Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-04-01

    Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

  19. Ectopic expression of Ralstonia solanacearum effector protein PopA early in invasion results in loss of virulence.

    PubMed

    Kanda, Ayami; Yasukohchi, Masahiko; Ohnishi, Kouhei; Kiba, Akinori; Okuno, Tetsuro; Hikichi, Yasufumi

    2003-05-01

    Ralstonia solanacearum OE1-1 (OE1-1) is pathogenic to tobacco. The type III-secreted effector protein popA of OE1-1 showed 97.6% identity to popA of R. solanacearum GMI1000, which is not pathogenic to tobacco. Reverse transcription-polymerase chain reaction analysis showed that popA in OE1-1 was expressed at 3 h after inoculation (HAI), but not before, in infiltrated-tobacco leaves. Pathogenicity analysis using a popABC operon-deleted mutant of OE1-1 (deltaABC) showed that popABC is not directly involved in the pathogenicity of OE1-1. When Papa, which constitutively expresses popA, was infiltrated into tobacco leaves, popA was expressed by 0.5 HAI. Papa could no longer multiply or spread in tobacco leaves and was no longer virulent. Moreover, the hypersensitive response (HR) and expression of HR-related genes were not induced in Papa-infiltrated leaves. Papa was also avirulent in a tobacco root-dipping inoculation assay. These results suggest that the expression of popA in Papa immediately after invasion triggers the suppression of bacterial proliferation and movement, resulting in loss of virulence. However, Papa retained its virulence when directly inoculated into xylem vessels. This result suggests that tobacco plants can recognize PopA when it is expressed early in disease development, and respond with an effective defense in the intercellular spaces.

  20. Enhanced expression of LINE-1-encoded ORF2 protein in early stages of colon and prostate transformation

    PubMed Central

    De Luca, Chiara; Guadagni, Fiorella; Sinibaldi-Vallebona, Paola; Sentinelli, Steno; Gallucci, Michele; Hoffmann, Andreas; Schumann, Gerald G.; Spadafora, Corrado; Sciamanna, Ilaria

    2016-01-01

    LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer. We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and tissues. We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively. Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker. PMID:26716650

  1. Growth pattern switch of renal cells and expression of cell cycle related proteins at the early stage of diabetic nephropathy

    SciTech Connect

    Zhang Yanling; Shi Yonghong; Liu Yaling; Dong Hui; Liu, Maodong; Li Ying; Duan Huijun

    2007-11-09

    Renal hypertrophy, partly due to cell proliferation and hypertrophy, has been found correlated to renal function deterioration in diabetes mellitus. We screened the up-regulated cell cycle related genes to investigate cell growth and the expression of cell cycle regulating proteins at the early stage of diabetic nephropathy using STZ-induced diabetic rats. Cyclin E, CDK{sub 2} and P{sup 27} were found significantly up-regulated in diabetic kidney. Increased cell proliferation in the kidney was seen at day 3, peaked at day 5, and returned to normal level at day 30. Cyclin E and CDK{sub 2} expression also peeked at day 5 and P{sup 27} activity peaked at day 14. These findings indicate that a hyperplastic growth period of renal cells is followed by a hypertrophic growth period at the early stage of diabetes. The growth pattern switch may be regulated by cell cycle regulating proteins, Cyclin E, CDK{sub 2}, and P{sup 27}.

  2. Altered gene expression in hippocampus and depressive-like behavior in young adult female mice by early protein malnutrition.

    PubMed

    Belluscio, L M; Alberca, C D; Pregi, N; Cánepa, E T

    2016-11-01

    Perinatal development represents a critical period in the life of an individual. A common cause of poor development is that which comes from undernutrition or malnutrition. In particular, protein deprivation during development has been shown to have deep deleterious effects on brain's growth and plasticity. Early-life stress has also been linked with an increased risk to develop different psychopathologies later in life. We have previously shown that perinatal protein malnutrition in mice leads to the appearance of anxiety-related behaviors in the adulthood. We also found evidence that the female offspring was more susceptible to the development of depression-related behaviors. In the present work, we further investigated this behavior together with its molecular bases. We focused our study on the hippocampus, as it is a structure involved in coping with stressful situations. We found an increase in immobility time in the forced swimming test in perinatally malnourished females, and an alteration in the expression of genes related with neuroplasticity, early growth response 1, calcineurin and c-fos. We also found that perinatal malnutrition causes a reduction in the number of neurons in the hippocampus. This reduction, together with altered gene expression, could be related to the increment in immobility time observed in the forced swimming test. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  3. Protein expression and oxygen consumption rate of early postmortem mitochondria relate to meat tenderness.

    PubMed

    Grabež, V; Kathri, M; Phung, V; Moe, K M; Slinde, E; Skaugen, M; Saarem, K; Egelandsdal, B

    2015-04-01

    Oxygen consumption rate (OCR) of muscle fibers from bovine semimembranosus muscle of 41 animals was investigated 3 to 4 h and 3 wk postmortem. Significant relations (P < 0.05) were found between OCR measurements and Warner-Bratzler shear force measurement. Muscles with high mitochondrial OCR after 3 to 4 h and low nonmitochondrial oxygen consumption gave more tender meat. Tender (22.92 ± 2.2 N/cm2) and tough (72.98 ± 7.2 N/cm2) meat samples (4 samples each), separated based on their OCR measurements, were selected for proteomic studies using mitochondria isolated approximately 2.5 h postmortem. Twenty-six differently expressed proteins (P < 0.05) were identified in tender meat and 19 in tough meat. In tender meat, the more prevalent antioxidant and chaperon enzymes may reduce reactive oxygen species and prolong oxygen removal by the electron transport system (ETS). Glycolytic, Krebs cycle, and ETS enzymes were also more abundant in tender meat

  4. Early Preclinical Changes in Hippocampal CREB-Binding Protein Expression in a Mouse Model of Familial Alzheimer's Disease.

    PubMed

    Ettcheto, Miren; Abad, Sonia; Petrov, Dmitry; Pedrós, Ignacio; Busquets, Oriol; Sánchez-López, Elena; Casadesús, Gemma; Beas-Zarate, Carlos; Carro, Eva; Auladell, Carme; Olloquequi, Jordi; Pallàs, Merce; Folch, Jaume; Camins, Antoni

    2017-07-27

    The molecular basis of memory loss in Alzheimer's disease (AD), the main cause of senile dementia, is under investigation. In the present study, we have focused on the early hippocampal memory-related changes in APPswe/PS1dE9 (APP/PS1) mice, a well-established mouse model of familial AD. It is well known that molecules like cAMP response element binding (CREB) and binding protein (CBP) play a crucial role in memory consolidation. We analyzed CBP on its transcriptional activity and protein levels, finding a significant downregulation of both of them at 3-month-old mice. In addition, the downregulation of this molecule was associated with a decrease on acetylation levels of histone H3 in the hippocampus of APP/PS1 mice. Moreover, the p-CREB levels, which are important for memory acquisition at 3 months in APP/PS1 mice, were downregulated. Furthermore, we suggest that early neuroinflammation, especially due to the Tnfα gene increased expression, could also be responsible to this process of memory loss. Given all the previously mentioned results, we propose that an early suitable treatment to prevent the evolution of the disease should include a combination of drugs, including anti-inflammatories, which may decrease glial activation and Tnfα levels, and phosphodiesterase inhibitors that increase cAMP levels.

  5. Phylogenetic analysis and seasonal cold acclimation-associated expression of early light-induced protein genes of Rhododendron catawbiense.

    PubMed

    Peng, Yanhui; Lin, Wuling; Wei, Hui; Krebs, Stephen L; Arora, Rajeev

    2008-01-01

    The early light-induced proteins (ELIPs) are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting Chl a/b-binding proteins. Recent evidence from physiological and genetic (mutant) studies supports a photoprotective function for ELIPs, particularly when green tissues are exposed to high light intensities at suboptimal temperatures. Broad-leaved evergreens belonging to genus Rhododendron are often exposed to a combination of low temperatures and high light in their natural habitat as the understory plants in deciduous forests and, therefore, are expected to employ photoprotective strategies during overwintering phase. Here we report analysis and characterization of previously identified ELIP expressed sequence tags (ESTs) from winter-collected Rhododendron catawbiense leaves. 5' or 3' rapid amplification of complementary DNA ends (RACEs) coupled with bioinformatic analyses were used to identify seven unique ELIPs from the 40 ESTs and were designated as RcELIP1-RcELIP7. Phylogenetic analysis revealed separate clustering of ELIP homologs from lower plants, monocots and eudicots (including RcELIPs) and further indicated an evolutionary divergence of ELIPs among angiosperms and gymnosperms. To gain insights into the cold acclimation (CA) physiology of rhododendrons, relative and absolute quantitative expression of RcELIPs was examined during seasonal CA of R. catawbiense leaves using real time reverse transcriptase-polymerase chain reaction. All seven RcELIPs were distinctly upregulated during the CA. It is postulated that RcELIPs expression constitutes an adaptive response to cold and high light in winter-adapted rhododendron leaves and perhaps plays a key role in the protection of photosynthetic apparatus from these stresses.

  6. Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses

    PubMed Central

    Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A.; Maines, Taronna R.

    2016-01-01

    ABSTRACT A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary

  7. Nuclear Expression of Hepatitis B Virus X Protein Is Associated with Recurrence of Early-Stage Hepatocellular Carcinomas: Role of Viral Protein in Tumor Recurrence

    PubMed Central

    Jin, Jing; Jung, Hae Yoen; Lee, Kyu Ho; Yi, Nam-Joon; Suh, Kyung-Suk; Jang, Ja-June; Lee, Kyoung-Bun

    2016-01-01

    Background: Hepatitis B virus (HBV) plays well-known roles in tumorigenesis of hepatocellular carcinoma (HCC) in infected patients. However, HBV-associated protein status in tumor tissues and the relevance to tumor behavior has not been reported. Our study aimed to examine the expression of HBV-associated proteins in HCC and adjacent nontumorous tissue and their clinicopathologic implication in HCC patients. Methods: HBV surface antigen (HBsAg), HBV core antigen (HBcAg), and HBV X protein (HBx) were assessed in 328 HBV-associated HCCs and in 155 matched nontumorous tissues by immunohistochemistry staining. Results: The positive rates of HBsAg and cytoplasmic HBx staining in tumor tissue were lower than those in nontumorous tissue (7.3% vs. 57.4%, p < .001; 43.4% vs. 81.3%, p < .001). Conversely, nuclear HBx was detected more frequently in tumors than in nontumorous tissue (52.1% vs. 30.3%, p < .001). HCCs expressing HBsAg, HBcAg, or cytoplasmic HBx had smaller size; lower Edmondson-Steiner (ES) nuclear grade, pT stage, and serum alpha-fetoprotein, and less angioinvasion than HCCs not expressing HBV-associated proteins. Exceptionally, nuclear HBx-positive HCCs showed higher ES nuclear grade and more frequent large-vessel invasion than did nuclear HBx-negative HCCs. In survival analysis, only nuclear HBx-positive HCCs had shorter disease-free survival than nuclear HBx-negative HCCs in pT1 and ES nuclear grade 1–2 HCC subgroup (median, 126 months vs. 35 months; p = .015). Conclusions: Our data confirmed that expression of normal HBV-associated proteins generally decreases in tumor cells in comparison to nontumorous hepatocytes, with the exception of nuclear HBx, which suggests that nuclear HBx plays a role in recurrence of well-differentiated and early-stage HCCs. PMID:27086597

  8. Expression of C-Reactive Protein and Serum Amyloid A in Early to Late Manifestations of Lyme Disease.

    PubMed

    Uhde, Melanie; Ajamian, Mary; Li, Xueting; Wormser, Gary P; Marques, Adriana; Alaedini, Armin

    2016-12-01

     Infection with Borrelia burgdorferi, the causative agent of Lyme disease, triggers host immune responses that affect the clinical outcome and are a source of biomarkers with diagnostic utility. Although adaptive immunity to B. burgdorferi has been extensively characterized, considerably less information is available about the development of innate acute-phase responses in Lyme disease. Our aim in this study was to evaluate the expression of C-reactive protein (CRP) and serum amyloid A (SAA), the prototype acute-phase response proteins, in the context of the varying manifestations associated with Lyme borreliosis.  Circulating concentrations of CRP and SAA in patients with a range of early to late objective manifestations of Lyme disease and in individuals with post-treatment Lyme disease syndrome were compared with those in healthy control groups.  CRP and SAA levels were significantly elevated in early localized and early disseminated Lyme disease but not in the later stages of active infection. Levels of CRP, but not SAA, were also found to be significantly increased in patients with antibiotic-refractory Lyme arthritis and in those with post-treatment Lyme disease syndrome.  These findings indicate that circulating CRP and SAA levels are highest when the concentration of spirochetes is greatest in skin and/or blood and that levels decline after the dissemination of the organism to extracutaneous sites in subsequent stages of infection. The data also suggest that antibiotic-refractory Lyme arthritis and post-treatment Lyme disease syndrome are associated with elevated CRP responses that are driven by inflammatory mechanisms distinct from those in active infection. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  9. Superovulation alters embryonic poly(A)-binding protein (Epab) and poly(A)-binding protein, cytoplasmic 1 (Pabpc1) gene expression in mouse oocytes and early embryos.

    PubMed

    Ozturk, Saffet; Yaba-Ucar, Aylin; Sozen, Berna; Mutlu, Derya; Demir, Necdet

    2016-03-01

    Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P<0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation.

  10. Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

    PubMed

    Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

    2016-12-15

    A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans.

  11. Correlation of tracheal smooth muscle function with structure and protein expression during early development.

    PubMed

    Cullen, Aaron B; Cooke, Peter H; Driska, Steven P; Wolfson, Marla R; Shaffer, Thomas H

    2007-05-01

    With increased survival of premature infants, understanding the impact of development on airway function and structure is imperative. Airway smooth muscle plays a primary role in the modulation of airway function. The purpose of this study is to correlate the functional maturation of airway smooth muscle during the perinatal period with structural alterations at the cellular, ultrastructural, and molecular levels. Length-tension and dose-response analyses were performed on tracheal rings acquired from preterm and term newborn lambs. Subsequent structural analyses included isolated airway smooth muscle cell length, electron microscopy, and myosin heavy chain isoform expression measurements. Functionally the compliance, contractility, and agonist sensitivity of the tracheal rings matured during preterm to term development. Structurally, isolated cell lengths and electron microscopic ultrastructure were not significantly altered during perinatal development. However, expression of myosin heavy chain isoforms increased significantly across the age range analyzed, correlating with the maturational increase in smooth muscle contractility. In conclusion, the developmental alterations in tracheal function appear due, in part, to enhanced smooth muscle myosin heavy chain expression. Copyright 2007 Wiley-Liss, Inc.

  12. Early weaning increases intestinal permeability, alters expression of cytokine and tight junction proteins, and activates mitogen-activated protein kinases in pigs.

    PubMed

    Hu, C H; Xiao, K; Luan, Z S; Song, J

    2013-03-01

    Although weaning stress has been reported to impair intestinal barrier function, the mechanisms have not yet been elucidated. In the present study, the intestinal morphology and permeability and mRNA expressions of tight junction proteins and cytokines in the intestine of piglets during the 2 wk after weaning were assessed. The phosphorylated (activated) ratios of p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular regulated kinases (ERK1/2) were determined to investigate whether mitogen-activated protein kinase (MAPK) signaling pathways are involved in the early weaning process. A shorter villus and deeper crypt were observed on d 3 and 7 postweaning. Although damaged intestinal morphology recovered to preweaning values on d 14 postweaning, the intestinal mucosal barrier, which was reflected by transepithelial electrical resistance (TER) and paracellular flux of dextran (4 kDa) in the Ussing chamber and tight junction protein expression, was not recovered. Compared with the preweaning stage (d 0), jejunal TER and mRNA expressions of occludin and claudin-1 on d 3, 7, and 14 postweaning and Zonula occludens-1 (ZO-1) mRNA on d 3 and 7 postweaning were reduced, and paracellular flux of dextran on d 3, 7, and 14 postweaning was increased. An increase (P < 0.05) of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA on d 3 and d 7 postweaning and an increase (P < 0.05) of interferon-γ (IFN-γ) mRNA on d 3 postweaning were observed compared with d 0. No significant increase of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) mRNA after weaning was observed. The phosphorylated (activated) ratios of JNK and p38 on d 3 and 7 postweaning and the phosphorylated ratio of ERK1/2 on d 3 postweaning were increased (P < 0.05) compared with d 0. The results indicated that early weaning induced sustained impairment in the intestinal barrier, decreased mRNA expression of tight junction proteins, and upregulated the expression of proinflammatory

  13. Differences in heat shock protein 70 expression during larval and early spat development in the Eastern oyster, Crassostrea virginica (Gmelin, 1791).

    PubMed

    Ueda, Nobuo; Boettcher, Anne

    2009-07-01

    For a variety of species, changes in the expression of heat shock proteins (HSP) have been linked to key developmental changes, i.e., gametogenesis, embryogenesis, and metamorphosis. Many marine invertebrates are known to have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis as they transition to the benthic life stage. A series of experiments were run to examine the expression of heat shock protein 70 (HSP 70) during larval and early spat (initial benthic phase) development in the Eastern oyster, Crassostrea virginica. In addition, the impact of thermal stress on HSP 70 expression during these early stages was studied. C. virginica larvae and spat expressed three HSP 70 isoforms, two constitutive, HSC 77 and HSC 72, and one inducible, HSP 69. We found differences in the expression of both the constitutive and inducible forms of HSP 70 among larval and early juvenile stages and in response to thermal stress. Low expression of HSP 69 during early larval and spat development may be associated with the susceptibility of these stages to environmental stress. Although developmental regulation of HSP 70 expression has been widely recognized, changes in its expression during settlement and metamorphosis of marine invertebrates are still unknown. The results of the current study demonstrated a reduction of HSP 70 expression during settlement and metamorphosis in the Eastern oyster, C. virginica.

  14. Early music exposure modifies GluR2 protein expression in rat auditory cortex and anterior cingulate cortex.

    PubMed

    Xu, Feng; Cai, Rui; Xu, Jinghong; Zhang, Jiping; Sun, Xinde

    2007-06-13

    GluR2, a major subunit in AMPA receptor, plays an important role in brain functional activity. We studied the effect of music exposure during development on the expression level of GluR2 proteins in the auditory cortex (AC) and anterior cingulate cortex (ACC) of SD rats. Rats were divided into three groups, Music1 (exposed to Nostalgy) group, Music2 (exposed to Wishmaster) group, and control (no music exposure) group. For music exposure groups, rats were exposed to music from postnatal day (PND) 14, and the expression levels of GluR2 proteins were determined at PND 28, 42 and 56. For the control group, the expression levels of GluR2 proteins were determined at PND1, 3, 5, 7, 9, 11, 14, 21, 28, 42, and 56. Results showed an age-dependent expression of GluR2 proteins in control rats. In AC, exposure to Music2 dramatically increased the expression of GluR2, while exposure to Music1 had no effect. In ACC, we found remarkable discrepancies in time-dependent expression of GluR2 between music exposed rats and control rats. These results indicate that exposure to music can modify the expression level of GluR2 protein in AC and ACC.

  15. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  16. The autoimmunity-associated BLK haplotype exhibits cis-regulatory effects on mRNA and protein expression that are prominently observed in B cells early in development

    PubMed Central

    Simpfendorfer, Kim R.; Olsson, Lina M.; Manjarrez Orduño, Nataly; Khalili, Houman; Simeone, Alyssa M.; Katz, Matthew S.; Lee, Annette T.; Diamond, Betty; Gregersen, Peter K.

    2012-01-01

    The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4+ and CD8+ T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development. PMID:22678060

  17. Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

    PubMed Central

    Voronina, Ekaterina; Lopez, Manuel; Juliano, Celina E.; Gustafson, Eric; Song, Jia L.; Extavour, Cassandra; George, Sophie; Oliveri, Paola; McClay, David; Wessel, Gary

    2009-01-01

    Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16 cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa-protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed, respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa-expression in this embryo is restricted early by translational regulation to the small micromere lineage. PMID:18191830

  18. Comparative proteomic analysis of differentially expressed proteins in the early milky stage of rice grains during high temperature stress

    PubMed Central

    Liao, Jiang-Lin; Zhou, Hui-Wen; Huang, Ying-Jin

    2014-01-01

    Rice yield and quality are adversely affected by high temperatures, and these effects are more pronounced at the ‘milky stage’ of the rice grain ripening phase. Identifying the functional proteins involved in the response of rice to high temperature stress may provide the basis for improving heat tolerance in rice. In the present study, a comparative proteomic analysis of paired, genetically similar heat-tolerant and heat-sensitive rice lines was conducted. Two-dimensional electrophoresis (2-DE) revealed a total of 27 differentially expressed proteins in rice grains, predominantly from the heat-tolerant lines. The protein profiles clearly indicated variations in protein expression between the heat-tolerant and heat-sensitive rice lines. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis revealed that 25 of the 27 differentially displayed proteins were homologous to known functional proteins. These homologous proteins were involved in biosynthesis, energy metabolism, oxidation, heat shock metabolism, and the regulation of transcription. Seventeen of the 25 genes encoding the differentially displayed proteins were mapped to rice chromosomes according to the co-segregating conditions between the simple sequence repeat (SSR) markers and the target genes in recombinant inbred lines (RILs). The proteins identified in the present study provide a basis to elucidate further the molecular mechanisms underlying the adaptation of rice to high temperature stress. PMID:24376254

  19. Effects of an Early Experience of Reward through Maternal Contact or its Denial on Laterality of Protein Expression in the Developing Rat Hippocampus

    PubMed Central

    Raftogianni, Androniki; Stamatakis, Antonios; Papadopoulou, Angeliki; Vougas, Konstantinos; Anagnostopoulos, Athanasios K.; Stylianopoulou, Fotini; Tsangaris, George Th.

    2012-01-01

    Laterality is a basic characteristic of the brain which is detectable early in life. Although early experiences affect laterality of the mature brain, there are no reports on their immediate neurochemical effects during neonatal life, which could provide evidence as to the mechanisms leading to the lateralized brain. In order to address this issue, we determined the differential protein expression profile of the left and right hippocampus of 13-day-old rat control (CTR) pups, as well as following exposure to an early experience involving either receipt (RER) or denial (DER) of the expected reward of maternal contact. Proteomic analysis was performed by 2-dimensional polyacrylamide gel electrophoresis (PAGE) followed by mass spectroscopy. The majority of proteins found to be differentially expressed either between the three experimental groups (DER, RER, CTR) or between the left and right hemisphere were cytoskeletal (34%), enzymes of energy metabolism (32%), and heat shock proteins (17%). In all three groups more proteins were up-regulated in the left compared to the right hippocampus. Tubulins were found to be most often up-regulated, always in the left hippocampus. The differential expression of β-tubulin, β-actin, dihydropyrimidinase like protein 1, glial fibrillary acidic protein (GFAP) and Heat Shock protein 70 revealed by the proteomic analysis was in general confirmed by Western blots. Exposure to the early experience affected brain asymmetry: In the RER pups the ratio of proteins up-regulated in the left hippocampus to those in the right was 1.8, while the respective ratio was 3.6 in the CTR and 3.4 in the DER. Our results could contribute to the elucidation of the cellular mechanisms mediating the effects of early experiences on the vulnerability for psychopathology, since proteins shown in our study to be differentially expressed (e.g. tubulins, dihydropyrimidinase like proteins, 14-3-3 protein, GFAP, ATP synthase, α-internexin) have also been

  20. Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

    PubMed

    Tsuji, Frederick I

    2010-08-01

    The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment.

  1. Expressive Education in Early Childhood.

    ERIC Educational Resources Information Center

    Mori, Kimie

    1996-01-01

    Presents a concise overview of early childhood music education in Japan. Japanese early childhood education stresses the natural development of childhood, as well as cultivation of expressive activities. Discusses teaching methods, creative activities, and educational guidelines (MJP)

  2. Beneficial Properties of Argon After Experimental Subarachnoid Hemorrhage: Early Treatment Reduces Mortality and Influences Hippocampal Protein Expression.

    PubMed

    Höllig, Anke; Weinandy, Agnieszka; Liu, Jingjin; Clusmann, Hans; Rossaint, Rolf; Coburn, Mark

    2016-07-01

    Until now, treatment ameliorating early brain injury following subarachnoid hemorrhage has been nonexistent. Here, we evaluate the neuroprotective properties of argon after experimental subarachnoid hemorrhage with mortality as the primary endpoint and functional outcome, as well as hippocampal cellular and molecular stress response as secondary endpoints. Randomized controlled animal study. University research laboratory. Ninety-eight male Sprague-Dawley rats. One hour after subarachnoid hemorrhage induction via endovascular perforation technique or sham surgery, a breathing gas mixture containing 50 vol% argon/50 vol% oxygen (argon group) or 50 vol% nitrogen/50 vol% oxygen (control group) was applied for 1 hour. The primary objective was mortality after subarachnoid hemorrhage. Additionally, outcome was assessed via 1) neurologic testing and 2) an open-field test 24 hours after subarachnoid hemorrhage, 3) protein analysis of hippocampal samples for hypoxia-inducible factor 1α and heme oxygenase 1, and 4) immunohistochemistry of hippocampal slices to quantify vital neurons. Animals were euthanized 6, 24, or 72 hours after subarachnoid hemorrhage or sham surgery. Occurrence of premature death (death prior to scheduled euthanasia) was assessed. Postconditioning with argon resulted in a reduction of risk with respect to premature death to 20.6% compared with the control group (95% CI, 4.39-96.7). Body weight was higher in the argon group over the entire observation period (p < 0.05). There was no difference in the neuroscore (p = 0.550). Expression of hypoxia-inducible factor 1α and heme oxygenase 1 in the hippocampus was increased in the argon group. Higher quantity of vital neurons in the hippocampal samples of the argon group was discovered 24 hours after subarachnoid hemorrhage. Argon application after experimental subarachnoid hemorrhage met the primary endpoint of reducing the risk of mortality. In addition, higher body weight indicating good overall

  3. The pipsqueak-domain proteins Distal antenna and Distal antenna-related restrict Hunchback neuroblast expression and early-born neuronal identity

    PubMed Central

    Kohwi, Minoree; Hiebert, Laurel S.; Doe, Chris Q.

    2011-01-01

    A fundamental question in brain development is how precursor cells generate a diverse group of neural progeny in an ordered manner. Drosophila neuroblasts sequentially express the transcription factors Hunchback (Hb), Krüppel (Kr), Pdm1/Pdm2 (Pdm) and Castor (Cas). Hb is necessary and sufficient to specify early-born temporal identity and, thus, Hb downregulation is essential for specification of later-born progeny. Here, we show that distal antenna (dan) and distal antenna-related (danr), encoding pipsqueak motif DNA-binding domain protein family members, are detected in all neuroblasts during the Hb-to-Cas expression window. Dan and Danr are required for timely downregulation of Hb in neuroblasts and for limiting the number of early-born neurons. Dan and Danr function independently of Seven-up (Svp), an orphan nuclear receptor known to repress Hb expression in neuroblasts, because Dan, Danr and Svp do not regulate each other and dan danr svp triple mutants have increased early-born neurons compared with either dan danr or svp mutants. Interestingly, misexpression of Hb can induce Dan and Svp expression in neuroblasts, suggesting that Hb might activate a negative feedback loop to limit its own expression. We conclude that Dan/Danr and Svp act in parallel pathways to limit Hb expression and allow neuroblasts to transition from making early-born neurons to late-born neurons at the proper time. PMID:21429984

  4. Proteomic Analysis on the Alteration of Protein Expression in the Early-Stage Placental Villous Tissue of Electromagnetic Fields Associated With Cell Phone Exposure

    PubMed Central

    Luo, Qiong; Jiang, Ying; Jin, Min

    2013-01-01

    Background: To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Methods: Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Results: Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified— procollagen–proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Conclusion: Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects. PMID:23420827

  5. Proteomic analysis on the alteration of protein expression in the early-stage placental villous tissue of electromagnetic fields associated with cell phone exposure.

    PubMed

    Luo, Qiong; Jiang, Ying; Jin, Min; Xu, Jian; Huang, He-Feng

    2013-09-01

    To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified- procollagen-proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects.

  6. Time course of immediate early gene protein expression in the spinal cord following conditioning stimulation of the sciatic nerve in rats.

    PubMed

    Bojovic, Ognjen; Panja, Debabrata; Bittins, Margarethe; Bramham, Clive R; Tjølsen, Arne

    2015-01-01

    Long-term potentiation induced by conditioning electrical stimulation of afferent fibers is a widely studied form of synaptic plasticity in the brain and the spinal cord. In the spinal cord dorsal horn, long-term potentiation is induced by a series of high-frequency trains applied to primary afferent fibers. Conditioning stimulation (CS) of sciatic nerve primary afferent fibers also induces expression of immediate early gene proteins in the lumbar spinal cord. However, the time course of immediate early gene expression and the rostral-caudal distribution of expression in the spinal cord have not been systematically studied. Here, we examined the effects of sciatic nerve conditioning stimulation (10 stimulus trains, 0.5 ms stimuli, 7.2 mA, 100 Hz, train duration 2 s, 8 s intervals between trains) on cellular expression of immediate early genes, Arc, c-Fos and Zif268, in anesthetized rats. Immunohistochemical analysis was performed on sagittal sections obtained from Th13- L5 segments of the spinal cord at 1, 2, 3, 6 and 12 h post-CS. Strikingly, all immediate early genes exhibited a monophasic increase in expression with peak increases detected in dorsal horn neurons at 2 hours post-CS. Regional analysis showed peak increases at the location between the L3 and L4 spinal segments. Both Arc, c-Fos and Zif268 remained significantly elevated at 2 hours, followed by a sharp decrease in immediate early gene expression between 2 and 3 hours post-CS. Colocalization analysis performed at 2 hours post-CS showed that all c-Fos and Zif268 neurons were positive for Arc, while 30% and 43% of Arc positive neurons were positive for c-Fos and Zif268, respectively. The present study identifies the spinal cord level and time course of immediate early gene (IEGP) expression of relevance for analysis of IEGPs function in neuronal plasticity and nociception.

  7. Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection.

    PubMed

    Gryspeerdt, Annick C; Vandekerckhove, Annelies P; Baghi, Hossein Bannazadeh; Van de Walle, Gerlinde R; Nauwynck, Hans J

    2012-08-01

    Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.

  8. Differential expression of proteins involved in energy production along the crypt-villus axis in early-weaning pig small intestine.

    PubMed

    Xiong, Xia; Yang, Huansheng; Tan, Bie; Yang, Chengbo; Wu, Miaomiao; Liu, Gang; Kim, Sung Woo; Li, Tiejun; Li, Lili; Wang, Junjun; Wu, Guoyao; Yin, Yulong

    2015-08-15

    Weaning of piglets reflects intestinal dysfunction and atrophy and affected the physiological state of enterocytes. However, few studies have defined physiological state of enterocytes along the crypt-villus axis in early-weaning piglets. A total of 16 piglets from 8 litters were used in the experiment. One group of piglets was nursed by sows until age 21 days, and another group was weaned at age 14 days and then fed creep feed instead of breast milk for 7 days. Piglets were killed at 21 days, and the jejunum segments were dissected. After sequential isolation of jejunum epithelial cells along the crypt-villus axis, their proteins were analyzed through the isobaric tags for relative and absolute quantification, and proteins involved in the mammalian target of rapamycin signaling pathway and proliferating cell nuclear antigen abundances in jejunal epithelial cells of weaning or suckling group were determined by Western blotting. The differential proteins in three cell fractions were identified and analyzed. The results showed that proteins involved in the tricarboxylic acid cycle, β-oxidation, and the glycolysis pathway were significantly downregulated in the upper and middle villus of the early-weaned group. However, proteins involved in glycolysis were significantly upregulated in crypt cells. In addition, Western blot analysis showed that the expression of mammalian target of rapamycin pathway-related proteins was decreased (P < 0.05) in the early-weaned group. The present results showed that early-weaning differentially affect the expression of proteins involved in energy production of enterocytes along the jejunal crypt-villus axis.

  9. Increased gene expression of growth associated protein-43 in skin of patients with early-stage peripheral neuropathies.

    PubMed

    Scheytt, Sarah; Riediger, Nadja; Braunsdorf, Silvia; Sommer, Claudia; Üçeyler, Nurcan

    2015-08-15

    Growth associated protein-43 (GAP-43) is one of the neural proteins associated with nerve injury that is upregulated after nerve injury. To investigate whether GAP-43 quantification in skin biopsies would differentiate subtypes of peripheral neuropathies, we analyzed GAP-43 expression in skin from the lateral thigh and the distal leg. We prospectively enrolled 130 patients with peripheral neuropathies and compared data with healthy controls. Intraepidermal nerve fiber density (IENFD) was determined using antibodies against protein gene product 9.5 (PGP 9.5); anti-GAP-43 antibodies were applied to visualize regenerating nerve fibers. PGP 9.5 and GAP-43 gene expression was analyzed using qRT-PCR. Patients with neuropathies had a generalized reduction of IENFD and GAP-43 immunoreactive fibers compared to controls (p<0.01). In contrast, cutaneous GAP-43 gene expression was increased in proximal skin in patients (p<0.05), particularly when disease duration was short (<3 years; p<0.01). While fiber density for both markers decreased with age in healthy skin (p<0.01), age-dependent reduction of skin innervation was absent in neuropathies. Diagnostic subgroups and neuropathic pain had no influence on skin innervation. We conclude that peripheral neuropathies lead to an initial increase in GAP-43 gene expression as a potential mechanism of regeneration, which is not sustained in neuropathies of long duration.

  10. Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy.

    PubMed

    Bojovic, Ognjen; Bramham, Clive R; Tjølsen, Arne

    2016-01-01

    Peripheral neuropathic pain is described as a pain state caused by an injury or dysfunction of the nervous system, and could have clinical manifestations such as hyperalgesia, allodynia and spontaneous pain. The development of neuropathic pain may depend on long-term forms of neuronal plasticity in the spinal cord (SC). Expression of the immediate early gene proteins (IEGPs) Arc, Zif268, and c-Fos are implicated in establishment of long-term potentiation (LTP) induced by conditioning stimulation (CS) of primary afferent fibres. However, the impact of the neuropathic state (Bennett's model) on CS-induced expression of IEGPs has not been studied. The aim of this study was to compare the levels of Arc, c-Fos and Zif268 immunoreactivity prior to and after conditioning stimulation in animals with developed neuropathic pain, with sham operated, non-ligated controls. Twenty-four animals were divided equally into the neuropathic and non-neuropathic groups. Neuropathic pain was induced in all animals by conducting a loose ligation of the sciatic nerve with Chromic Catgut 4.0 sutures 7 days prior to conditioning stimulation or sham operation. The loose ligation was performed by placing sutures around the sciatic nerve compressing the nerve slightly just enough to reduce but not completely diminish the perineural circulation. A state of neuropathy was confirmed by a significant decrease in mechanical withdrawal threshold measured by von Frey's fibres. Immunohistochemical analysis was performed on transverse sections obtained from the L3-L5 segments of the SC at 2 and 6h post-CS and IEGP positive cells were counted in lamina I and II of the dorsal horn. During statistical analyses, the groups were compared by means of analysis of variance (univariate general linear model). If significant differences were found, each set of animals was compared with the sham group with post hoc Tukey's multiple comparison test. Strikingly, all IEGPs exhibited a significant increase in

  11. Knockdown of the intraflagellar transport protein IFT46 stimulates selective gene expression in mouse chondrocytes and affects early development in zebrafish.

    PubMed

    Gouttenoire, Jérôme; Valcourt, Ulrich; Bougault, Carole; Aubert-Foucher, Elisabeth; Arnaud, Estelle; Giraud, Lionel; Mallein-Gerin, Frédéric

    2007-10-19

    Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.

  12. Protein synthesis inhibitors enhance the expression of mRNAs for early inducible inflammatory genes via mRNA stabilization.

    PubMed

    Yamazaki, Soh; Takeshige, Koichiro

    2008-02-01

    Expression of inflammatory genes is regulated at multiple steps, including transcriptional activation and mRNA stabilization. During an investigation into the requirement of de novo protein synthesis for the induction of inflammatory genes, it was revealed that protein synthesis inhibitors unexpectedly potentiated the induction of mRNAs for primary response genes, while the inhibitors suppressed the induction of secondary inducible genes as previously described. Stimulus-induced nuclear translocation and promoter recruitment of NF-kappaB, which is responsible for the transcriptional activation of many inflammatory genes, were largely unaffected by the inhibitors. Instead, these inhibitors prolonged the half-lives of all of the primary inducible mRNAs tested. Thus, these findings emphasize the important contribution of regulated mRNA longevity to gene expression induced by pro-inflammatory stimulation.

  13. Early-Life Exposure to Lead (Pb) Alters the Expression of microRNA that Target Proteins Associated with Alzheimer's Disease.

    PubMed

    Masoud, Anwar M; Bihaqi, Syed W; Machan, Jason T; Zawia, Nasser H; Renehan, William E

    2016-01-01

    There is a growing recognition of the impact of environmental toxins on the epigenetic regulation of gene expression, including the genes that play a critical role in neural development, neural function, and neurodegeneration. We have shown previously that exposure to the heavy metal lead (Pb) in early life results in a latent over-expression of AD-related proteins in rodents and primates. The present study provides evidence that early postnatal exposure to Pb also alters the expression of select miRNA. Mice were exposed to 0.2% Pb acetate from Postnatal Day 1 (PND 1, first 24 h after birth) to PND 20 via their mother's milk. Brain tissue was harvested at PND 20, 180, or 700, and miRNA were isolated and quantified by qPCR. This exposure produced a transient increase (relative to control) in the expression of miR-106b (binds to AβPP mRNA), miR-29b (targets the mRNA for the transcription factor SP1) and two miRNAs (miR-29b and miR-132) that have the ability to inhibit translation of proteins involved in promoter methylation. The expression of miR-106b decreased over time in the Pb-exposed animals and was significantly less than the levels exhibited by the control animals at PND700. The level of miR-124, which binds to SP1 mRNA, was also reduced (relative to controls) at PND700. In summary, we show that exposure to the heavy metal Pb in early life has a significant impact on the short- and long-term expression of miRNA that target epigenetic mediators and neurotoxic proteins.

  14. Regulation of desmocollin gene expression in the epidermis: CCAAT/enhancer-binding proteins modulate early and late events in keratinocyte differentiation.

    PubMed Central

    Smith, Conrad; Zhu, Kuichun; Merritt, Anita; Picton, Rhian; Youngs, Denise; Garrod, David; Chidgey, Martyn

    2004-01-01

    Desmocollins (Dscs) are desmosomal cadherins that exhibit differentiation-specific patterns of expression in the epidermis. Dsc3 expression is strongest in basal cell layers, whereas Dsc1 is largely confined to upper, terminally differentiating strata. To understand better the processes by which Dsc expression is regulated in the epidermis, we have isolated Dsc3 and Dsc1 5'-flanking DNAs and analysed their activity in primary keratinocytes. In the present study, we found that transcription factors of the CCAAT/enhancer-binding protein family play a role in the regulation of expression of both Dscs and, in so doing, implicate this class of transcription factors in both early and late events in keratinocyte differentiation. We show that Dscs are differentially regulated by C/EBP (CCAAT/enhancer-binding protein) family members, with Dsc3 expression being activated by C/EBPbeta but not C/EBPalpha, and the reverse being the case for Dsc1. Expression of both Dscs is activated by another family member, C/EBPdelta. These results show for the first time how desmosomal cadherin gene expression is regulated and provide a mechanism for the control of other differentiation-specific genes in the epidermis. PMID:15030314

  15. Maternal Low-Protein Diet Modulates Glucose Metabolism and Hepatic MicroRNAs Expression in the Early Life of Offspring †

    PubMed Central

    Zheng, Jia; Xiao, Xinhua; Zhang, Qian; Wang, Tong; Yu, Miao; Xu, Jianping

    2017-01-01

    Emerging studies revealed that maternal protein restriction was associated with increased risk of type 2 diabetes mellitus in adulthood. However, the mechanisms of its effects on offspring, especially during early life of offspring, are poorly understood. Here, it is hypothesized that impaired metabolic health in offspring from maternal low-protein diet (LPD) is associated with perturbed miRNAs expression in offspring as early as the weaning age. We examined the metabolic effects on the C57BL/6J mice male offspring at weaning from dams fed with LPD or normal chow diet (NCD) throughout pregnancy and lactation. Maternal LPD feeding impaired metabolic health in offspring. Microarray profiling indicated that mmu-miR-615, mmu-miR-124, mmu-miR-376b, and mmu-let-7e were significantly downregulated, while, mmu-miR-708 and mmu-miR-879 were upregulated in LPD offspring. Bioinformatic analysis showed target genes were mapped to inflammatory-related pathways. Serum tumor necrosis factor-α (TNF-α) levels were higher and interleukin 6 (IL-6) had a tendency to be elevated in the LPD group. Finally, both mRNA and protein levels of IL-6 and TNF-α were significantly increased in the LPD group. Our findings provide novel evidence that maternal LPD can regulate miRNAs expression, which may be associated with chronic inflammation status and metabolic health in offspring as early as the weaning age. PMID:28264458

  16. Maternal Low-Protein Diet Modulates Glucose Metabolism and Hepatic MicroRNAs Expression in the Early Life of Offspring †.

    PubMed

    Zheng, Jia; Xiao, Xinhua; Zhang, Qian; Wang, Tong; Yu, Miao; Xu, Jianping

    2017-02-27

    Emerging studies revealed that maternal protein restriction was associated with increased risk of type 2 diabetes mellitus in adulthood. However, the mechanisms of its effects on offspring, especially during early life of offspring, are poorly understood. Here, it is hypothesized that impaired metabolic health in offspring from maternal low-protein diet (LPD) is associated with perturbed miRNAs expression in offspring as early as the weaning age. We examined the metabolic effects on the C57BL/6J mice male offspring at weaning from dams fed with LPD or normal chow diet (NCD) throughout pregnancy and lactation. Maternal LPD feeding impaired metabolic health in offspring. Microarray profiling indicated that mmu-miR-615, mmu-miR-124, mmu-miR-376b, and mmu-let-7e were significantly downregulated, while, mmu-miR-708 and mmu-miR-879 were upregulated in LPD offspring. Bioinformatic analysis showed target genes were mapped to inflammatory-related pathways. Serum tumor necrosis factor-α (TNF-α) levels were higher and interleukin 6 (IL-6) had a tendency to be elevated in the LPD group. Finally, both mRNA and protein levels of IL-6 and TNF-α were significantly increased in the LPD group. Our findings provide novel evidence that maternal LPD can regulate miRNAs expression, which may be associated with chronic inflammation status and metabolic health in offspring as early as the weaning age.

  17. Primary structure and developmental expression of Bufo arenarum cellular nucleic acid-binding protein: changes in subcellular localization during early embryogenesis.

    PubMed

    Armas, P; Cabada, M O; Calcaterra, N B

    2001-02-01

    A Bufo arenarum cellular nucleic acid-binding protein (bCNBP) full-length cDNA was cloned. bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser-rich region. Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes. Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior. One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription. Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes. In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm. At mid-blastula stage, CNBP was mainly detected in the epiblast. At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining. Nuclei in this layer were stained even stronger than the cytoplasm. Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.

  18. PRDA1, a novel chloroplast nucleoid protein, is required for early chloroplast development and is involved in the regulation of plastid gene expression in Arabidopsis.

    PubMed

    Qiao, Jiangwei; Li, Jun; Chu, Wen; Luo, Meizhong

    2013-12-01

    Chloroplast development requires accurate spatio-temporal expression of plastid genes. The regulation of plastid genes mediated by plastid-encoded RNA polymerase (PEP) is rather complex, and its related mechanism remains largely unclear. Here, we report the identification of a novel protein that is essential for plant development, PEP-Related Development Arrested 1 (PRDA1). Knock-out of PRDA1 in Arabidopsis (prda1 mutant) caused a seedling-lethal, albino phenotype and arrested the development of leaf chloroplasts. Localization analysis showed that PRDA1 was specifically targeted to chloroplasts and co-localized with chloroplast nucleoids, revealing that PRDA1 is a chloroplast nucleoid-associated protein. Gene expression analyses revealed that the PEP-dependent plastid transcript levels were greatly reduced in prda1. PRDA1 was co-expressed with most of the PEP-associated proteins. Protein interaction assays showed that PRDA1 clearly interacts with MRL7 and FSD2, both of which have been verified as essential for PEP-related chloroplast development. Reactive oxygen species scavenging through dimethylthiourea markedly alleviated the cotyledon-albino phenotypes of PRDA1 and MRL7 RNA interference seedlings. These results demonstrate that PRDA1 is required for early chloroplast development and involved in the regulation of plastid gene expression.

  19. Immediate early gene expression in cat visual cortex during and after the critical period: differences between EGR-1 and Fos proteins.

    PubMed

    Kaplan, I V; Guo, Y; Mower, G D

    1996-02-01

    Immediate early gene (IEG) expression in the cat visual cortex is highly responsive to visual input and may initiate genetic mechanisms responsible for neuronal plasticity. The present study used immunohistochemical methods to address two issues regarding IEG expression in response to visual input. One was to define the differential response of distinct IEG families by comparing EGR-1 (also termed zif-268, NGFI-A, and Krox-24) and Fos proteins. The second was to determine whether IEG expression, in addition to reflecting neural activity, is related to the state of plasticity by comparing young and adult visual cortex. Immunoreactivity of the two IEG proteins was compared between 5-week-old and adult cats under three conditions of visual input: ambient light to assess basal levels of expression, 1 week of darkness to assess the effect of reduced activity, and exposure to light after 1 week of darkness to determine rapid changes in expression as a result of visual input. At both ages, there were marked differences in the expression of the two IEG proteins. EGR-1 responded to visual input with sustained changes in its level of expression. It showed high basal levels, reduced expression in darkness, and a rapid return to high constitutive levels with the introduction of light. Fos showed a markedly different profile. It had very low basal expression which was not demonstrably affected by darkness and its principal response was a marked transient induction upon exposure to light after darkness. These unique changes in expression highlight the complex response across IEGs to environmental input and suggest a genetic "on/off' signaling mechanism. There were marked differences in the laminar distribution of EGR-1 and Fos proteins between young and adult cats. In young animals, cells in all visual cortical layers showed high levels of EGR-1 and Fos proteins. In adults, immunostaining was largely specific to cells located above and below layer IV and only very faint labeling

  20. Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

    PubMed

    Lippolis, Rosa; Siciliano, Rosa Anna; Pacelli, Consiglia; Ferretta, Anna; Mazzeo, Maria Fiorella; Scacco, Salvatore; Papa, Francesco; Gaballo, Antonio; Dell'Aquila, Claudia; De Mari, Michele; Papa, Sergio; Cocco, Tiziana

    2015-09-01

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

  1. Complement regulatory proteins in early human fetal life: CD59, membrane co-factor protein (MCP) and decay-accelerating factor (DAF) are differentially expressed in the developing liver.

    PubMed Central

    Simpson, K L; Houlihan, J M; Holmes, C H

    1993-01-01

    The human fetus appears to be capable of protecting itself from maternal complement (C) from an early stage in development by expressing the C regulatory proteins decay-accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 on fetally derived trophoblast at the feto-maternal interface. In this study we have examined the ontogeny of these proteins within the fetus itself and have focused on the liver which represents a major site of haemopoiesis during development. Immunostaining revealed that DAF, MCP and CD59 are all expressed from at least 6 weeks of gestation in the liver but that these proteins display distinct distribution patterns. CD59 was broadly distributed both within the epithelial and haemopoietic compartments, but expression of C3 convertase regulators was more restricted. DAF expression was limited to isolated cells within haemopoietic nests and the epithelium was DAF-negative. Although MCP expression on haemopoietic cells was also limited, by contrast with DAF the developing hepatic epithelium was strongly MCP-positive. Typical CD59 and MCP components were observed in fetal liver extracts by immunoblotting, although liver MCP components consistently migrated 4000-5000 MW ahead of those observed on placental trophoblast. Differences in the distribution of these proteins were also observed between the fetal and adult liver. In particular, by comparison with fetal hepatic epithelium, there was an apparent loss of MCP expression from adult hepatocytes. Thus, MCP appears to be developmentally regulated in the human liver and is expressed in the absence of DAF on the early hepatic epithelium. Overall, this study suggests that C regulatory proteins, and in particular CD59 and MCP, are required from the very early stages of gestation within the fetus itself. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7505254

  2. Detection of early gene expression changes during activation of human primary lymphocytes by in vitro synthesis of proteins from polysome-associated mRNAs

    PubMed Central

    Miyamoto, Suzanne; Qin, Jun; Safer, Brian

    2001-01-01

    The rapid increase in protein synthesis during the mitogenic stimulation of human peripheral blood lymphocyte is the result of global and specific translational control mechanisms. To study some of these mechanisms, we examined the in vitro translatability of mRNAs associated with the polyribosome fraction. Polyribosome fractions were isolated from lymphocytes after activation with ionomycin and the phorbol ester PMA. The associated PAmRNAs were translated in the presence of mRNA-depleted rabbit reticulocyte lysate and [35S]Met, and the protein products were analyzed by SDS–PAGE and autoradiography. There was little synthesis of protein from the PAmRNAs isolated from unactivated T cells, but the PAmRNAs isolated from activated T cells showed a rapid increase in translatability. Translation of the PAmRNAs was sensitive to edeine and m7GTP, suggesting their cap-dependent translation. With activation, the majority of proteins showed increasing in vitro translation, but two proteins, p72 and p33, were found to have increased synthesis within 30 min, which decreased in 1 h. Transcription inhibitors were used to ascertain if regulation of their expression was transcriptional or translational. To identify these proteins, we used biotinylated lysine during the in vitro translation reaction, and we extracted the biotinylated protein by using streptavidin magnetic beads. The protein product was analyzed by mass spectrometry. p33 was identified as a prohibitin-like protein (BAP37), but the identification of p72 was not found in the databases. The distinct up-regulation and down-regulation of their protein expression suggest their tightly controlled regulation during early T cell activation. PMID:11266628

  3. Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

    PubMed

    Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

    2017-05-01

    Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    PubMed

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

  5. Effect of Exposure to UV-C Irradiation and Monochloramine on Adenovirus Serotype 2 Early Protein Expression and DNA Replication▿

    PubMed Central

    Sirikanchana, Kwanrawee; Shisler, Joanna L.; Mariñas, Benito J.

    2008-01-01

    The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20°C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants. PMID:18424543

  6. Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication.

    PubMed

    Sirikanchana, Kwanrawee; Shisler, Joanna L; Mariñas, Benito J

    2008-06-01

    The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20 degrees C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.

  7. DELAYED GREENING 238, a Nuclear-Encoded Chloroplast Nucleoid Protein, Is Involved in the Regulation of Early Chloroplast Development and Plastid Gene Expression in Arabidopsis thaliana.

    PubMed

    Wang, Menglong; Jiang, Lan; Da, Qingen; Liu, Jun; Feng, Dongru; Wang, Jinfa; Wang, Hong-Bin; Jin, Hong-Lei

    2016-12-01

    Chloroplast development is an essential process for plant growth that is regulated by numerous proteins. Plastid-encoded plastid RNA polymerase (PEP) is a large complex that regulates plastid gene transcription and chloroplast development. However, many proteins in this complex remain to be identified. Here, through large-scale screening of Arabidopsis mutants by Chl fluorescence imaging, we identified a novel protein, DELAYED GREENING 238 (DG238), which is involved in regulating chloroplast development and plastid gene expression. Loss of DG238 retards plant growth, delays young leaf greening, affects chloroplast development and lowers photosynthetic efficiency. Moreover, blue-native PAGE (BN-PAGE) and Western blot analysis indicated that PSII and PSI protein levels are reduced in dg238 mutants. DG238 is mainly expressed in young tissues and is regulated by light signals. Subcellular localization analysis showed that DG238 is a nuclear-encoded chloroplast nucleoid protein. More interestingly, DG238 was co-expressed with FLN1, which encodes an essential subunit of the PEP complex. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays showed that DG238 can also interact with FLN1. Taken together, these results suggest that DG238 may function as a component of the PEP complex that is important for the early stage of chloroplast development and helps regulate PEP-dependent plastid gene expression. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    PubMed

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  9. Autoinduction of Protein Expression

    PubMed Central

    Fox, Brian G.; Blommel, Paul G.

    2017-01-01

    This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included. PMID:19365792

  10. Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line

    SciTech Connect

    Jeang, K.T.; Cho, M.S.; Hayward, G.S.

    1984-10-01

    A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The authors isolated a clonal Ltk/sup +/ cell line which expressed the /sup 35/methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH/sub 2/p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH/sub 2/l198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

  11. Isolation rearing attenuates social interaction-induced expression of immediate early gene protein products in the medial prefrontal cortex of male and female rats.

    PubMed

    Wall, Vanessa L; Fischer, Eva K; Bland, Sondra T

    2012-10-10

    Early life adversity and stress in humans have been related to a number of psychological disorders including anxiety, depression, and addiction. The present study used isolation rearing, a well-characterized animal model of early life adversity, to examine its effects on social behavior and immediate early gene (IEG) expression produced by exposure to a novel social experience. Male and female rats were housed in same-sex groups or in isolation for 4 weeks beginning at weaning and were tested during late adolescence. The protein products of the IEGs c-fos and Arc, as well as the neurotrophic factor BDNF were assessed in medial prefrontal cortex (mPFC) subregions (anterior cingulate, prelimbic and infralimbic) using immunohistochemistry. Aggressive and non-aggressive behaviors during novel social exposure were also assessed. Exposure to a novel conspecific produced increases in Arc and c-fos activation in the mPFC of group reared animals in a sex- and subregion-dependent fashion compared to no social exposure controls, but this increase was blunted or absent in isolated animals. Isolates engaged in more social interactions and more aggressive behavior than group reared rats. Sex differences in some behaviors as well as in Arc and BDNF expression were observed. These results indicate that isolation rearing alters IEG activation in the mPFC produced by exposure to a novel conspecific, in addition to changing social behavior, and that these effects depend in part on sex.

  12. Elfin is expressed during early heart development.

    PubMed

    Kotaka, M; Lau, Y M; Cheung, K K; Lee, S M; Li, H Y; Chan, W Y; Fung, K P; Lee, C Y; Waye, M M; Tsui, S K

    Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.

  13. Protein expression in liposomes.

    PubMed

    Oberholzer, T; Nierhaus, K H; Luisi, P L

    1999-08-02

    Compartmentalization is one of the key steps in the evolution of cellular structures and, so far, only few attempts have been made to model this kind of "compartmentalized chemistry" using liposomes. The present work shows that even such complex reactions as the ribosomal synthesis of polypeptides can be carried out in liposomes. A method is described for incorporating into 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) liposomes the ribosomal complex together with the other components necessary for protein expression. Synthesis of poly(Phe) in the liposomes is monitored by trichloroacetic acid of the (14)C-labelled products. Control experiments carried out in the absence of one of the ribosomal subunits show by contrast no significant polypeptide expression. This methodology opens up the possibility of using liposomes as minimal cell bioreactors with growing degree of synthetic complexity, which may be relevant for the field of origin of life as well as for biotechnological applications. Copyright 1999 Academic Press.

  14. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    SciTech Connect

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O'Callaghan, Dennis J. . E-mail: docall@lsuhsc.edu

    2007-06-20

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed.

  15. Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein.

    PubMed

    Azimzadeh, Agnes M; Kelishadi, Sean S; Ezzelarab, Mohamed B; Singh, Avneesh K; Stoddard, Tiffany; Iwase, Hayato; Zhang, Tianshu; Burdorf, Lars; Sievert, Evelyn; Avon, Chris; Cheng, Xiangfei; Ayares, David; Horvath, Keith A; Corcoran, Philip C; Mohiuddin, Muhammad M; Barth, Rolf N; Cooper, David K C; Pierson, Richard N

    2015-01-01

    We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

  16. Loss of ARID1A protein expression occurs as an early event in ovarian clear-cell carcinoma development and frequently coexists with PIK3CA mutations.

    PubMed

    Yamamoto, Sohei; Tsuda, Hitoshi; Takano, Masashi; Tamai, Seiichi; Matsubara, Osamu

    2012-04-01

    ARID1A is a recently identified tumor suppressor gene that is mutated in ∼50% of ovarian clear-cell carcinomas. This mutation is associated with loss of ARID1A protein expression as assessed by immunohistochemistry. The present study aimed at determining the timing of the loss of ARID1A protein expression during the development of ovarian clear-cell carcinoma and assessing its relevance in correlation to PIK3CA gene mutations. A total of 42 clear-cell carcinoma cases with adjacent putative precursor lesions (endometriosis-associated carcinoma cases (n=28) and (clear-cell) adenofibroma-associated carcinoma cases (n=14)) were selected and subjected to immunohistochemical analysis for ARID1A protein expression and direct genomic DNA sequencing of exons 9 and 20 of the PIK3CA gene. ARID1A immunoreactivity was deficient in 17 (61%) of the 28 endometriosis-associated carcinomas and 6 (43%) of the 14 adenofibroma-associated carcinomas. Among the precursor lesions adjacent to the 23 ARID1A-deficient carcinomas, 86% of the non-atypical endometriosis (12 of 14) and 100% of the atypical endometriosis (14 of 14), benign (3 of 3), and borderline (6 of 6) clear-cell adenofibroma components were found to be ARID1A deficient. In contrast, in the 19 patients with ARID1A-intact carcinomas, all of the adjacent precursor lesions retained ARID1A expression regardless of their types and cytological atypia. Analysis of 22 solitary endometrioses and 10 endometrioses distant from ARID1A-deficient carcinomas showed that all of these lesions were diffusely immunoreactive for ARID1A. Among the 42 clear-cell carcinomas, somatic mutations of PIK3CA were detected in 17 (40%) tumors and majority (71%) of these were ARID1A-deficient carcinomas. These results suggest that loss of ARID1A protein expression occurs as a very early event in ovarian clear-cell carcinoma development, similar to the pattern of PIK3CA mutation recently reported by our group, and frequently coexists (not mutually

  17. Differential effect of early antibiotic intervention on bacterial fermentation patterns and mucosal gene expression in the colon of pigs under diets with different protein levels.

    PubMed

    Zhang, Chuanjian; Yu, Miao; Yang, Yuxiang; Mu, Chunlong; Su, Yong; Zhu, Weiyun

    2017-03-01

    The study aimed to evaluate the effects of early antibiotic intervention (EAI) on bacterial fermentation patterns and mucosal immune markers in the colon of pigs with different protein level diets. Eighteen litters of piglets at day (d) 7 were fed creep feed without or with growth promoting antibiotics until d 42. At d 42, pigs within each group were further randomly assigned to a normal- or low-crude protein (CP) diet. At d 77 and d 120, five pigs per group were slaughtered for analyzing colonic bacteria, metabolites, and mucosal gene expressions. Results showed that low-CP diet increased propionate and butyrate concentrations at d 77 but reduced ammonia and phenol concentrations (P < 0.05). EAI increased p-cresol and indole concentrations under normal-CP diet at d 77 (P < 0.05). Low-CP diet significantly affected (P < 0.05) some bacteria groups (Firmicutes, Clostridium cluster IV, Clostridium cluster XIVa, Escherichia coli, and Lactobacillus), but EAI showed limited effects. Low-CP diet down-regulated gene expressions of pro-inflammatory cytokines, toll-like receptor (TLR4), myeloid differentiating factor 88 (MyD88), and nuclear factor-κB p65 (NF-κB p65) (P < 0.05). EAI up-regulated mRNA expressions of interleukin-8 (IL-8) and interferon-γ (IFN-γ) under normal-CP diet at d 77 (P < 0.05). Furthermore, reductions of E. coli and ammonia under low-CP diet were positively correlated with down-regulated gene expressions of pro-inflammatory cytokines, which were positively correlated with the down-regulated TLR4-MyD88-NF-κB signaling pathway. In conclusion, EAI had short-term effects under normal-CP diet with increased aromatic amino acid fermentation and gene expressions of pro-inflammatory cytokines. Low-CP diet markedly reduced protein fermentation, modified microbial communities, and down-regulated gene expressions of pro-inflammatory cytokines possibly via down-regulating TLR4-MyD88-NF-κB signaling pathway.

  18. Growth hormone receptor, insulin-like growth factor (IGF)-1, and IGF-binding protein-2 expression in the reproductive tissues of early postpartum dairy cows.

    PubMed

    Rhoads, M L; Meyer, J P; Kolath, S J; Lamberson, W R; Lucy, M C

    2008-05-01

    The growth hormone/insulin-like growth factor (IGF) system plays a critical endocrine role controlling nutrient metabolism in dairy cattle. In liver, growth hormone receptor (GHR) and IGF-1 are dynamically regulated by lactation and energy balance. Less is known about the regulation of GHR, IGF-1, and IGF-binding protein mRNA in reproductive tissues (uterus, ovarian follicle, and corpus luteum). The objective was to determine expression patterns for GHR, IGF-1, and IGF-binding protein (IGFBP)-2 mRNA in the liver, uterus, dominant follicle, and corpus luteum in Holstein cows (n = 21) sampled at 3 times during early lactation. The first postpartum ovulation was induced with an injection of GnRH within 15 d of calving. Nine days after ovulation [23 +/- 1 d postpartum; 20 d in milk (DIM)], the liver, uterus, dominant follicle, and corpus luteum were biopsied. Prostaglandin F(2alpha) and GnRH were injected 7 and 9 d after each biopsy to synchronize the second (41 +/- 1 d postpartum; 40 DIM) and third (60 +/- 1 d postpartum; 60 DIM) tissue collections. Total RNA was isolated and used for mRNA analysis by real-time quantitative reverse transcription PCR. Liver had more GHR, IGF-1, and IGFBP-2 mRNA than the reproductive tissues that were tested. Gene expression for GHR, IGF-1, and IGFPB-2 within tissues did not change across the sampling interval (20 to 60 DIM). The only detected change in gene expression across days was for cyclophilin in uterus (increased after 20 DIM). Parity had an effect on gene expression for GHR in corpus luteum. Neither level of milk production nor body condition score affected the amount of GHR, IGF-1, or IGFBP-2 mRNA in the respective tissues. The repeatability of gene expression within a tissue was 0.25 to 0.5 for most genes. In most instances, expression of a single gene within a tissue was correlated with other genes in the same tissue but was not correlated with the same gene in a different tissue. We did not find evidence for major changes

  19. Changes in heat shock protein 70 expression and blood characteristics in transported broiler chickens as affected by housing and early age feed restriction.

    PubMed

    Al-Aqil, A; Zulkifli, I

    2009-07-01

    An experiment was conducted to determine the effects of 2 types of housing systems and early age feed restriction on heat shock protein (hsp) 70 expression and blood parameters in broiler chickens subjected to road transportation. On d 1, female chicks were housed either in windowless environmentally controlled chambers (temperature was set at 32 degrees C on d 1 and gradually reduced to 23 degrees C by d 21; CH) or in conventional open-sided houses (OH) with cyclic temperatures (minimum, 24 degrees C; maximum, 34 degrees C). Equal number of chicks from each housing system were subjected to either ad libitum feeding or 60% feed restriction on d 4, 5, and 6 (FR). On d 42, all of the birds were crated and transported for 6 h. Birds raised in OH had smaller increases in heterophil:lymphocyte ratios and plasma corticosterone concentrations than those of CH. Subjecting birds to FR dampened heterophil:lymphocyte ratios and corticosterone reactions to transportation. After 4 h of transportation, the OH birds had greater hsp 70 expression than their CH counterparts. Within the CH, the FR chicks showed higher hsp 70 density than those of the ad libitum-fed group. Except for glucose, housing system had a negligible effect on serum levels of cholesterol, potassium, and chloride. Collectively, the results suggest that the improved tolerance to transport stress in OH and FR chicks could be associated with better hsp 70 expression.

  20. Low Ki67/high ATM protein expression in malignant tumors predicts favorable prognosis in a retrospective study of early stage hormone receptor positive breast cancer.

    PubMed

    Feng, Xiaolan; Li, Haocheng; Kornaga, Elizabeth N; Dean, Michelle; Lees-Miller, Susan P; Riabowol, Karl; Magliocco, Anthony M; Morris, Don; Watson, Peter H; Enwere, Emeka K; Bebb, Gwyn; Paterson, Alexander

    2016-12-27

    This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (< 4 cm) and fewer than four positive lymph nodes. 532 formalin-fixed paraffin-embedded specimens of resected primary breast tumors were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC.

  1. Low Ki67/high ATM protein expression in malignant tumors predicts favorable prognosis in a retrospective study of early stage hormone receptor positive breast cancer

    PubMed Central

    Feng, Xiaolan; Li, Haocheng; Kornaga, Elizabeth N.; Dean, Michelle; Lees-Miller, Susan P.; Riabowol, Karl; Magliocco, Anthony M.; Morris, Don; Watson, Peter H.; Enwere, Emeka K.; Bebb, Gwyn; Paterson, Alexander

    2016-01-01

    Introduction This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (< 4 cm) and fewer than four positive lymph nodes. Methods 532 formalin-fixed paraffin-embedded specimens of resected primary breast tumors were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Results Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). Conclusions These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC. PMID:27741524

  2. Auxin-Regulated Genes Encoding Cell Wall-Modifying Proteins Are Expressed during Early Tomato Fruit Growth

    PubMed Central

    Catalá, Carmen; Rose, Jocelyn K.C.; Bennett, Alan B.

    2000-01-01

    An expansin gene, LeExp2, was isolated from auxin-treated, etiolated tomato (Lycopersicon esculentum cv T5) hypocotyls. LeExp2 mRNA expression was restricted to the growing regions of the tomato hypocotyl and was up-regulated during incubation of hypocotyl segments with auxin. The pattern of expression of LeExp2 was also studied during tomato fruit growth, a developmental process involving rapid cell enlargement. The expression of genes encoding a xyloglucan endotransglycosylase (LeEXT1) and an endo-1,4-β-glucanase (Cel7), which, like LeExp2, are auxin-regulated in etiolated hypocotyls (C. Catalá, J.K.C. Rose, A.B. Bennett [1997] Plant J 12: 417–426), was also studied to examine the potential for synergistic action with expansins. LeExp2 and LeEXT1 genes were coordinately regulated, with their mRNA accumulation peaking during the stages of highest growth, while Cel7 mRNA abundance increased and remained constant during later stages of fruit growth. The expression of LeExp2, LeEXT1, and Cel7 was undetectable or negligible at the onset of and during fruit ripening, which is consistent with a specific role of these genes in regulating cell wall loosening during fruit growth, not in ripening-associated cell wall disassembly. PMID:10677445

  3. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    PubMed Central

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O’Callaghan, Dennis J.

    2007-01-01

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5′untranslated region (UTR), a 285 base pair open reading frame (ORF) and a poly adenylation (A) signal (Holden et al., 1992 DNA Seq 3, 143-52). Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed. PMID:17306852

  4. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway.

    PubMed

    Szlachcic, Wojciech J; Switonski, Pawel M; Krzyzosiak, Wlodzimierz J; Figlerowicz, Marek; Figiel, Maciej

    2015-09-01

    Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life.

  5. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway

    PubMed Central

    Szlachcic, Wojciech J.; Switonski, Pawel M.; Krzyzosiak, Wlodzimierz J.; Figlerowicz, Marek; Figiel, Maciej

    2015-01-01

    ABSTRACT Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life. PMID:26092128

  6. Leptospira Protein Expression During Infection

    USDA-ARS?s Scientific Manuscript database

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  7. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.

    PubMed

    Chishti, M A; Yang, D S; Janus, C; Phinney, A L; Horne, P; Pearson, J; Strome, R; Zuker, N; Loukides, J; French, J; Turner, S; Lozza, G; Grilli, M; Kunicki, S; Morissette, C; Paquette, J; Gervais, F; Bergeron, C; Fraser, P E; Carlson, G A; George-Hyslop, P S; Westaway, D

    2001-06-15

    We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.

  8. Differential expression of serum glycodelin and insulin-like growth factor binding protein 1 in early pregnancy.

    PubMed

    Douglas, Nataki C; Thornton, Melvin H; Nurudeen, Sahadat K; Bucur, Maria; Lobo, Rogerio A; Sauer, Mark V

    2013-11-01

    This prospective study evaluated whether serum glycodelin and insulin-like growth factor binding protein 1 (IGFBP-1) predict the likelihood of embryo implantation in recipients undergoing donor egg in vitro fertilization. We measured glycodelin and IGFBP-1 at 6 points from lining check to lutenizing hormone (LH) + 31. β-Human chorionic gonadotropin levels were first measured at LH + 17. The recipients were divided into those without embryo implantation (group 1, n = 6) and those with successful implantation (group 2, n = 30). Although this is a negative study in that neither glycodelin nor IGFBP-1 alone reflected endometrial (EM) receptivity, the glycodelin/IGFBP-1 ratio on the day of blastocyst transfer was higher in recipients who achieved pregnancy (P = .05). At LH + 17, glycodelin was higher (P = .04), and IGFBP-1 was lower (P = .004) in recipients who achieved pregnancy when compared to those who did not. These observations are likely due to EM changes induced by successful embryo implantation.

  9. Morphological analysis of the early development of telencephalic and diencephalic gonadotropin-releasing hormone neuronal systems in enhanced green fluorescent protein-expressing transgenic medaka lines.

    PubMed

    Takahashi, Akiko; Islam, M Sadiqul; Abe, Hideki; Okubo, Kataaki; Akazome, Yasuhisa; Kaneko, Takeshi; Hioki, Hiroyuki; Oka, Yoshitaka

    2016-03-01

    Teleosts possess two or three paralogs of gonadotropin-releasing hormone (GnRH) genes: gnrh1, gnrh2, and gnrh3. Some species have lost the gnrh1 and/or gnrh3 genes, whereas gnrh2 has been completely conserved in the teleost species analyzed to date. In most teleosts that possess gnrh1, GnRH1 peptide is the authentic GnRH that stimulates gonadotropin release, whereas GnRH2 and GnRH3, if present, are neuromodulatory. Progenitors of GnRH1 and GnRH3 neurons originate from olfactory placodes and migrate to their destination during early development. However, because of the relatively low affinity/specificity of generally available antibodies that recognize GnRH1 or GnRH3, labeling of these neurons has only been possible using genetic manipulation. We used a model teleost, medaka, which possesses all three paralogous gnrh genes, to analyze development of forebrain GnRH neurons composed of GnRH1 and GnRH3 neurons. Here, we newly generated transgenic medaka lines that express enhanced green fluorescent protein under the control of promoters for gnrh1 or gnrh3, to detect GnRH neurons and facilitate immunohistochemical analysis of the neuronal morphology. We used a combination of immunohistochemistry and three-dimensional confocal microscopy image reconstructions to improve identification of neurites from GnRH1 or GnRH3 neuronal populations with greater precision. This led us to clearly identify the hypophysiotropic innervation of GnRH1 neurons residing in the ventral preoptic area (vPOA) from as early as 10 days post hatching. Furthermore, these analyses also revealed retinopetal projections of nonhypophysiotropic GnRH1 neurons in vPOA, prominent during early developmental stages, and multiple populations of GnRH3 neurons with different origins and migratory pathways.

  10. Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

    SciTech Connect

    Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica; Bagnasco, Luca; Orecchia, Paola; Carnemolla, Barbara; Patrone, Eligio; Balbi, Cecilia

    2009-01-15

    Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.

  11. Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

    PubMed

    Salem, K A; Adrian, T E; Qureshi, M A; Parekh, K; Oz, M; Howarth, F C

    2012-12-01

    There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0

  12. Differential Expression of Serum Glycodelin and Insulin-Like Growth Factor Binding Protein 1 in Early Pregnancy

    PubMed Central

    Thornton, Melvin H.; Nurudeen, Sahadat K.; Bucur, Maria; Lobo, Rogerio A.; Sauer, Mark V.

    2013-01-01

    This prospective study evaluated whether serum glycodelin and insulin-like growth factor binding protein 1 (IGFBP-1) predict the likelihood of embryo implantation in recipients undergoing donor egg in vitro fertilization. We measured glycodelin and IGFBP-1 at 6 points from lining check to lutenizing hormone (LH) + 31. β-Human chorionic gonadotropin levels were first measured at LH + 17. The recipients were divided into those without embryo implantation (group 1, n = 6) and those with successful implantation (group 2, n = 30). Although this is a negative study in that neither glycodelin nor IGFBP-1 alone reflected endometrial (EM) receptivity, the glycodelin/IGFBP-1 ratio on the day of blastocyst transfer was higher in recipients who achieved pregnancy (P = .05). At LH + 17, glycodelin was higher (P = .04), and IGFBP-1 was lower (P = .004) in recipients who achieved pregnancy when compared to those who did not. These observations are likely due to EM changes induced by successful embryo implantation. PMID:23585335

  13. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  14. Reduced expression of the immediate-early protein IE0 enables efficient replication of Autographa californica multiple nucleopolyhedrovirus in poorly permissive Spodoptera littoralis cells.

    PubMed

    Lu, Liqun; Du, Quansheng; Chejanovsky, Nor

    2003-01-01

    Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.

  15. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    PubMed

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  16. Obestatin induction of early-response gene expression in gastrointestinal and adipose tissues and the mediatory role of G protein-coupled receptor, GPR39.

    PubMed

    Zhang, Jian V; Jahr, Holger; Luo, Chin-Wei; Klein, Cynthia; Van Kolen, Kristof; Ver Donck, Luc; De, Ananya; Baart, Esther; Li, Jing; Moechars, Dieder; Hsueh, Aaron J W

    2008-06-01

    Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.

  17. Early Detection of Baculovirus Expression and Infection in Lepidopteran Larvae Fed Occlusion Bodies of an AcMNPV Recombinant Carrying a Red Fluorescent Protein Gene

    USDA-ARS?s Scientific Manuscript database

    A method has been devised utilizing a baculovirus recombinant (AcMNPV hsp70Red) carrying a red fluorescent protein (RFP) gene under the early heat shock promoter (hsp70) to assess potential infectivity of larvae fed occlusion bodies. A time study was employed whereby first and third instars of Trich...

  18. A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

    PubMed Central

    Tyndall, C; La Mantia, G; Thacker, C M; Favaloro, J; Kamen, R

    1981-01-01

    Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed. Images PMID:6275353

  19. Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

    PubMed

    Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Rehfeldt, Charlotte; Metges, Cornelia C

    2011-12-01

    Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

  20. A TSPO-related protein localizes to the early secretory pathway in Arabidopsis, but is targeted to mitochondria when expressed in yeast.

    PubMed

    Vanhee, Celine; Guillon, Stéphanie; Masquelier, Danièle; Degand, Hervé; Deleu, Magali; Morsomme, Pierre; Batoko, Henri

    2011-01-01

    AtTSPO is a TspO/MBR domain-protein potentially involved in multiple stress regulation in Arabidopsis. As in most angiosperms, AtTSPO is encoded by a single, intronless gene. Expression of AtTSPO is tightly regulated both at the transcriptional and post-translational levels. It has been shown previously that overexpression of AtTSPO in plant cell can be detrimental, and the protein was detected in the endoplasmic reticulum (ER) and Golgi stacks, contrasting with previous findings and suggesting a mitochondrial subcellular localization for this protein. To ascertain these findings, immunocytochemistry and ABA induction were used to demonstrate that, in plant cells, physiological levels of AtTSPO colocalized with AtArf1, a mainly Golgi-localized protein in plant cells. In addition, fluorescent protein-tagged AtTSPO was targeted to the secretory pathway and did not colocalize with MitoTracker-labelled mitochondria. These results suggest that the polytopic membrane protein AtTSPO is cotranslationally targeted to the ER in plant cells and accumulates in the Trans-Golgi Network. Heterologous expression of AtTSPO in Saccharomyces cerevisiae, yeast devoid of TSPO-related protein, resulted in growth defects. However, subcellular fractionation and immunoprecipitation experiments showed that AtTSPO was targeted to mitochondria where it colocalized and interacted with the outer mitochondrial membrane porin VDAC1p, reminiscent of the subcellular localization and activity of mammalian translocator protein 18 kDa TSPO. The evolutionarily divergent AtTSPO appears therefore to be switching its sorting mode in a species-dependent manner, an uncommon peculiarity for a polytopic membrane protein in eukaryotic cells. These results are discussed in relation to the recognition and organelle targeting mechanisms of polytopic membrane proteins in eukaryotic cells.

  1. A TSPO-related protein localizes to the early secretory pathway in Arabidopsis, but is targeted to mitochondria when expressed in yeast

    PubMed Central

    Vanhee, Celine; Guillon, Stéphanie; Masquelier, Danièle; Degand, Hervé; Deleu, Magali; Morsomme, Pierre; Batoko, Henri

    2011-01-01

    AtTSPO is a TspO/MBR domain-protein potentially involved in multiple stress regulation in Arabidopsis. As in most angiosperms, AtTSPO is encoded by a single, intronless gene. Expression of AtTSPO is tightly regulated both at the transcriptional and post-translational levels. It has been shown previously that overexpression of AtTSPO in plant cell can be detrimental, and the protein was detected in the endoplasmic reticulum (ER) and Golgi stacks, contrasting with previous findings and suggesting a mitochondrial subcellular localization for this protein. To ascertain these findings, immunocytochemistry and ABA induction were used to demonstrate that, in plant cells, physiological levels of AtTSPO colocalized with AtArf1, a mainly Golgi-localized protein in plant cells. In addition, fluorescent protein-tagged AtTSPO was targeted to the secretory pathway and did not colocalize with MitoTracker-labelled mitochondria. These results suggest that the polytopic membrane protein AtTSPO is cotranslationally targeted to the ER in plant cells and accumulates in the Trans-Golgi Network. Heterologous expression of AtTSPO in Saccharomyces cerevisiae, yeast devoid of TSPO-related protein, resulted in growth defects. However, subcellular fractionation and immunoprecipitation experiments showed that AtTSPO was targeted to mitochondria where it colocalized and interacted with the outer mitochondrial membrane porin VDAC1p, reminiscent of the subcellular localization and activity of mammalian translocator protein 18 kDa TSPO. The evolutionarily divergent AtTSPO appears therefore to be switching its sorting mode in a species-dependent manner, an uncommon peculiarity for a polytopic membrane protein in eukaryotic cells. These results are discussed in relation to the recognition and organelle targeting mechanisms of polytopic membrane proteins in eukaryotic cells. PMID:20847098

  2. The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection.

    PubMed Central

    Tollefson, A E; Scaria, A; Saha, S K; Wold, W S

    1992-01-01

    We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit. Images PMID:1316473

  3. Maize Brittle stalk2 Encodes a COBRA-Like Protein Expressed in Early Organ Development But Required for Tissue Flexibility at Maturity1[C][OA

    PubMed Central

    Sindhu, Anoop; Langewisch, Tiffany; Olek, Anna; Multani, Dilbag S.; McCann, Maureen C.; Vermerris, Wilfred; Carpita, Nicholas C.; Johal, Gurmukh

    2007-01-01

    analyses, levels of saponifiable hydroxycinnamates are elevated in bk2 leaves and stems. As Bk2 is highly expressed during early development, well before the onset of the brittle phenotype, we propose that Bk2 functions in a patterning of lignin-cellulosic interactions that maintain organ flexibility rather than having a direct role in cellulose biosynthesis. PMID:17932309

  4. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  5. Forkhead box protein P3 (Foxp3) expression serves as an early chronic inflammation marker of squamous cell differentiation and aggressive pathology of urothelial carcinomas in neurological patients.

    PubMed

    Phé, Véronique; Rouprêt, Morgan; Cussenot, Olivier; Chartier-Kastler, Emmanuel; Gamé, Xavier; Compérat, Eva

    2015-04-01

    To establish whether the expression of forkhead box protein P3 (Foxp3) provides specific diagnostic information about neurological patients with urothelial carcinoma of the bladder (UCB). UCB tissue samples from neurological patients were retrieved and compared with control samples. The expression of Foxp3 was analysed via immunohistochemistry of microarray tissue sections. The correlation between Foxp3 expression, histological parameters and tumour stage was assessed. Overall, 20 UCB tissue samples and 20 others without UCB from neurological patients, and 46 UCB tissue samples from non-neurological patients were analysed. The distribution of pT of UCB in the neurological patients was as follows: one low-grade pTa (5%), three high-grade pTa (15%), three pT1(15%), one pT2(5%), seven pT3(35%) and five pT4(25%). Squamous cell differentiation was seen in nine UCB samples (45%). Foxp3 expression was detected in tumour tissues, including one pTa high grade, one pT1, one pT2, five pT3 and five pT4 tumours. Foxp3 was expressed in 11/13 muscle-invasive tumours. All tumours displaying squamous cell differentiation expressed Foxp3. Foxp3 was not expressed in the pT3 tumours that displayed sarcomatoid and micropapillary properties. Among the bladder samples without UCB from neurological patients, no expression of Foxp3 was observed. Among the UCB samples from the non-neurological patients, only seven displayed squamous cell differentiation. All tumours that displayed squamous cell differentiation expressed Foxp3, including one pTa high grade, four pT3 and two pT4 tumours. Other tumours displaying urothelial differentiation did not express Foxp3. The expression of Foxp3 correlated to squamous cell differentiation in neurological (P = 0.004) and non-neurological UCB tissue (P < 0.001). In neurological, but not non-neurological UCB tissue, the expression of Foxp3 correlated with the muscle-invasive stage (P = 0.022). Elevated expression of Foxp3 appears to be a characteristic of

  6. Early socio-emotional experience induces expression of the immediate-early gene Arc/arg3.1 (activity-regulated cytoskeleton-associated protein/activity-regulated gene) in learning-relevant brain regions of the newborn chick.

    PubMed

    Bock, J; Thode, C; Hannemann, O; Braun, K; Darlison, M G

    2005-01-01

    We have cloned a full-length complementary DNA from the chicken (Gallus gallus domesticus), which encodes a polypeptide that exhibits approximately 75% identity to the product of the mammalian gene Arc (activity-regulated cytoskeleton-associated protein), also known as arg3.1 (activity-regulated gene). Since this gene is an immediate-early gene that has been suggested to play a role in synaptic plasticity and learning and memory processes, its expression has been analyzed in a juvenile form of learning, namely, filial imprinting. Our results demonstrate that Arc/arg3.1 mRNA is detectable in the newborn chick brain, and that at this early age the level of this transcript can be altered by brief sensory/emotional experience. After postnatal exposure to a novel 30-min auditory imprinting stimulus, Arc/arg3.1 mRNA was found to be significantly increased in two higher associative areas, the mesopallium intermediomediale (P = 0.002) and the nidopallium dorso-caudale (P = 0.031), compared with naïve controls. The transcript level was also significantly elevated after imprinting in Area L pallii (P=0.045), which is analogous to the mammalian auditory cortex. In addition, increases were seen in the medio-rostral nidopallium/mesopallium (P = 0.054), which is presumed to be the analog of the mammalian prefrontal cortex, and the hyperpallium intercalatum (P = 0.054), but these did not quite reach significance. We discuss these data in the light of those obtained in an earlier study, in the same paradigm, for the avian immediate-early gene, zenk (an acronym for zif-268, egr-1, ngfi-a and krox-24, which are different names for the orthologous mammalian gene). We conclude that, although both the Arc/arg3.1 and zenk genes are induced by auditory imprinting, they are significantly up-regulated in different learning-relevant brain regions. It is, therefore, evident that they must be activated by different mechanisms.

  7. Effect of supplemental concentrate during the dry period or early lactation on rumen epithelium gene and protein expression in dairy cattle during the transition period.

    PubMed

    Dieho, K; van Baal, J; Kruijt, L; Bannink, A; Schonewille, J T; Carreño, D; Hendriks, W H; Dijkstra, J

    2017-09-01

    We previously reported 2 experiments with rumen-cannulated Holstein-Friesian dairy cows showing that during the transition period, rumen papillae surface area, and fractional absorption rate of volatile fatty acids (VFA) increase after calving. However, supplemental concentrate during the dry period and rate of increase of concentrate allowance during lactation affected papillae surface area, but not VFA absorption. Here we report the changes in gene and protein expression in rumen papillae related to tissue growth and VFA utilization. The lactation experiment treatment consisted of a rapid [RAP; 1.0 kg of dry matter (DM)/d; n = 6] or gradual (GRAD; 0.25 kg of DM/d; n = 6) increase of concentrate allowance (up to 10.9 kg of DM/d), starting at 4 d postpartum (pp). The dry period experiment treatment consisted of 3.0 kg of DM/d of concentrate (n = 4) or no concentrate (n = 5) during the last 28 d of the dry period. Real-time quantitative PCR analysis of rumen papillae showed that the expression of apoptosis-related genes was neither affected by day nor its interaction with treatment for both experiments. Expression of epithelial transporter genes was not affected by day or treatment in the lactation experiment, except for NBC1. In the dry period experiment, expression of MCT1, NBC1, DRA, NHE2, NHE3, and UT-B generally decreased after calving. A day and treatment interaction was observed for ATP1A1 in the dry period experiment, with greater expression at 18 and 8 d antepartum for concentrate than no concentrate. Generally, expression of VFA metabolism-related genes was not affected by day or its interaction with treatment. In the lactation experiment, immunoblotting of 5 selected genes showed that protein expression of DRA and PCCA was greater at 16 d pp compared with 3 and 44 d pp. Expression of NHE2 was greater, and that of ATP1A1 lower, at 16 and 44 d pp compared with 3 d pp, suggesting alterations in intracellular pH regulation and sodium homeostasis. Both MCT1

  8. Pre- and early-postnatal nutrition modify gene and protein expressions of muscle energy metabolism markers and phospholipid Fatty Acid composition in a muscle type specific manner in sheep.

    PubMed

    Hou, Lei; Kongsted, Anna H; Ghoreishi, Seyed M; Takhtsabzy, Tasnim K; Friedrichsen, Martin; Hellgren, Lars I; Kadarmideen, Haja N; Vaag, Allan; Nielsen, Mette O

    2013-01-01

    We previously reported that undernutrition in late fetal life reduced whole-body insulin sensitivity in adult sheep, irrespective of dietary exposure in early postnatal life. Skeletal muscle may play an important role in control of insulin action. We therefore studied a range of putative key muscle determinants of insulin signalling in two types of skeletal muscles (longissimus dorsi (LD) and biceps femoris (BF)) and in the cardiac muscle (ventriculus sinister cordis (VSC)) of sheep from the same experiment. Twin-bearing ewes were fed either 100% (NORM) or 50% (LOW) of their energy and protein requirements during the last trimester of gestation. From day-3 postpartum to 6-months of age (around puberty), twin offspring received a high-carbohydrate-high-fat (HCHF) or a moderate-conventional (CONV) diet, whereafter all males were slaughtered. Females were subsequently raised on a moderate diet and slaughtered at 2-years of age (young adults). The only long-term consequences of fetal undernutrition observed in adult offspring were lower expressions of the insulin responsive glucose transporter 4 (GLUT4) protein and peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC1α) mRNA in BF, but increased PGC1α expression in VSC. Interestingly, the HCHF diet in early postnatal life was associated with somewhat paradoxically increased expressions in LD of a range of genes (but not proteins) related to glucose uptake, insulin signalling and fatty acid oxidation. Except for fatty acid oxidation genes, these changes persisted into adulthood. No persistent expression changes were observed in BF and VSC. The HCHF diet increased phospholipid ratios of n-6/n-3 polyunsaturated fatty acids in all muscles, even in adults fed identical diets for 1½ years. In conclusion, early postnatal, but not late gestation, nutrition had long-term consequences for a number of determinants of insulin action and metabolism in LD. Tissues other than muscle may account for reduced whole

  9. Pronecrotic Mixed Lineage Kinase Domain-Like Protein Expression Is a Prognostic Biomarker in Patients With Early-Stage Resected Pancreatic Adenocarcinoma

    PubMed Central

    Colbert, Lauren E.; Fisher, Sarah B.; Hardy, Claire W.; Hall, William A.; Saka, Burcu; Shelton, Joseph W.; Petrova, Aleksandra V.; Warren, Matthew D.; Pantazides, Brooke G.; Gandhi, Khanjan; Kowalski, Jeanne; Kooby, David A.; El-Rayes, Bassel F.; Staley, Charles A.; Volkan Adsay, N.; Curran, Walter J.; Landry, Jerome C.; Maithel, Shishir K.; Yu, David S.

    2015-01-01

    Background Mixed lineage kinase domain-like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC). Methods Tissue from 80 patients was collected from a prospectively maintained database of patients with PAC who underwent pancreaticoduodenectomy between January 2000 and October 2008. Immunohistochemistry analysis was performed and scored using an established scoring system. Kaplan-Meier survival curves were generated for recurrence-free survival (RFS) and overall survival (OS) for all patients and for patients receiving adjuvant chemotherapy. MLKL scores were correlated with RFS and OS using univariate and multivariate Cox regression analyses incorporating clinically relevant covariates. Results The median age of the patients was 63 years and 53% were men. Low MLKL expression was associated with decreased OS (6 months vs 17 months; P=.006). In the subset of 59 patients who received adjuvant chemotherapy, low MLKL expression was associated with decreased RFS (5 months vs 15 months; P=.006) and decreased OS (6 months vs 19 months; P<.0001). On multivariate analysis, low MLKL expression was associated with poor OS in all patients (hazards ratio, 4.6 [95% confidence interval, 1.6-13.8]; P=.006) and in patients receiving adjuvant chemotherapy (hazards ratio, 8.1 [95% confidence interval, 2.2-29.2]; P=.002). Conclusions Low expression of MLKL is associated with decreased OS in patients with resected PAC and decreased RFS and OS in the subset of patients with resected PAC who receive adjuvant chemotherapy. The use of this biomarker in patients with PAC may provide important prognostic information. PMID:23720157

  10. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection.

    PubMed Central

    McMahan, L; Schaffer, P A

    1990-01-01

    mutant, which specifies an ICP27 peptide lacking the repressing region between residues 327 and 407, is able to (i) repress early gene expression, consistent with the repressing ability of the d3 mutation in transient expression assays, (ii) induce the synthesis of significant but reduced levels of delayed-early (gamma 1) proteins and no gamma 2 proteins (thus vd3 exhibits a late protein phenotype intermediate between that of the wild-type virus and 5dl 1.2), and (iii) confer altered electrophoretic mobility on ICP4, demonstrating a role for ICP27 in the posttranslational modification of this essential regulatory protein.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2161950

  11. Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi.

    PubMed

    Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo; Chávez-Mardones, Jacqueline; Maldonado-Aguayo, Waleska

    2014-02-15

    The couch potato (CPO) protein is a key biomolecule involved in regulating diapause through the RNA-binding process of the peripheral and central nervous systems in insects and also recently discovered in a few crustacean species. As such, ectoparasitic copepods are interesting model species that have no evidence of developmental arrest. The present study is the first to report on the cloning of a putative CPO gene from the salmon louse Caligus rogercresseyi (CrCPO), as identified by high-throughput transcriptome sequencing. In addition, the transcription expression in larvae and adults was evaluated using quantitative real-time PCR. The CrCPO cDNA sequence showed 3261 base pairs (bp), consisting of 713bp of 5' UTR, 1741bp of 3' UTR, and an open reading frame of 807bp encoding for 268 amino acids. The highly conserved RNA binding regions RNP2 (LFVSGL) and RNP1 (SPVGFVTF), as well the dimerization site (LEF), were also found. Furthermore, eight single nucleotide polymorphisms located in the untranslated regions and one located in the coding region were detected. Gene transcription analysis revealed that CrCPO has ubiquitous expression across larval stages and in adult individuals, with the highest expression from nauplius to copepodid stages. The present study suggests a putative biological function of CrCPO associated with the development of the nervous system in salmon lice and contributes molecular evidence for candidate genes related to host-parasite interactions. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Seedling Lethal1, a pentatricopeptide repeat protein lacking an E/E+ or DYW domain in Arabidopsis, is involved in plastid gene expression and early chloroplast development.

    PubMed

    Pyo, Young Jae; Kwon, Kwang-Chul; Kim, Anna; Cho, Myeon Haeng

    2013-12-01

    Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E(+) or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development.

  13. Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

    PubMed Central

    Pawlik, Timothy M; Hawke, David H; Liu, Yanna; Krishnamurthy, Savitri; Fritsche, Herbert; Hunt, Kelly K; Kuerer, Henry M

    2006-01-01

    Background Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. Methods Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. Results 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. Conclusion ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer. PMID:16542425

  14. Low ATM protein expression in malignant tumor as well as cancer-associated stroma are independent prognostic factors in a retrospective study of early-stage hormone-negative breast cancer.

    PubMed

    Feng, Xiaolan; Li, Haocheng; Dean, Michelle; Wilson, Holly E; Kornaga, Elizabeth; Enwere, Emeka K; Tang, Patricia; Paterson, Alexander; Lees-Miller, Susan P; Magliocco, Anthony M; Bebb, Gwyn

    2015-05-03

    The serine/threonine protein kinase ataxia telangiectasia mutated (ATM) is critical in maintaining genomic integrity. Upon DNA double-strand breaks, ATM phosphorylates key downstream proteins including p53 and BRCA1/2, thereby orchestrating complex signaling pathways involved in cell cycle arrest, DNA repair, senescence and apoptosis. Although sporadic mutation of ATM occurs rarely in breast cancer, the status of its protein expression and its clinical significance in breast cancer remain not well established. Our study was designed to investigate the influence of ATM protein in both tumor and cancer-associated stroma on clinical outcome in hormone-positive (HPBC) and hormone-negative (HNBC) early-stage breast cancer (EBC). Tissue microarrays (TMAs), containing formalin-fixed, paraffin-embedded resected tumors from two cohorts of patients (HPBC cohort: n=130; HNBC cohort: n=168) diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, were analyzed for ATM protein expression using fluorescence immunohistochemistry (IHC) and automated quantitative analysis (AQUA). ATM expression levels were measured within the tumor as a whole (tATM) as indicated by pan-cytokeratin expression, tumor nuclear compartment (nATM) as indicated by both DAPI and pan-cytokeratin-positive results, and cancer-associated stroma (csATM) as indicated by vimentin-positive and pan-cytokeratin-negative results. ATM expression levels within these compartments were correlated with clinical outcome. While tATM and nATM were significantly lower in tumors compared to normal breast epithelial tissues, csATM was significantly higher than the corresponding normal tissue compartment. In addition, the median expression level of both tATM and nATM were two- to threefold lower (P<0.001) in HNBC than in HPBC. In both HNBC and HPBC cohorts, patients with low tATM, nATM and csATM tumors had significantly poorer survival outcomes than those with a high tATM, nATM and csATM, but this effect was more pronounced in

  15. Data Mining for Expressivity of Recombinant Protein Expression

    NASA Astrophysics Data System (ADS)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

    We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.

  16. ES2, a gene deleted in DiGeorge syndrome, encodes a nuclear protein and is expressed during early mouse development, where it shares an expression domain with a Goosecoid-like gene.

    PubMed

    Lindsay, E A; Harvey, E L; Scambler, P J; Baldini, A

    1998-04-01

    ES2 is a gene deleted in DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) which has homologs in species as distant as Caenorhabditis elegans and Drosophila . The function of ES2 is unknown, and the predicted protein sequence does not contain motifs which suggest a particular role in the developmental defects present in DGS and VCFS. Here we show that the mouse homolog, Es2 , is transcribed in two forms resulting from the use of alternative polyadenylation signals. Structural analysis programs predict that the Es2 -encoded peptide has a coiled-coil domain, and transfection experiments with an Es2 -green fluorescent protein (GFP) fusion construct show that the peptide is recruited into the nucleus. Es2 is highly expressed during mouse embryogenesis from E7 onwards. In situ hybridization with an RNA probe revealed that the gene is widely expressed; however, relatively higher expression was detected in the nervous system, with a particularly high area of expression in a sub-region of the pons. The Es2 expression domain in the pons is shared with a Goosecoid-like gene ( Gscl) which is located upstream of Es2 , and raises the possibility that the two genes share regulatory elements and/or interact in this region of the developing brain. This finding suggests that different genes in the deleted region may be functionally related and might explain the occurrence of the characteristic phenotype in patients with non-overlapping genetic lesions.

  17. Krüppel-like factor 4 is widely expressed in the mouse male and female reproductive tract and responds as an immediate early gene to activation of the protein kinase A in TM4 Sertoli cells.

    PubMed

    Godmann, M; Kosan, C; Behr, R

    2010-04-01

    Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor critically involved in cell proliferation, differentiation, and carcinogenesis. Recently, KLF4 has also been used for the generation of induced pluripotent stem cells. In this study, we analyzed Klf4 expression in different mouse tissues using northern blot analysis and immunohistochemistry. Focusing on the male and female reproductive tract, we showed for the first time that KLF4 is expressed in the epithelia of the murine uterus and the vagina. In the male reproductive tract, we detected KLF4 in the epithelia of the epididymis, ductus deferens, coagulating gland, and the penis. As KLF4 is strongly inducible by FSH signaling in Sertoli cells and as this transcription factor is also involved in Sertoli cell development, we employed the mouse Sertoli cell line TM4 as a model system to investigate i) the induction kinetics of Klf4 upon activation of the cAMP/protein kinase A pathway by forskolin and ii) the effects of Klf4 induction on TM4 cell cycle progression. Interestingly, Klf4 mRNA and protein were rapidly but transiently induced, reaching peak levels after 90-120 min and declining to basal levels within 4 h. Compared with the inducible cAMP early repressor, an immediate early response gene, the induction kinetics of Klf4 is much faster. In conclusion, Klf4 is an immediate early gene in TM4 cells and its expression in several epithelia of the male and female reproductive tract suggests an important role of Klf4 in mouse reproductive functions.

  18. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  19. Expression of potato RNA-binding proteins StUBA2a/b and StUBA2c induces hypersensitive-like cell death and early leaf senescence in Arabidopsis

    PubMed Central

    Na, Jong-Kuk; Kim, Jae-Kwang; Kim, Dool-Yi; Assmann, Sarah M.

    2015-01-01

    The Arabidopsis thaliana genome encodes three RNA-binding proteins (RBPs), UBP1-associated protein 2a (UBA2a), UBA2b, and UBA2c, that contain two RNA-recognition motif (RRM) domains. They play important roles in wounding response and leaf senescence, and are homologs of Vicia faba abscisic-acid-activated protein kinase-interacting protein 1 (VfAKIP1). The potato (Solanum tuberosum) genome encodes at least seven AKIP1-like RBPs. Here, two potato RBPs have been characterized, StUBA2a/b and StUBA2c, that are homologous to VfAKIP1 and Arabidopsis UBA2s. Transient expression of StUBA2s induced a hypersensitive-like cell death phenotype in tobacco leaves, and an RRM-domain deletion assay of StUBA2s revealed that the first RRM domain is crucial for the phenotype. Unlike overexpression of Arabidopsis UBA2s, constitutive expression of StUBA2a/b in Arabidopsis did not cause growth arrest and lethality at the young seedling stage, but induced early leaf senescence. This phenotype was associated with increased expression of defence- and senescence-associated genes, including pathogen-related genes (PR) and a senescence-associated gene (SAG13), and it was aggravated upon flowering and ultimately resulted in a shortened life cycle. Leaf senescence of StUBA2a/b Arabidopsis plants was enhanced under darkness and was accompanied by H2O2 accumulation and altered expression of autophagy-associated genes, which likely cause cellular damage and are proximate causes of the early leaf senescence. Expression of salicylic acid signalling and biosynthetic genes was also upregulated in StUBA2a/b plants. Consistent with the localization of UBA2s-GFPs and VfAKIP1-GFP, soluble-modified GFP-StUBA2s localized in the nucleus within nuclear speckles. StUBA2s potentially can be considered for transgenic approaches to induce potato shoot senescence, which is desirable at harvest. PMID:25944928

  20. Adenovirus Early Proteins and Host Sumoylation

    PubMed Central

    Sohn, Sook-Young

    2016-01-01

    ABSTRACT The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system. PMID:27651358

  1. Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

    PubMed Central

    Heston, Lee; El-Guindy, Ayman; Countryman, Jill; Dela Cruz, Charles; Delecluse, Henri-Jacques; Miller, George

    2006-01-01

    The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding

  2. Functional analysis of chloroplast early light inducible proteins (ELIPs)

    SciTech Connect

    Wetzel, Carolyn M

    2005-02-22

    The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identified and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.

  3. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  4. Infant avoidance training alters cellular activation patterns in prefronto-limbic circuits during adult avoidance learning: I. Cellular imaging of neurons expressing the synaptic plasticity early growth response protein 1 (Egr1).

    PubMed

    Gröger, Nicole; Mannewitz, Anja; Bock, Jörg; de Schultz, Tony Fernando; Guttmann, Katja; Poeggel, Gerd; Braun, Katharina

    2017-04-08

    Both positive feedback learning and negative feedback learning are essential for adapting and optimizing behavioral performance. There is increasing evidence in humans and animals that the ability of negative feedback learning emerges postnatally. Our work in rats, using a two-way active avoidance task (TWA) as an experimental paradigm for negative feedback learning, revealed that medial and lateral prefrontal regions of infant rats undergo dramatic synaptic reorganization during avoidance training, resulting in improved avoidance learning in adulthood. The aim of this study was to identify changes of cellular activation patterns during the course of training and in relation to infant pretraining. We applied a quantitative cellular imaging technique using the immunocytochemical detection of the activity marker early growth response protein 1 (Egr1) as a candidate contributing to learning-induced synaptic plasticity. We found region-specific cellular activity patterns, which indicate that during the acquisition phase, Egr1 expression is specifically elevated in cellular ensembles of the orbitofrontal, dorsal anterior cingulate and hippocampal CA1 region. During memory retrieval Egr1 expression is elevated in cellular ensembles of the dentate gyrus. Moreover, we, for the first time, show here that TWA training during infancy alters adult learning- and memory-related patterns of Egr1 expression in these brain regions. It is tempting to speculate that during infant learning, specific Egr1-expressing cellular ensembles are "tagged" representing long-term memory formation, and that these cell ensembles may be reactivated during adult learning.

  5. Evolution, diversification, and expression of KNOX proteins in plants

    PubMed Central

    Gao, Jie; Yang, Xue; Zhao, Wei; Lang, Tiange; Samuelsson, Tore

    2015-01-01

    The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification. PMID:26557129

  6. Identification, cloning, and expression analysis of three putative Lymantria dispar nuclear polyhedrosis virus immediate early genes

    Treesearch

    James M. Slavicek; Nancy Hayes-Plazolles

    1991-01-01

    Viral immediate early gene products are usually regulatory proteins that control expression of other viral genes at the transcriptional level or are proteins that are part of the viral DNA replication complex. The identification and functional characterization of the immediate early gene products of Lymantria dispar nuclear polyhedrosis virus (LdNPV...

  7. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  8. Spatiotemporal expression profiling of proteins in rat sciatic nerve regeneration using reverse phase protein arrays

    PubMed Central

    2012-01-01

    Background Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide. Results The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and β-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate. Conclusions In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite

  9. Effect of adiponectin on the steroidogenic acute regulatory protein, P450 side chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase gene expression, progesterone and androstenedione production by the porcine uterus during early pregnancy.

    PubMed

    Smolinska, N; Dobrzyn, K; Kiezun, M; Szeszko, K; Maleszka, A; Kaminski, T

    2016-06-01

    Adiponectin and its receptors are expressed in the human and porcine uterus and this endocrine system has important role in the regulation of reproductive processes. The expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (HSD3B1) were observed in the human and porcine uterus during the oestrous cycle and pregnancy. The de novo synthesis of steroids in the uterus might be a crucial factor for effective implantation and maintenance of pregnancy. We hypothesized that adiponectin modulates the expression of key enzymes in the synthesis of the steroids: StAR, P450 side chain cleavage enzyme (CYP11A1) and HSD3B1, as well as progesterone (P4) and androstenedione (A4) secretion by the porcine uterus. Endometrial and myometrial explants harvested from gilts (n = 5) on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and on days 10 to 11 of the oestrous cycle were cultured in vitro in the presence of adiponectin (1, 10 μg/ml), adiponectin with insulin (10 ng/ml) and insulin alone (10 ng/ml). Gene expression was examined by real-time PCR, and the secretion of the steroids was determined by radioimmunoassay. The content of StAR, CYP11A1 and HSD3B1 mRNAs and the secretion of P4 and A4 was modulated by adiponectin in endometrial and myometrial tissue explants during early pregnancy and the oestrous cycle. In this action adiponectin interacted with insulin. Insulin itself also regulated the steroidogenic activity of the porcine uterus. ere we reported, for the first time, the expression of CYP11A1 genes in the porcine endometrium and myometrium. Our novel findings indicate that adiponectin affects basal and insulin-stimulated expression of key steroidogenic genes and production of steroid hormones by the porcine uterus during maternal recognition of pregnancy and implantation.

  10. Protein structure protection commits gene expression patterns.

    PubMed

    Chen, Jianping; Liang, Han; Fernández, Ariel

    2008-01-01

    Gene co-expressions often determine module-defining spatial and temporal concurrences of proteins. Yet, little effort has been devoted to tracing coordinating signals for expression correlations to the three-dimensional structures of gene products. We performed a global structure-based analysis of the yeast and human proteomes and contrasted this information against their respective transcriptome organizations obtained from comprehensive microarray data. We show that protein vulnerability quantifies dosage sensitivity for metabolic adaptation phases and tissue-specific patterns of mRNA expression, determining the extent of co-expression similarity of binding partners. The role of protein intrinsic disorder in transcriptome organization is also delineated by interrelating vulnerability, disorder propensity and co-expression patterns. Extremely vulnerable human proteins are shown to be subject to severe post-transcriptional regulation of their expression through significant micro-RNA targeting, making mRNA levels poor surrogates for protein-expression levels. By contrast, in yeast the expression of extremely under-wrapped proteins is likely regulated through protein aggregation. Thus, the 85 most vulnerable proteins in yeast include the five confirmed prions, while in human, the genes encoding extremely vulnerable proteins are predicted to be targeted by microRNAs. Hence, in both vastly different organisms protein vulnerability emerges as a structure-encoded signal for post-transcriptional regulation. Vulnerability of protein structure and the concurrent need to maintain structural integrity are shown to quantify dosage sensitivity, compelling gene expression patterns across tissue types and temporal adaptation phases in a quantifiable manner. Extremely vulnerable proteins impose additional constraints on gene expression: They are subject to high levels of regulation at the post-transcriptional level.

  11. Effects of early age feed restriction and heat conditioning on heat shock protein 70 expression, resistance to infectious bursal disease, and growth in male broiler chickens subjected to heat stress.

    PubMed

    Liew, P K; Zulkifli, I; Hair-Bejo, M; Omar, A R; Israf, D A

    2003-12-01

    The effects of early age feed restriction and heat conditioning on heat shock protein (HSP) 70 expression, antibody production, resistance to infectious bursal disease (IBD), and growth of heat-stressed male broiler chickens were investigated. Chicks were divided into 4 groups: 60% feed restriction on d 4,5, and 6 (FR); exposure to 36 +/- 1 degrees C for 1 h from d 1 to 21 (HT); combination of FR and HT (FRHT); and control. From d 35 to 50, heat stress was induced by exposing birds to 38 +/- 1 degrees C and 80% RH for 2 h/d. On d 36, each bird was administered 10 times the normal dose of live IBD vaccine. After heat exposure, the FRHT birds had higher HSP 70 density (d 41) and weight gain (from d 35 to 49) and lower bursal histological score (BHS) (d 51) than their HT and control counterparts. The HSP 70 expression and BHS of FR birds were not significantly different from those of the other 3 groups during the heat exposure period. Heat shock protein 70 and BHS data were negatively correlated (r = -0.33, P = 0.0008). We concluded that FRHT could improve weight gain and resistance to IBD in male broiler chickens under heat stress conditions. The improved heat tolerance and disease resistance in FRHT birds could be attributed to better HSP 70 response.

  12. In situ expression of heat-shock proteins and 3-nitrotyrosine in brains of young rats exposed to a WiFi signal in utero and in early life.

    PubMed

    Aït-Aïssa, Saliha; de Gannes, Florence Poulletier; Taxile, Murielle; Billaudel, Bernard; Hurtier, Annabelle; Haro, Emmanuelle; Ruffié, Gilles; Athané, Axel; Veyret, Bernard; Lagroye, Isabelle

    2013-06-01

    The bioeffects of exposure to Wireless High-Fidelity (WiFi) signals on the developing nervous systems of young rodents was investigated by assessing the in vivo and in situ expression levels of three stress markers: 3-Nitrotyrosine (3-NT), an oxidative stress marker and two heat-shock proteins (Hsp25 and Hsp70). These biomarkers were measured in the brains of young rats exposed to a 2450 MHz WiFi signal by immunohistochemistry. Pregnant rats were first exposed or sham exposed to WiFi from day 6 to day 21 of gestation. In addition three newborns per litter were further exposed up to 5 weeks old. Daily 2-h exposures were performed blind in a reverberation chamber and whole-body specific absorption rate levels were 0, 0.08, 0.4 and 4 W/kg. 3-NT and stress protein expression was assayed in different areas of the hippocampus and cortex. No significant difference was observed among exposed and sham-exposed groups. These results suggest that repeated exposure to WiFi during gestation and early life has no deleterious effects on the brains of young rats.

  13. Expressive writing in early breast cancer survivors.

    PubMed

    Craft, Melissa A; Davis, Gail C; Paulson, René M

    2013-02-01

    This article is the report of a study aimed at determining whether or not expressive writing improves the quality-of-life of early breast cancer survivors. An additional aim is the investigation of whether or not the type of writing prompt makes a difference in results. The risk of distress can extend well beyond the time of a breast cancer diagnosis. Emotional expression may assist in dealing with this. Randomized controlled study. Participants (n = 120) were randomized into one of four groups: a control group (no writing) or one of three expressive writing groups: breast cancer trauma, any self-selected trauma and facts related to breast cancer. Participants wrote 20 minutes a day for 4 consecutive days. Their quality-of-life was measured, using the 'Functional Assessment of Cancer Therapy-Breast Cancer Version', at baseline and at 1 month and 6 months after writing. Paired t-tests, multivariate analysis of variance and multiple regression were used to analyse the data of the 97 participants who completed the journaling assignment and at least the first assessment, collected in 2006. Intention-to-treat analysis was used. Expressive writing about one's breast cancer, breast cancer trauma and facts related to breast cancer, significantly improved the quality-of-life outcome. Expressive writing, focusing the instructions on writing about one's living and dealing with a diagnosis of breast cancer, is recommended for early breast cancer survivors as a feasible and easily implemented treatment approach to improve quality-of-life. © 2012 Blackwell Publishing Ltd.

  14. Protein Expression Dynamics During Postnatal Mouse Brain Development

    PubMed Central

    Laeremans, Annelies; Van de Plas, Babs; Clerens, Stefan; Van den Bergh, Gert; Arckens, Lutgarde; Hu, Tjing-Tjing

    2013-01-01

    We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation. PMID:25157209

  15. Biased gene expression in early honeybee larval development

    PubMed Central

    2013-01-01

    Background Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ from each other in physiology, behaviour and life span. Results To understand how these trajectories are established we have generated a comprehensive atlas of gene expression throughout larval development. We found substantial differences in gene expression between worker and queen-destined larvae at 6 hours after hatching. Some of these early changes in gene expression are maintained throughout larval development, indicating that caste-specific developmental trajectories are established much earlier than previously thought. Within our gene expression data we identified processes that potentially underlie caste differentiation. Queen-destined larvae have higher expression of genes involved in transcription, translation and protein folding early in development with a later switch to genes involved in energy generation. Using RNA interference, we were able to demonstrate that one of these genes, hexamerin 70b, has a role in caste differentiation. Both queen and worker developmental trajectories are associated with the expression of genes that have alternative splice variants, although only a single variant of a gene tends to be differentially expressed in a given caste. Conclusions Our data, based on the biases in gene expression early in development together with published data, supports the idea that caste development in the honeybee consists of two phases; an initial biased phase of development, where larvae can still switch to the other caste by differential feeding, followed by commitment to a particular developmental trajectory. PMID:24350621

  16. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  17. Expression of the immediate early IE180 protein under the control of the hTERT and CEA tumor-specific promoters in recombinant pseudorabies viruses: Effects of IE180 protein on promoter activity and apoptosis induction.

    PubMed

    Lerma, L; Alcalá, S; Piñero, C; Torres, M; Martin, B; Lim, F; Sainz, B; Tabarés, E

    2016-01-15

    Since the pseudorabies virus (PRV) genome encodes for a single immediate-early protein, IE180, we reasoned that this strong transactivating protein could represent a key regulatory switch that could be genetically manipulated in order to alter its tropism towards cancer cells. We therefore initiated studies to test whether the human telomerase reverse transcriptase (hTERT) and carcinoembryonic antigen (CEA) tumor promoters could functionally replace the IE180 promoter. We show that both promoters can functionally substitute the IE180 promoter in plasmid constructs and recombinant viruses, and observed that IE180 differentially auto-regulated each promoter tested, with PRV IE180 negatively regulating the hTERT promoter but positively hyper-activating the CEA promoter. Interestingly, we also observed that the recombinant PRV-TER and PRV-CEA viruses preferentially replicated in diverse cancer cell lines compared to control non-cancer cells, and the PRV-CEA was capable of additionally inducing a profound apoptotic phenotype which we correlated to the overexpression of IE180. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. A-TWinnipeg: Pathogenesis of rare ATM missense mutation c.6200C>A with decreased protein expression and downstream signaling, early-onset dystonia, cancer, and life-threatening radiotoxicity.

    PubMed

    Nakamura, Kotoka; Fike, Francesca; Haghayegh, Sara; Saunders-Pullman, Rachel; Dawson, Angelika J; Dörk, Thilo; Gatti, Richard A

    2014-07-01

    We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important.

  19. A-TWinnipeg: Pathogenesis of rare ATM missense mutation c.6200C>A with decreased protein expression and downstream signaling, early-onset dystonia, cancer, and life-threatening radiotoxicity

    PubMed Central

    Nakamura, Kotoka; Fike, Francesca; Haghayegh, Sara; Saunders-Pullman, Rachel; Dawson, Angelika J; Dörk, Thilo; Gatti, Richard A

    2014-01-01

    We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important. PMID:25077176

  20. Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins

    PubMed Central

    Lisak, Robert P; Benjamins, Joyce A; Bealmear, Beverly; Nedelkoska, Liljana; Yao, Bin; Land, Susan; Studzinski, Diane

    2007-01-01

    Background In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells. Methods To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M) on CNS glia after 6 hours of treatment. Results In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells. Conclusion Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system. PMID:18088439

  1. Heterologous and cell free protein expression systems.

    PubMed

    Farrokhi, Naser; Hrmova, Maria; Burton, Rachel A; Fincher, Geoffrey B

    2009-01-01

    In recognition of the fact that a relatively small percentage of 'named' genes in databases have any experimental proof for their annotation, attention is shifting towards the more accurate assignment of functions to individual genes in a genome. The central objective will be to reduce our reliance on nucleotide or amino acid sequence similarities as a means to define the functions of genes and to annotate genome sequences. There are many unsolved technical difficulties associated with the purification of specific proteins from extracts of biological material, especially where the protein is present in low abundance, has multiple isoforms or is found in multiple post-translationally modified forms. The relative ease with which cDNAs can be cloned has led to the development of methods through which cDNAs from essentially any source can be expressed in a limited range of suitable host organisms, so that sufficient levels of the encoded proteins can be generated for functional analysis. Recently, these heterologous expression systems have been supplemented by more robust prokaryotic and eukaryotic cell-free protein synthesis systems. In this chapter, common host systems for heterologous expression are reviewed and the current status of cell-free expression systems will be presented. New approaches to overcoming the special problems encountered during the expression of membrane-associated proteins will also be addressed. Methodological considerations, including the characteristics of codon usage in the expressed DNA, peptide tags that facilitate subsequent purification of the expressed proteins and the role of post-translational modifications, are examined.

  2. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  3. Expression of clock proteins in developing tooth.

    PubMed

    Zheng, Li; Papagerakis, Silvana; Schnell, Santiago D; Hoogerwerf, Willemijntje A; Papagerakis, Petros

    2011-01-01

    Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

    Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

  5. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  6. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  7. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  8. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  9. Cytomegalovirus immediate early proteins promote stemness properties in glioblastoma

    PubMed Central

    Soroceanu, Liliana; Matlaf, Lisa; Khan, Sabeena; Akhavan, Armin; Singer, Eric; Bezrookove, Vladimir; Decker, Stacy; Ghanny, Saleena; Hadaczek, Piotr; Bengtsson, Henrik; Ohlfest, John; Luciani-Torres, Maria-Gloria; Harkins, Lualhati; Perry, Arie; Guo, Hong; Soteropoulos, Patricia; Cobbs, Charles S

    2015-01-01

    Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with onconcogenic properties. Here, we show how HCMV IE are preferentially expressed in glioma stem-like cells (GSC), where they co-localize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSC that are endogenously infected with HCMV, attenuating IE expression by an RNA-i-based strategy, was sufficient to inhibit tumorsphere formation, Sox2 expression, cell cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSC elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSC, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and pro-inflammatory cytokines, resembling the therapeutically-resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miRNA-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSC infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared to mock-infected human GSC. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in glioblastoma cells. PMID:26239477

  10. Contingency Table Browser − prediction of early stage protein structure

    PubMed Central

    Kalinowska, Barbara; Krzykalski, Artur; Roterman, Irena

    2015-01-01

    The Early Stage (ES) intermediate represents the starting structure in protein folding simulations based on the Fuzzy Oil Drop (FOD) model. The accuracy of FOD predictions is greatly dependent on the accuracy of the chosen intermediate. A suitable intermediate can be constructed using the sequence-structure relationship information contained in the so-called contingency table − this table expresses the likelihood of encountering various structural motifs for each tetrapeptide fragment in the amino acid sequence. The limited accuracy with which such structures could previously be predicted provided the motivation for a more indepth study of the contingency table itself. The Contingency Table Browser is a tool which can visualize, search and analyze the table. Our work presents possible applications of Contingency Table Browser, among them − analysis of specific protein sequences from the point of view of their structural ambiguity. PMID:26664034

  11. Contingency Table Browser - prediction of early stage protein structure.

    PubMed

    Kalinowska, Barbara; Krzykalski, Artur; Roterman, Irena

    2015-01-01

    The Early Stage (ES) intermediate represents the starting structure in protein folding simulations based on the Fuzzy Oil Drop (FOD) model. The accuracy of FOD predictions is greatly dependent on the accuracy of the chosen intermediate. A suitable intermediate can be constructed using the sequence-structure relationship information contained in the so-called contingency table - this table expresses the likelihood of encountering various structural motifs for each tetrapeptide fragment in the amino acid sequence. The limited accuracy with which such structures could previously be predicted provided the motivation for a more indepth study of the contingency table itself. The Contingency Table Browser is a tool which can visualize, search and analyze the table. Our work presents possible applications of Contingency Table Browser, among them - analysis of specific protein sequences from the point of view of their structural ambiguity.

  12. Alteration of gene expression by alcohol exposure at early neurulation

    PubMed Central

    2011-01-01

    Background We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Result Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. Conclusion This study revealed a set of genes vulnerable to alcohol exposure and genes that were

  13. ESPRESSO: a system for estimating protein expression and solubility in protein expression systems.

    PubMed

    Hirose, Shuichi; Noguchi, Tamotsu

    2013-05-01

    Recombinant protein technology is essential for conducting protein science and using proteins as materials in pharmaceutical or industrial applications. Although obtaining soluble proteins is still a major experimental obstacle, knowledge about protein expression/solubility under standard conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we present a computational approach to estimate the probability of protein expression and solubility for two different protein expression systems: in vivo Escherichia coli and wheat germ cell-free, from only the sequence information. It implements two kinds of methods: a sequence/predicted structural property-based method that uses both the sequence and predicted structural features, and a sequence pattern-based method that utilizes the occurrence frequencies of sequence patterns. In the benchmark test, the proposed methods obtained F-scores of around 70%, and outperformed publicly available servers. Applying the proposed methods to genomic data revealed that proteins associated with translation or transcription have a strong tendency to be expressed as soluble proteins by the in vivo E. coli expression system. The sequence pattern-based method also has the potential to indicate a candidate region for modification, to increase protein solubility. All methods are available for free at the ESPRESSO server (http://mbs.cbrc.jp/ESPRESSO). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  15. Heart Failure Protein May Signal Early Brain Damage

    MedlinePlus

    ... 162447.html Heart Failure Protein May Signal Early Brain Damage Higher levels indicated potential trouble, study showed ... a specific heart disease protein are associated with brain damage, a new study suggests. N-terminal Pro- ...

  16. Effect of chronic thyroxine treatment on IGF-I, IGF-II and IGF-binding protein expression in mammary gland and liver during pregnancy and early lactation in rats.

    PubMed

    Rosato, Roberto; Lindenbergh-Kortleve, Dicky; Neck, Johan van; Drop, Stenvert; Jahn, Graciela

    2002-05-01

    Hyperthyroidism in rats produces organ hypertrophy and increases in circulating IGF-I and IGF-binding protein (IGFBP)-3. Chronic treatment with thyroxine (T(4)) during pregnancy advances parturition, blocks lactation and changes several hormone receptors in mammary gland and liver. Since IGFs are implicated in mammary and liver growth and in differentiation, we studied the effects of hyperthyroidism, induced by daily injections of T(4) (0.25 mg/kg). Using quantitative RT-PCR and in situ hybridization, the gene expression of IGF-I, IGF-II and the IGFBPs was determined in mammary gland and liver of rats at estrus and days 7, 14 and 21 of pregnancy (G7, G14, G21), day 1 postpartum (L1) and 3 days after removing the litter (L4). Circulating levels of IGF-I, tri-iodothyronine (T(3)), PRL and GH were measured. T(4) treatment (HT) increased circulating T(3) save on G21, did not change serum IGF-I, increased PRL on G21 and decreased GH on L1. PRL decreased on L1 because of the absence of lactation. Hepatic IGF-I mRNA was low during pregnancy and increased on L4. HT advanced this increase to L1. In controls, liver IGFBP-3 mRNA levels decreased from G14 to G21, whereas IGFBP-4 showed an inverse pattern. HT lowered IGFBP-3 mRNA and increased IGFBP-4. Increases in mammary concentrations of IGF-I, IGFBP-3 and IGFBP-4 mRNAs were seen on G21. HT delayed these peaks to L1. Mammary IGF-II and IGFBP-2 mRNA levels were high on G7 and G14, and fell afterwards, with HT having no effects. IGFBP-5 mRNA decreased during pregnancy and increased on L1. HT increased IGFBP-5 levels in early pregnancy and on L1. IGF-I mRNA localized to connective and epithelial mammary tissue, while IGFBP-2 and IGFBP-5 mRNA was only in epithelial cells. These results imply a role for IGF-I, IGFBP-3 and IGFBP-4 in terminal mammary development, while IGF-II and IGFBP-2 may be implicated in early growth. IGFBP-5 has been implicated in mammary apoptosis, and the HT-induced increase may play a role in the premature

  17. Streamlined expressed protein ligation using split inteins.

    PubMed

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  18. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  19. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

    PubMed Central

    Stenberg, R M; Fortney, J; Barlow, S W; Magrane, B P; Nelson, J A; Ghazal, P

    1990-01-01

    trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression. Images PMID:2157043

  20. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    PubMed Central

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  1. Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis.

    PubMed

    Choi, Ivy Y; Karpus, Olga N; Turner, Jason D; Hardie, Debbie; Marshall, Jennifer L; de Hair, Maria J H; Maijer, Karen I; Tak, Paul P; Raza, Karim; Hamann, Jörg; Buckley, Christopher D; Gerlag, Danielle M; Filer, Andrew

    2017-01-01

    Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

  2. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  3. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  4. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  5. PARP-1 protein expression in glioblastoma multiforme

    PubMed Central

    Galia, A.; Calogero, A.E.; Condorelli, R.A.; Fraggetta, F.; La Corte, C.; Ridolfo, F.; Bosco, P.; Castiglione, R.; Salemi, M.

    2012-01-01

    One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

  6. Expression and regulation of Foxa2 in the rat uterus during early pregnancy

    PubMed Central

    YAMAGAMI, Kazuki; YAMAUCHI, Nobuhiko; KUBOTA, Kaiyu; NISHIMURA, Sho; CHOWDHURY, Vishwajit Sur; YAMANAKA, Kenichi; TAKAHASHI, Masashi; TABATA, Shoji; HATTORI, Masa-aki

    2014-01-01

    The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein. PMID:25262775

  7. Cathepsin D expression in early cutaneous malignant melanoma.

    PubMed

    Bartenjev, I; Rudolf, Z; Stabuc, B; Vrhovec, I; Perkovic, T; Kansky, A

    2000-08-01

    The aspartic proteinase cathepsin D is believed to be associated with proteolytic processes leading to the invasion and seeding of tumor cells. An association between cathepsin D tissue concentration and aggressiveness of tumors has been detected in different cancer types, as well as in metastatic melanoma. The concentration of cathepsin D was measured immunoradiometrically (ELSA-CATH-D kit, CIS Bio International) in the cytosols of 51 primary cutaneous melanomas (with Breslow index < 4 mm) to estimate the tissue concentrations of cathepsin D in early cutaneous melanoma. A significantly elevated concentration of cathepsin D was measured in the tumor cytosols as compared to adjacent normal tissue (44.2 vs. 14.7 pmol/mg of total protein, P < 0.001). Our results indicate that cathepsin D is expressed at high levels by melanoma cells. The extremely high expression of cathepsin D in two of our patients, with later progression of the disease over a 42-month follow-up period, suggests a possible correlation between the cathepsin D tissue concentration and the prognosis of primary cutaneous malignant melanoma.

  8. Gene Expression Changes and Early Events in Cotton Fibre Development

    PubMed Central

    Lee, Jinsuk J.; Woodward, Andrew W.; Chen, Z. Jeffrey

    2007-01-01

    Background Cotton is the dominant source of natural textile fibre and a significant oil crop. Cotton fibres, produced by certain species in the genus Gossypium, are seed trichomes derived from individual cells of the epidermal layer of the seed coat. Cotton fibre development is delineated into four distinct and overlapping developmental stages: fibre initiation, elongation, secondary wall biosynthesis and maturation. Scope Recent advances in gene expression studies are beginning to provide new insights into a better understanding of early events in cotton fibre development. Fibre cell development is a complex process involving many pathways, including various signal transduction and transcriptional regulation components. Several analyses using expressed sequence tags and microarray have identified transcripts that preferentially accumulate during fibre development. These studies, as well as complementation and overexpression experiments using cotton genes in arabidopsis and tobacco, indicate some similar molecular events between trichome development from the leaf epidermis and fibre development from the ovule epidermis. Specifically, MYB transcription factors regulate leaf trichome development in arabidopsis and may regulate seed trichome development in cotton. In addition, transcript profiling and ovule culture experiments both indicate that several phytohormones and other signalling pathways mediate cotton fibre development. Auxin and gibberellins promote early stages of fibre initiation; ethylene- and brassinosteroid-related genes are up-regulated during the fibre elongation phase; and genes associated with calmodulin and calmodulin-binding proteins are up-regulated in fibre initials. Additional genomic data, mutant and functional analyses, and genome mapping studies promise to reveal the critical factors mediating cotton fibre cell development. PMID:17905721

  9. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum.

    PubMed

    Silvestrini, Francesco; Lasonder, Edwin; Olivieri, Anna; Camarda, Grazia; van Schaijk, Ben; Sanchez, Massimo; Younis Younis, Sumera; Sauerwein, Robert; Alano, Pietro

    2010-07-01

    Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent development of the elongated gametocyte. We developed a protocol to obtain for the first time highly purified preparations of early gametocytes using a transgenic line expressing a green fluorescent protein from the onset of gametocytogenesis. We determined the cellular proteome (1427 proteins) of this parasite stage by high accuracy tandem mass spectrometry and newly determined the proteomes of asexual trophozoites and mature gametocytes, identifying altogether 1090 previously undetected parasite proteins. Quantitative label-free comparative proteomics analysis determined enriched protein clusters for the three parasite developmental stages. Gene set enrichment analysis on the 251 proteins enriched in the early gametocyte proteome revealed that proteins putatively exported and involved in erythrocyte remodeling are the most overrepresented protein set in these stages. One-tenth of the early gametocyte-enriched proteome is constituted of putatively exported proteins, here named PfGEXPs (P. falciparum gametocyte-exported proteins). N-terminal processing and N-acetylation at a conserved leucine residue within the Plasmodium export element pentamotif were detected by mass spectrometry for three such proteins in the early but not in the mature gametocyte sample, further supporting a specific role in protein export in early gametocytogenesis. Previous reports and results of our experiments confirm that the three proteins are indeed exported in the erythrocyte cytoplasm. This work indicates that protein export profoundly marks early sexual differentiation in P. falciparum, probably contributing to host cell remodeling in this phase of the life cycle, and that gametocyte

  10. Soluble expression and complex formation of proteins required for HCMV DNA replication using the SFV expression system.

    PubMed

    McCue, L A; Anders, D G

    1998-08-01

    Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms-a precursor and a product-indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.

  11. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    PubMed

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  12. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  13. Differential regulation of dentin matrix protein 1 expression during odontogenesis.

    PubMed

    Lu, Yongbo; Zhang, Shubin; Xie, Yixia; Pi, Yuli; Feng, Jian Q

    2005-01-01

    Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development. 2005 S. Karger AG, Basel

  14. Prion Protein Expression Regulates Embryonic Stem Cell Pluripotency and Differentiation

    PubMed Central

    Miranda, Alberto; Pericuesta, Eva

    2011-01-01

    Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis. PMID:21483752

  15. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    PubMed

    Miranda, Alberto; Pericuesta, Eva; Ramírez, Miguel Ángel; Gutierrez-Adan, Alfonso

    2011-04-04

    Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  16. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  17. Trypanosoma cruzi expresses diverse repetitive protein antigens.

    PubMed Central

    Hoft, D F; Kim, K S; Otsu, K; Moser, D R; Yost, W J; Blumin, J H; Donelson, J E; Kirchhoff, L V

    1989-01-01

    We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship. Images PMID:2659529

  18. Wheat germ systems for cell-free protein expression.

    PubMed

    Harbers, Matthias

    2014-08-25

    Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

  19. AGL15, a MADS domain protein expressed in developing embryos.

    PubMed Central

    Heck, G R; Perry, S E; Nichols, K W; Fernandez, D E

    1995-01-01

    To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo. PMID:7549483

  20. Sphedgehog is expressed by pigment cell precursors during early gastrulation in Strongylocentrotus purpuratus.

    PubMed

    Egaña, Ana L; Ernst, Susan G

    2004-10-01

    We have sequenced the Sphedgehog (Sphh) gene from the sea urchin Strongylocentrotus purpuratus. Sphh transcripts are detected first at the mesenchyme blastula stage, and they accumulate throughout early embryogenesis. The Sphh protein is produced by precursor pigment cells during early and midgastrulation. NiCl2 inhibits pigment cell differentiation in sea urchins. Here, we show that, in S. purpuratus, nickel affects a process(es) between 17 and 24 hr of development, corresponding to the time period when Sphh mRNA is first detected. However, nickel treatment does not alter the early expression of Sphh.

  1. Elevated nuclear CCND1 expression confers an unfavorable prognosis for early stage lung adenocarcinoma patients

    PubMed Central

    Xu, Ping; Zhao, Mengyang; Liu, Zhen; Liu, Yiyi; Chen, Yiyu; Luo, Rongcheng; Fang, Weiyi

    2015-01-01

    Purposes: To examine the expression pattern of CCND1 and analyze the correlation of its nuclear expression with clinicopathologic features and prognosis in lung adenocarcinoma. Methods: CCND1 mRNA and protein levels in lung adenocarcinoma tissues were examined. The relationship between nuclear CCND1 protein expression and clinical features including survival prognosis was analyzed. Results: CCND1 mRNA levels were markedly increased in lung adenocarcinoma (P=0.0019). Western blot analysis confirmed increased nuclear CCND1 protein expression in lung adenocarcinoma specimens. Immunohistochemistry analysis confirmed that CCND1 protein was predominantly nuclear localized in lung adenocarcinoma cells and significantly elevated relative to normal lung tissues (P<0.001). Furthermore, high levels of nuclear CCND1 were positively correlated with clinical stage (P=0.026). Patients with nuclear CCND1 expression had a significantly shorter overall survival time than did patients with low expression. Interestingly, nuclear CCND1 expression in clinical stage I+II, but not clinical stage III, was shown associated with poor prognosis and shorter overall survival time for lung adenocarcinoma patients by strata analysis. Finally, nuclear CCND1 expression tended to be an independent prognostic indicator (P=0.087) for lung adenocarcinoma patient survival. Conclusion: Increased nuclear CCND1 is a potential unfavorable prognostic factor for lung adenocarcinoma patients, especially those with clinical early stage (stage I+II). PMID:26884860

  2. Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

    2010-02-15

    Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing.

  3. [Expression of promyelocytic leukaemia protein in Bowen's disease, skin squamous cell carcinoma and basal cell carcinoma].

    PubMed

    Wang, Qiongyu; Ma, Huiqun; Wang, Shijie; Ma, Yunyun; Zou, Xingwei; Li, Ruilian

    2013-07-01

    To investigate the expression of promyelocytic leukaemia (PML) protein of PML protein in Bowen's disease (BD), skin squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) and explore the role of PML in the pathogenesis of these diseases. PML protein in normal skin tissues and lesions of Bowen's disease, SCC and BCC were detected with immunohistochemistry. Normal skin tissues did not express PML protein. In BCC, PML showed rather low expressions in the skin lesions (8.69% in cell nuclei and 4.35% in cytoplasm). The lesions in BD and SCC (grade I and II) showed obvious overexpression of PML protein in the cell nuclei and cytoplasm, and its expression in the cell nuclei of these lesions was significantly higher than that in grade III-IV SCC. PML protein may play an important role in the early stage of SCC, and its overexpression may contribute to the carcinogenesis and metastasis of SCC.

  4. Heat-shock protein expression in canine corneal wound healing.

    PubMed

    Peterson, Cornelia W M; Carter, Renee T; Bentley, Ellison; Murphy, Christopher J; Chandler, Heather L

    2016-05-01

    Heat-shock proteins, particularly the 70-kDa member (Hsp70), have been implicated in facilitating wound healing in multiple tissues. Expression and localization of three HSPs were assessed in normal and wounded canine corneas to elucidate a role in epithelial healing. Paraffin-embedded normal corneas, acute and repeatedly abraded corneas, and keratectomies of spontaneous chronic corneal epithelial defects (SCCEDs) were subjected to routine immunohistochemistry for Hsp27, 47, and 70 expression. Ex vivo corneal defects were created and treated with anti-HSPs or IgG controls, and wound healing was monitored. Primary cultures of canine corneal stromal fibroblasts and corneal epithelial cells were treated with exogenous Hsp70, and an artificial wound was created in vitro to monitor restoration of the monolayer. Normal canine corneas exhibited constitutive expression of all HSPs evaluated. Inducible expression was demonstrated in acutely wounded tissues, and expression in the chronically abraded corneas was relocalized. All HSP expression was below the limits of detection in the epithelium of SCCED samples. Inhibition of HSPs in culture resulted in delayed wound healing when compared to controls. Hsp70-treated fibroblasts demonstrated significantly (P < 0.001) increased migration and proliferation compared to the vehicle control; however, there was no significant effect of exogenous Hsp70 on corneal epithelial cells. These findings suggest that HSPs are induced in the normal canine cornea during re-epithelialization. Hsp70 expression is likely important for inducing the cytoarchitectural remodeling, migration, and proliferation necessary early in the canine corneal healing response, and suppressed expression may contribute to the pathophysiology of nonhealing defects. © 2015 American College of Veterinary Ophthalmologists.

  5. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  6. Analysis on Gene Expression Profile in Oncospheres and Early Stage Metacestodes from Echinococcus multilocularis

    PubMed Central

    Dang, Zhisheng; Suzuki, Yutaka; Horiuchi, Terumi; Yagi, Kinpei; Kouguchi, Hirokazu; Irie, Takao; Kim, Kyeongsoon; Oku, Yuzaburo

    2016-01-01

    Alveolar echinococcosis is a worldwide zoonosis of great public health concern. Analysis of genome data for Echinococcus multilocularis has identified antigen families that can be used in diagnostic assays and vaccine development. However, little gene expression data is available for antigens of the egg and early larval stages. To address this information gap, we used a Next-Generation Sequencing approach to investigate three different stages (non-activated and activated oncospheres, and early stage metacestodes) of E. multilocularis (Nemuro strain). Transcriptome data analysis revealed that some diagnostic antigen gp50 isoforms and the antigen Eg95 family dominated in activated oncospheres, and the antigen B family dominated in early stage metacestodes. Furthermore, heat shock proteins and antigen II/3 are constantly expressed in the three stages. The expression pattern of various known antigens in E. multilocularis may give fundamental information for choosing candidate genes used in diagnosis and vaccine development. PMID:27092774

  7. Motor memory in childhood: early expression of consolidation phase gains.

    PubMed

    Ashtamker, Lilach; Karni, Avi

    2013-11-01

    Are children faster than adults in consolidating procedural knowledge? In adults, the expression of the full benefits of motor practice requires a few hours of consolidation and sleep. Here we show that, although the processes generating the delayed gains continued beyond the first few hours post-training, children expressed significant gains as early as 1 h post-training, in the awake state. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Expression and regulation of androgen receptor in the mouse uterus during early pregnancy and decidualization.

    PubMed

    Xu, Jingjie; Li, Mo; Zhang, Lu; Xiong, Hao; Lai, Lidan; Guo, Meijun; Zong, Teng; Zhang, Dalei; Yang, Bei; Wu, Lei; Tang, Min; Kuang, Haibin

    2015-11-01

    The androgen receptor (AR) is a ligand-activated transcription factor that is important for both the male and female reproductive systems. The expression and regulation of AR in the uterine endometrium during early pregnancy and decidualization remain relatively under-investigated, so we sought to immunohistochemically examine the spatiotemporal expression of AR in mouse uteri during the peri-implantation period as well as in response to specific steroid hormones. AR protein was found in the nuclei of uterine stromal cells starting on pregnancy Days 1 and 2, with its abundance increasing on Days 3 and 4. From pregnancy Days 5 to 9, however, the expression of AR markedly declined in stromal zones of uteri. No signal was detected in the decidualized cells surrounding the site of embryo implantation; moreover, no AR immunostaining was observed in decidualized uterine cells in an artificial oil-induced model of decidualization. Progesterone significantly inhibited AR protein expression, whereas estrogen dramatically elevated AR abundance in the stroma of ovariectomized mouse uteri. Taken together, our results are the first to demonstrate that decidualization and progesterone significantly inhibited the AR protein expression in vivo, whereas estrogen increased AR protein levels in the stromal cells of mouse uteri. These responses might be advantageous for the proliferation and differentiation of uterine stroma and for embryo implantation during early pregnancy.

  9. Molecular analysis of LEFTY-expressing cells in early human embryoid bodies.

    PubMed

    Dvash, Tamar; Sharon, Nadav; Yanuka, Ofra; Benvenisty, Nissim

    2007-02-01

    Human ESCs (HESCs) are self-renewing pluripotent cell lines that are derived from the inner cell mass of blastocyst-stage embryos. These cells can produce terminally differentiated cells representing the three embryonic germ layers. We thus hypothesized that during the course of in vitro differentiation of HESCs, progenitor-like cells are transiently formed. We demonstrated that LEFTY proteins, which are known to play a major role during mouse gastrulation, are transiently expressed during HESC differentiation. Moreover, LEFTY proteins seemed to be exclusively expressed by a certain population of cells in the early human embryoid bodies that does not overlap with the population expressing the ESC marker OCT4. We also showed that LEFTY expression is regulated at the cellular transcription level by molecular labeling of LEFTY-positive cells. A DNA microarray analysis of LEFTY-overexpressing cells revealed a signature of cell surface markers such as CADHERIN 2 and 11. Expression of LEFTY controlled by NODAL appears to have a substantial role in mesodermal origin cell population establishment, since inhibition of NODAL activity downregulated expression not only of LEFTY A and LEFTY B but also of BRACHYURY, an early mesodermal marker. In addition, other mesodermal lineage-related genes were downregulated, and this was accompanied by an upregulation in ectoderm-related genes. We propose that during the initial step of HESC differentiation, mesoderm progenitor-like cells appear via activation of the NODAL pathway. Our analysis suggests that in vitro differentiation of HESCs can model early events in human development.

  10. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    PubMed

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

  11. Seedling Lethal1, a Pentatricopeptide Repeat Protein Lacking an E/E+ or DYW Domain in Arabidopsis, Is Involved in Plastid Gene Expression and Early Chloroplast Development1[C][W

    PubMed Central

    Pyo, Young Jae; Kwon, Kwang-Chul; Kim, Anna; Cho, Myeon Haeng

    2013-01-01

    Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E+ or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development. PMID:24144791

  12. Early Gene Expression Changes in the Retinal Ganglion Cell Layer of a Rat Glaucoma Model

    PubMed Central

    Guo, Ying; Johnson, Elaine C.; Cepurna, William O.; Dyck, Jennifer A.; Doser, Tom

    2011-01-01

    Purpose. To identify patterns of early gene expression changes in the retinal ganglion cell layer (GCL) of a rodent model of chronic glaucoma. Methods. Prolonged elevation of intraocular pressure (IOP) was produced in rats by episcleral vein injection of hypertonic saline (N = 30). GCLs isolated by laser capture microdissection were grouped by grading of the nerve injury (<25% axon degeneration for early injury; >25% for advanced injury). Gene expression was determined by cDNA microarray of independent GCL RNA samples. Quantitative PCR (qPCR) was used to further examine the expression of selected genes. Results. By array analysis, 533 GCL genes (225 up, 308 down) were significantly regulated in early injury. Compared to only one major upregulated gene class of metabolism regulation, more were downregulated, including mitochondria, ribosome, proteasome, energy pathways, protein synthesis, protein folding, and synaptic transmission. qPCR confirmed an early upregulation of Atf3. With advanced injury, 1790 GCL genes were significantly regulated (997 up, 793 down). Altered gene categories included upregulated protein synthesis, immune response, and cell apoptosis and downregulated dendrite morphogenesis and axon extension. Of all the early changed genes, 50% were not present in advanced injury. These uniquely affected genes were mainly associated with upregulated transcription regulation and downregulated protein synthesis. Conclusions. Early GCL gene responses to pressure-induced injury are characterized by an upregulation of Atf3 and extensive downregulation in genes associated with cellular metabolism and neuronal functions. Most likely, these changes represent those specific to RGCs and are thus potentially important for enhancing RGC survival in glaucoma. PMID:21051717

  13. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  14. Changes in ovarian protein expression during primordial follicle formation in the hamster.

    PubMed

    Mukherjee, Anindit; Reisdorph, Nichole; Guda, Chttibabu; Pandey, Sanjit; Roy, Shyamal K

    2012-01-02

    Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4-7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and

  15. Regulation of viral and cellular promoter activity by polyomavirus early proteins.

    PubMed Central

    Pannuti, A; La Mantia, G; Lania, L

    1987-01-01

    The chloramphenicol-acetyl-transferase (CAT) expression system has been utilized to study the ability of the polyomavirus (Py) early proteins, the 100K large T, the 55K middle T and 22K small T-antigens, to activate a variety of eukaryotic promoters (the SV40 early, the alpha 2(1) collagen, the rabbit beta-globin, the polyomavirus early and the H-2 class I) in both transient and stable expression assays. We have found that either the complete polyomavirus early region or a plasmid expressing only the 55K middle T-antigen are capable of stimulating the expression of all the promoter-CAT plasmids in transient co-transfection experiments in both NIH-3T3 and Rat-2 cells. Conversely, the Py early proteins do not stimulate the transcription of most of the promoter-CAT genes stably introduced in the cell chromosomes, with the exception of H-2 class I promoter, when stimulation of transcription has been observed upon infection with recombinant retrovirus encoding the Py middle T-antigen. Images PMID:3029721

  16. Effects of immunosuppressive treatment on protein expression in rat kidney

    PubMed Central

    Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

    2014-01-01

    The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

  17. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  18. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  19. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  20. Strain engineering for improved expression of recombinant proteins in bacteria

    PubMed Central

    2011-01-01

    Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins. PMID:21569582

  1. Loss of expression of DNA repair enzyme MGMT in oral leukoplakia and early oral squamous cell carcinoma. A prognostic tool?

    PubMed

    Rodríguez, María J; Acha, Amelia; Ruesga, María T; Rodríguez, Carlos; Rivera, José M; Aguirre, José M

    2007-01-08

    MGMT is a specific DNA repair enzyme that removes alkylating lesions and therefore plays an important role in maintaining normal cell physiology and genomic stability. Loss of expression of MGMT is associated with increased carcinogenic risk and sensitivity to methylating agents in different types of tumours. The expression of MGMT was immunohistochemically assessed in 12 normal oral mucosa, 38 oral leukoplakias and 33 early oral squamous cell carcinomas. The results were correlated with clinicopathological data. We found a significant loss of MGMT protein expression from leukoplakia when compared with early squamous cell carcinoma. We also observed a statistically significant relationship between smoking and the loss of MGMT protein expression. Loss of MGMT expression could be considered an early event in oral carcinogenesis with possible prognostic implications.

  2. Characterization of early stages of human B cell development by gene expression profiling.

    PubMed

    Hystad, Marit E; Myklebust, June H; Bø, Trond H; Sivertsen, Einar A; Rian, Edith; Forfang, Lise; Munthe, Else; Rosenwald, Andreas; Chiorazzi, Michael; Jonassen, Inge; Staudt, Louis M; Smeland, Erlend B

    2007-09-15

    We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

  3. Dietary Soy Protein Inhibits DNA Damage and Cell Survival of Colon Epithelial Cells through Attenuated Expression of Fatty Acid Synthase

    USDA-ARS?s Scientific Manuscript database

    Dietary intake of soy protein decreases tumor incidence in rat models of chemically induced colon cancer. We hypothesized that decreased expression of Fatty Acid Synthase (FASN) underlies, in part, the tumor preventive effects of soy protein, since FASN over-expression characterizes early tumorigene...

  4. Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditions

    PubMed Central

    Da Silva, Carla Alexandra; Kassel, Olivier; Lebouquin, Renaud; Lacroix, Emmanuel Jean; Frossard, Nelly

    2003-01-01

    Stem cell factor (SCF) is a major growth factor for mast cells, promoting their differentiation and chemotaxis. Its expression is regulated by glucocorticoids in inflammatory conditions, showing an early increased protein expression, before the expected anti-inflammatory decrease (Da Silva et al., Br. J. Pharmacol. 2002:135,1634). We here evaluated the early kinetic of SCF expression regulated by interleukin (IL)-1β, budesonide and the combination of both in human lung fibroblasts in culture. Budesonide potentiated the IL-1β-enhanced expression of SCF mRNA (+103%) and protein (+98%) very shortly after treatment (at 30 min and 1 h, respectively). A gentle downregulation followed. This potentiating effect of budesonide was related to increased SCF mRNA stability and SCF gene transcription. Deletion of a κB-like site that we identified in the first intron of the SCF gene, in a luciferase reporter system, abolished the potentiation by budesonide, as well as the effect of IL-1β alone, as compared to the wild-type construction activity. All budesonide-induced effects were glucocorticoid-receptor dependent, since they were reproduced by dexamethasone and blocked by RU486. IL-1β+budesonide did not affect the relative expression of the soluble and membrane-bound forms of SCF. In conclusion, our results clearly show that glucocorticoids act very early to adversely increase the expression of SCF mRNA and protein in the inflammatory conditions created by IL-1β, and that this effect involves increased mRNA stability and increased gene expression through activation of the NF-κB-like responsive element. PMID:14662725

  5. Expression of Glucocorticoid Receptor and Early Growth Response Gene 1 during Postnatal Development of Two Inbred Strains of Mice Exposed to Early Life Stress

    PubMed Central

    Navailles, Sylvia; Zimnisky, Ross; Schmauss, Claudia

    2010-01-01

    Early life stress can elicit profound changes in adult gene expression and behavior. One consequence of early life stress is a decreased expression of glucocorticoid receptors (GRs) in the frontal cortex and hippocampus. However, neither the time of onset nor the mechanism(s) leading to decreased GR expression during postnatal development are known. The present study used two inbred strains of mice that differ in their behavioral responsiveness to stress (Balb/c and C57Bl/6), exposed them to an established paradigm of early life stress (infant maternal separation), and measured their expression of frontal cortical and hippocampal GRs and the putative transcriptional activator of the GR gene, early growth response gene (egr)-1, at defined stages of postnatal development. In both strains, real-time RT-PCR experiments revealed that decreased expression of GR in adolescence and adulthood is, in fact, preceded by increased GR expression during early life stress exposure. Thus, the early life stress-induced disruption of the normal stress-hyporesponsive period during infancy is accompanied by increased GR expression. Moreover, chronic treatment with the antidepressant drug fluoxetine during adolescence or adulthood reversed the effect of early life stress on adult GR mRNA expression. In contrast to the strain-independent effect of early life stress on GR expression, however, changes in egr-1 expression occurred only in Balb/c mice, and unlike the biphasic developmental changes in GR mRNA expression, egr-1 mRNA was decreased throughout postnatal development. Moreover, there was no consistent overlap of anatomic regions affected by decreased GR and egr-1 protein expression. Thus, in Balb/c mice, changes in GR and egr-1 expression can independently contribute to the phenotypes resulting from early life stress exposure. These findings illustrate that the impact of early life stress on gene expression changes is modulated by the genetic background and that the persistent

  6. Increased expression of neutrophil-related genes in patients with early sepsis-induced ARDS.

    PubMed

    Kangelaris, Kirsten Neudoerffer; Prakash, Arun; Liu, Kathleen D; Aouizerat, Bradley; Woodruff, Prescott G; Erle, David J; Rogers, Angela; Seeley, Eric J; Chu, Jeffrey; Liu, Tom; Osterberg-Deiss, Thomas; Zhuo, Hanjing; Matthay, Michael A; Calfee, Carolyn S

    2015-06-01

    The early sequence of events leading to the development of the acute respiratory distress syndrome (ARDS) in patients with sepsis remains inadequately understood. The purpose of this study was to identify changes in gene expression early in the course of illness, when mechanisms of injury may provide the most relevant treatment and prognostic targets. We collected whole blood RNA in critically ill patients admitted from the Emergency Department to the intensive care unit within 24 h of admission at a tertiary care center. Whole genome expression was compared in patients with sepsis and ARDS to patients with sepsis alone. We selected genes with >1 log2 fold change and false discovery rate <0.25, determined their significance in the literature, and performed pathway analysis. Several genes were upregulated in 29 patients with sepsis with ARDS compared with 28 patients with sepsis alone. The most differentially expressed genes included key mediators of the initial neutrophil response to infection: olfactomedin 4, lipocalin 2, CD24, and bactericidal/permeability-increasing protein. These gene expression differences withstood adjustment for age, sex, study batch, white blood cell count, and presence of pneumonia or aspiration. Pathway analysis demonstrated overrepresentation of genes involved in known respiratory and infection pathways. These data indicate that several neutrophil-related pathways may be involved in the early pathogenesis of sepsis-related ARDS. In addition, identifiable gene expression differences occurring early in the course of sepsis-related ARDS may further elucidate understanding of the neutrophil-related mechanisms in progression to ARDS.

  7. Expression profiling of the mouse early embryo: Reflections and Perspectives

    PubMed Central

    Ko, Minoru S. H.

    2008-01-01

    Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

  8. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  9. The small GTP-binding protein rab4 is associated with early endosomes

    SciTech Connect

    van der Sluijs, P.; Hull, M.; Mellman, I. ); Zahraoui, A.; Tavitian, A. ); Goud, B. )

    1991-07-15

    Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. The authors demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of {approx} 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing {sup 125}-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. They also localized rab4p to tansferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4p cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular tansport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.

  10. BPI-fold (BPIF) containing/plunc protein expression in human fetal major and minor salivary glands.

    PubMed

    Alves, Daniel Berretta Moreira; Bingle, Lynne; Bingle, Colin David; Lourenço, Silvia Vanessa; Silva, Andréia Aparecida; Pereira, Débora Lima; Vargas, Pablo Agustin

    2017-01-16

    The aim of this study was to determine expression, not previously described, of PLUNC (palate, lung, and nasal epithelium clone) (BPI-fold containing) proteins in major and minor salivary glands from very early fetal tissue to the end of the second trimester and thus gain further insight into the function of these proteins. Early fetal heads, and major and minor salivary glands were collected retrospectively and glands were classified according to morphodifferentiation stage. Expression of BPI-fold containing proteins was localized through immunohistochemistry. BPIFA2, the major BPI-fold containing protein in adult salivary glands, was detected only in the laryngeal pharynx; the lack of staining in salivary glands suggested salivary expression is either very late in development or is only in adult tissues. Early expression of BPIFA1 was seen in the trachea and nasal cavity with salivary gland expression only seen in late morphodifferentiation stages. BPIFB1 was seen in early neural tissue and at later stages in submandibular and sublingual glands. BPIFA1 is significantly expressed in early fetal oral tissue but BPIFB1 has extremely limited expression and the major salivary BPIF protein (BPIFA2) is not produced in fetal development. Further studies, with more sensitive techniques, will confirm the expression pattern and enable a better understanding of embryonic BPIF protein function.

  11. Expression Pattern of Early Growth Response Gene 1 during Olive Flounder (Paralichthys olivaceus) Embryonic Development

    PubMed Central

    Yang, Hyun; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Kim, Kyung-Kil

    2014-01-01

    The early growth response protein 1 (Egr-1) is a widely reported zinc finger protein and a well known transcription factor encoded by the Egr-1 gene, which plays key roles in many aspects of vertebrate embryogenesis and in adult vertebrates. The Egr-1 expression is important in the formation of the gill vascular system in flounders, which develops during the post-hatching phase and is essential for survival during the juvenile period. However, the complete details of Egr-1 expression during embryo development in olive flounder are not available. We assessed the expression patterns of Egr-1 during the early development of olive flounders by using reverse transcription polymerase chain reaction (RT-PCR) analysis. Microscopic observations showed that gill filament formation corresponded with the Egr-1 expression. Thus, we showed that Egr-1 plays a vital role in angiogenesis in the gill filaments during embryogenesis. Further, Egr-1 expression was found to be strong at 5 days after hatching (DAH), in the development of the gill vascular system, and this strong expression level was maintained throughout all the development stages. Our findings have important implications with respect to the biological role of Egr-1 and evolution of the first respiratory blood vessels in the gills of olive flounder. Further studies are required to elucidate the Egr-1-mediated stress response and to decipher the functional role of Egr-1 in developmental stages. PMID:25949193

  12. Dynamic changes in stanniocalcin gene expression in the mouse uterus during early implantation.

    PubMed

    Stasko, S E; DiMattia, G E; Wagner, G F

    2001-03-28

    Blastocyst implantation is accompanied by dramatic changes in gene expression to facilitate decidualization and remodelling of uterine architecture. Stanniocalcin (STC) is a new mammalian polypeptide hormone with roles in ion transport, reproduction and development. Here we report dynamic changes in STC mRNA and protein distributions in the early post-implantation mouse uterus. In the non-pregnant state, STC gene expression was confined to the uterine lumenal epithelium. Following implantation STC gene expression shifted to mesometrial stromal cells bordering the uterine lumen. Between E6.5-E8.5 expression shifted once more to cells of the mesometrial lateral sinusoids, and then declined thereafter. Intriguingly immunoreactive STC did not entirely co-localize with areas of high STC gene activity and instead appeared to accumulate in presumptive targets of the hormone (uterine epithelium, stromal and decidual cells, trophoblastic giant cells). STC is only the fourth gene identified as being expressed mesometrially in the uterus following implantation.

  13. SSAO/VAP-1 protein expression during mouse embryonic development.

    PubMed

    Valente, Tony; Solé, Montse; Unzeta, Mercedes

    2008-09-01

    SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation.

  14. Protein-guided RNA dynamics during early ribosome assembly.

    PubMed

    Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2014-02-20

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  15. Protein-guided RNA dynamics during early ribosome assembly

    PubMed Central

    Kim, Hajin; Abeysirigunawardena, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-01-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. PMID:24522531

  16. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  17. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.

    PubMed

    Hayashi, Kokoro; Kojima, Chojiro

    2010-11-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  18. Early childhood stuttering III: initial status of expressive language abilities.

    PubMed

    Watkins, R V; Yairi, E; Ambrose, N G

    1999-10-01

    This investigation evaluated the expressive language abilities of 84 preschool-age children who stuttered, 62 who recovered from stuttering, and 22 who persisted in stuttering. The participants were identical to those identified in E. Yairi and N. G. Ambrose (1999) and E. Paden, E. Yairi, and N. G. Ambrose (1999). A range of lexical, morphological, and syntactic measures--calculated from spontaneous language samples of approximately 250-300 utterances in length collected relatively near stuttering onset--were used to examine the children's expressive language skills. For the purpose of analysis and comparison to normative data, children were grouped into three age intervals, in terms of the age at which they entered the study (2- to 3-year-olds, 3- to 4-year-olds, and 4- to 5-year-olds). Findings revealed similarity in the expressive language abilities of children whose stuttering persisted as opposed to abated at all age intervals. In addition, persistent and recovered stutterers displayed expressive language abilities near or above developmental expectations, based on comparison with normative data, at all age intervals. Children who entered the study at the youngest age level consistently demonstrated expressive language abilities well above normative expectations; this pattern was found for both persistent and recovered groups. These findings provide relatively limited information to assist in the early differentiation of persistence in or recovery from stuttering, but they do shed light on theoretical issues regarding the nature and character of early stuttering and potential associations with language learning.

  19. In vitro manipulation of early mouse embryos induces HIV1-LTRlacZ transgene expression.

    PubMed

    Vernet, M; Cavard, C; Zider, A; Fergelot, P; Grimber, G; Briand, P

    1993-12-01

    We report here that the transcriptional activity of early mouse embryos is affected by their manipulation and culture in vitro, using transgenic embryos that express the reporter gene lacZ. We examined the pattern of expression of the lacZ gene fused to the human immunodeficiency virus type 1 long terminal repeat during the preimplantation stages. Transgene expression is induced as early as the two-cell stage in embryos developed in vitro, while there is no constitutive expression at the same stage in embryos developed in vivo. We have established a relation between this inducible expression occurring in vitro and an oxidative stress phenomenon. Indeed, when the culture medium is supplemented with antioxidants such N-acetyl-cysteine or CuZn-superoxide dismutase the transgene expression is markedly reduced. We also present evidence that the transgene expression in vitro coincides with the onset of the embryonic genome activation as attested by the synthesis of the 70 x 10(3) M(r) protein complex. Therefore, this transgene expression could prove to be a useful tool in our understanding of the molecular mechanisms involved in this crucial developmental event.

  20. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

    PubMed

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

  1. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation. PMID:26378917

  2. Comparative Transcriptome Analysis Reveals Early Pregnancy-Specific Genes Expressed in Peripheral Blood of Pregnant Sows

    PubMed Central

    Zhu, Shien; Shi, Wenqing; Hu, Maishun; Fu, Xiangwei; Wang, Chuduan; Wang, Yachun; Zhang, Qin; Yu, Ying

    2014-01-01

    Early and accurate diagnosis of pregnancy is important for effective management of an economical pig farm. Besides the currently available methods used in early diagnosis of sows, circulating nucleic acids in peripheral blood may contain some early pregnancy-specific molecular markers. For the first time, microarray analysis of peripheral blood from pregnant sows versus non-pregnant sows identified 127 up-regulated and 56 down-regulated genes at day 14 post-insemination. Gene Ontology annotation grouped the total differently expressed genes into 3 significantly enriched terms, cell surface receptor linked signal transduction, G-protein coupled receptor protein signaling pathway and regulation of vesicle-mediated transport. Signaling pathway analysis revealed the only one significantly changed pathway was arachidonic acid metabolism. Of the differently expressed genes, nine (including LPAR3, RXFP4, GALP, CBR1, CBR2, GPX6, USP18, LHB and NR5A1) were found to exert function related to early pregnancy processes. This study provides a clue that differentially abundant RNAs in maternal peripheral blood can help to identify the molecular markers of early pregnancy in pigs. PMID:25479131

  3. Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

    PubMed Central

    Sauvegarde, Caroline; Paul, Delphine; Bridoux, Laure; Jouneau, Alice; Degrelle, Séverine; Hue, Isabelle; Rezsohazy, René; Donnay, Isabelle

    2016-01-01

    Background We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. Results The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. Conclusions Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals. PMID:27798681

  4. Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation.

    PubMed

    Tada, H; Lashgari, M; Rappaport, J; Khalili, K

    1989-01-01

    We analyzed control sequences of the human papovavirus JC virus (JCV) to define the cis-acting elements that regulate specific expression of the viral early region genes in glial cells. Nuclear run-on transcription, S1 analysis, and chloramphenicol acetyltransferase enzyme activity in a transient transfection assay established that the cell type-specific expression of JCV early genes is determined at the transcriptional level. Using DNase footprinting analysis of nuclear proteins prepared from glial and nonglial cells, we located four regions within the JCV control sequences that specifically interacted with the proteins. In glial cells, all four domains contributed to the specific expression of a heterologous promoter, whereas in nonglial cells, two protein-binding regions showed no effect on basal transcriptional activity and the other two domains significantly downregulated transcription of the promoter. We conclude that cell type-specific transcription of the JCV early promoter is under both positive and negative regulation in eucaryotic cells.

  5. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  6. Store-Operated Ca2+ Channels in Mesangial Cells Inhibit Matrix Protein Expression.

    PubMed

    Wu, Peiwen; Wang, Yanxia; Davis, Mark E; Zuckerman, Jonathan E; Chaudhari, Sarika; Begg, Malcolm; Ma, Rong

    2015-11-01

    Accumulation of extracellular matrix derived from glomerular mesangial cells is an early feature of diabetic nephropathy. Ca(2+) signals mediated by store-operated Ca(2+) channels regulate protein production in a variety of cell types. The aim of this study was to determine the effect of store-operated Ca(2+) channels in mesangial cells on extracellular matrix protein expression. In cultured human mesangial cells, activation of store-operated Ca(2+) channels by thapsigargin significantly decreased fibronectin protein expression and collagen IV mRNA expression in a dose-dependent manner. Conversely, inhibition of the channels by 2-aminoethyl diphenylborinate significantly increased the expression of fibronectin and collagen IV. Similarly, overexpression of stromal interacting molecule 1 reduced, but knockdown of calcium release-activated calcium channel protein 1 (Orai1) increased fibronectin protein expression. Furthermore, 2-aminoethyl diphenylborinate significantly augmented angiotensin II-induced fibronectin protein expression, whereas thapsigargin abrogated high glucose- and TGF-β1-stimulated matrix protein expression. In vivo knockdown of Orai1 in mesangial cells of mice using a targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV, and mice showed significant mesangial expansion compared with controls. Similarly, in vivo knockdown of stromal interacting molecule 1 in mesangial cells by recombinant adeno-associated virus-encoded shRNA markedly increased collagen IV protein expression in renal cortex and caused mesangial expansion in rats. These results suggest that store-operated Ca(2+) channels in mesangial cells negatively regulate extracellular matrix protein expression in the kidney, which may serve as an endogenous renoprotective mechanism in diabetes.

  7. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    USDA-ARS?s Scientific Manuscript database

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  8. Cell-free protein synthesis as a promising expression system for recombinant proteins.

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

    Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

  9. Sonic hedgehog expression during early tooth development in Suncus murinus.

    PubMed

    Miyado, Mami; Ogi, Hidenao; Yamada, Gen; Kitoh, Junzo; Jogahara, Takamichi; Oda, Sen-Ichi; Sato, Iwao; Miyado, Kenji; Sunohara, Masataka

    2007-11-16

    Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.

  10. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  11. Isoforms of the nuclear envelope protein Nurim are differentially expressed during heart development in mice.

    PubMed

    Zhang, Wan; Bai, Tianyu; Zhang, Shuai; Xu, Shiqiang; Chen, Hengling; Li, Chenhong

    2017-09-05

    To date, transcript variants of the nuclear envelope protein Nurim and their expression profiles in mice have never been elucidated. In this study, we determined that the primary Nurim variant a was abundantly expressed in mouse heart, liver, spleen and kidney. The protein level of isoform a is initiated at an early stage of heart formation and demonstrated a significant increase in expression throughout embryonic heart development. Interestingly, Nurim b is also up-regulated from E12.5 to E18.5 in different individuals. Our research represents the first report on alternative splicing variants of mouse Nurim and their differential expression profile during embryonic development. These studies suggest a potential role for Nurim in early heart morphogenesis and should help further elucidate the function of Nurim. Copyright © 2017. Published by Elsevier B.V.

  12. [Expression of myeloid cell triggering receptor-1 in monocytes at early post-burn stage].

    PubMed

    Min, An-jie; Zhang, Pi-hong; Xiao, Mu-zhang; Ren, Li-cheng; Zhang, Ming-hua; Yue, Li-qing; Huang, Xiao-yuan

    2007-08-01

    To investigate the expression of triggering receptor expressed on myeloid cells (TREM-1) in monocytes of burn patients at early post-burn stage, and its significance. The monocytes of 8 healthy volunteers (A group), 29 patients with mild and moderate burn (B group), and 9 patients with severe and very serious burns (C group) were isolated from the blood, and the THEM-1 mRNA and protein expression were determined by semi-quantitative RT-PCR and flow cytometry, respectively. The plasma levels of TNF-alpha, IL-1beta were determined by ELISA method. The value of TREM-1 mRNA expression in A, B and C groups were 0.74 +/- 0.13, 1.24 +/- 0.09, and 1.46 +/-0.07, respectively, and the expression rates on cell surface in the 3 groups were (9 +/- 4)%, (51 +/- 6)%, and (71 +/- 7)%, respectively, and there were significant differences among the three groups (P = 0.000). the plasma levels of TNF-alpha and IL-1beta in B and C groups were obviously higher than that in A group (P = 0.000), and they were positively correlated to TREM-1 expression (rs = 0.68, 0.72, P = 0.000). Increased expression of TREM-1 in monocytes of burn patients at early post-burn stage is correlated with the release of inflammatory factors, indicating that TREM-1 might contribute to the onset and development of acute inflammatory response after burns.

  13. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  14. The switch from early to late transcription in phage GA-1: characterization of the regulatory protein p4G.

    PubMed

    Horcajadas, J A; Monsalve, M; Rojo, F; Salas, M

    1999-07-30

    The transcription program of the Bacillus phage GA-1, a distant relative of phage Phi29, has been studied. Transcription of the GA-1 genome occurred in two stages, early and late. Early genes were expressed from two promoters equivalent to the Phi29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the Phi29 late A3 promoter. The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased significantly. The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s). An open reading frame was found in the GA-1 genome coding for a protein showing a 53 % similarity to Phi29 regulatory protein p4, and was named p4G. In Phi29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it. A binding site for protein p4Gwas localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter. In vitro, protein p4Gprevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in Phi29, had no effect on the expression of the late A3 promoter: RNA polymerase could efficiently bind and initiate transcription from the A3 promoter in the absence of protein p4G. Therefore, activation of late transcription occurs differently in GA-1 and Phi29. We propose that protein p4Gis an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain. Copyright 1999 Academic Press.

  15. Expression of the orexin system in the porcine uterus, conceptus and trophoblast during early pregnancy.

    PubMed

    Smolinska, N; Kiezun, M; Dobrzyn, K; Szeszko, K; Maleszka, A; Kaminski, T

    2015-11-01

    Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.

  16. EARLY SENESCENCE1 Encodes a SCAR-LIKE PROTEIN2 That Affects Water Loss in Rice.

    PubMed

    Rao, Yuchun; Yang, Yaolong; Xu, Jie; Li, Xiaojing; Leng, Yujia; Dai, Liping; Huang, Lichao; Shao, Guosheng; Ren, Deyong; Hu, Jiang; Guo, Longbiao; Pan, Jianwei; Zeng, Dali

    2015-10-01

    The global problem of drought threatens agricultural production and constrains the development of sustainable agricultural practices. In plants, excessive water loss causes drought stress and induces early senescence. In this study, we isolated a rice (Oryza sativa) mutant, designated as early senescence1 (es1), which exhibits early leaf senescence. The es1-1 leaves undergo water loss at the seedling stage (as reflected by whitening of the leaf margin and wilting) and display early senescence at the three-leaf stage. We used map-based cloning to identify ES1, which encodes a SCAR-LIKE PROTEIN2, a component of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous complex involved in actin polymerization and function. The es1-1 mutants exhibited significantly higher stomatal density. This resulted in excessive water loss and accelerated water flow in es1-1, also enhancing the water absorption capacity of the roots and the water transport capacity of the stems as well as promoting the in vivo enrichment of metal ions cotransported with water. The expression of ES1 is higher in the leaves and leaf sheaths than in other tissues, consistent with its role in controlling water loss from leaves. GREEN FLUORESCENT PROTEIN-ES1 fusion proteins were ubiquitously distributed in the cytoplasm of plant cells. Collectively, our data suggest that ES1 is important for regulating water loss in rice. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  18. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  19. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  20. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  1. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  2. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

    2005-01-01

    Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

  3. Engineering Cells to Improve Protein Expression

    PubMed Central

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J.

    2014-01-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

  4. Engineering cells to improve protein expression.

    PubMed

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J

    2014-06-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Early Impact of Fontan Operation on Enteric Protein Loss

    PubMed Central

    Patel, Jyoti K.; Loomes, Kathleen M.; Goldberg, David J.; Mercer-Rosa, Laura; Dodds, Kathryn; Rychik, Jack

    2016-01-01

    Background Protein losing enteropathy (PLE) is a challenging complication after Fontan operation. Subclinical enteric protein loss may precede development of overt PLE. We evaluated the acute effects of Fontan circulation on enteric protein loss and mesenteric vascular resistance. Methods A prospective cohort study was performed evaluating enteric protein loss in children undergoing Fontan operation. Stool alpha-1-antitrypsin (A1AT) concentration was measured in the pre-operative, early post-operative, and intermediate post-operative (3–9 months) periods. The intestinal circulation was characterized by Doppler-derived resistance indices of the superior mesenteric artery, and serum albumin and protein levels were obtained. Results We enrolled 33 subjects at a median age at operation of 3.0 (2.5–3.3) years. No clinical PLE was observed. Six of the 93 stool A1AT samples obtained were elevated (>54 mg/dl), with two abnormal samples at each of the three time points. Two of the five subjects with elevated stool A1AT values had significant hemodynamic disturbances requiring intervention (junctional bradycardia or tricuspid stenosis). There was no difference in superior mesenteric artery resistance in the pre-operative versus early post-operative period (p=0.9). Serum albumin levels were lower in the early post-operative period compared to the pre-operative period (3.2 mg/dl [IQR 2.9–3.5] vs. 4.1 mg/dl [IQR 3.4–4.5], p=0.01) but did not correlate with abnormal stool A1AT concentration or superior mesenteric artery resistance indices. Conclusions The Fontan operation does not commonly result in acute development of increased enteric protein loss. However, increased enteric protein loss may occur in children before or after Fontan operation, particularly when hemodynamic disturbances are present. PMID:26652137

  6. Expression of heat shock protein genes in insect stress responses

    USDA-ARS?s Scientific Manuscript database

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  7. Differential expression of prostaglandin E receptor subtype EP2 in rat uterus during early pregnancy.

    PubMed

    Shi, J J; Ma, X H; Diao, H L; Ni, H; Xu, L B; Zhu, H; Yang, Z M

    2005-10-01

    PGE2 is essential for mammalian female reproduction. This study was to examine the expression of EP2 gene in the rat uterus during early pregnancy, delayed implantation and artificial decidualization by in situ hybridization and immunohistochemistry. There was no detectable EP2 mRNA expression in the uterus from days 1 to 4 of pregnancy (day 1 = day of vaginal sperm). A low level of EP2 immunostaining was observed in the luminal and glandular epithelium from days 1 to 4 of pregnancy. Both EP2 mRNA and protein expression were highly detected in the luminal epithelium at implantation sites on day 6 of pregnancy. EP2 expression decreased from day 7 of pregnancy and was undetectable on days 8 and 9 of pregnancy. After delayed implantation was terminated by estrogen treatment and the embryo implanted, both EP2 mRNA and protein expression were strongly observed in the luminal epithelium at the implantation site. There was no detectable EP2 expression in both control and decidualized uteri. In conclusion, these data suggest that EP2 expression at implantation site may play an important role during embryo implantation in rats.

  8. Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

    PubMed Central

    Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

    2013-01-01

    Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

  9. Emerging technology of in situ cell free expression protein microarrays.

    PubMed

    Nand, Amita; Gautam, Anju; Pérez, Javier Batista; Merino, Alejandro; Zhu, Jinsong

    2012-02-01

    Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.

  10. Altered representation of facial expressions after early visual deprivation

    PubMed Central

    Gao, Xiaoqing; Maurer, Daphne; Nishimura, Mayu

    2013-01-01

    We investigated the effects of early visual deprivation on the underlying representation of the six basic emotions. Using multi-dimensional scaling (MDS), we compared the similarity judgments of adults who had missed early visual input because of bilateral congenital cataracts to control adults with normal vision. Participants made similarity judgments of the six basic emotional expressions, plus neutral, at three different intensities. Consistent with previous studies, the similarity judgments of typical adults could be modeled with four underlying dimensions, which can be interpreted as representing pleasure, arousal, potency and intensity of expressions. As a group, cataract-reversal patients showed a systematic structure with dimensions representing pleasure, potency, and intensity. However, an arousal dimension was not obvious in the patient group's judgments. Hierarchical clustering analysis revealed a pattern in patients seen in typical 7-year-olds but not typical 14-year-olds or adults. There was also more variability among the patients than among the controls, as evidenced by higher stress values for the MDS fit to the patients' data and more dispersed weightings on the four dimensions. The findings suggest an important role for early visual experience in shaping the later development of the representations of emotions. Since the normal underlying structure for emotion emerges postnatally and continues to be refined until late childhood, the altered representation of emotion in adult patients suggests a sleeper effect. PMID:24312071

  11. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  12. Identification of serum protein markers for early diagnosis of pregnancy in buffalo.

    PubMed

    Buragohain, Lukumoni; Nanda, Trilok; Ghosh, Arnab; Ghosh, Mayukh; Kumar, Rajesh; Kumar, Sunil; Gupta, Sambhu Sharan; Bharali, Arpita; Mohanty, Ashok K; Singh, Inderjeet; Balhara, Ashok Kumar

    2017-08-01

    Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0-35 post-artificial insemination and from 12 inseminated non-pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two-dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up-regulated proteins as anti-testosterone antibody light chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over-expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B-cell epitopes were predicted for all these five proteins. An antibody cocktail-based approach involving antibodies against all these five up-regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science. © 2016 Japanese Society of Animal Science.

  13. Elevated nuclear S100P expression is associated with poor survival in early breast cancer patients.

    PubMed

    Maciejczyk, Adam; Łacko, Aleksandra; Ekiert, Marcin; Jagoda, Ewa; Wysocka, Teresa; Matkowski, Rafał; Hałoń, Agnieszka; Györffy, Balázs; Lage, Hermann; Surowiak, Paweł

    2013-04-01

    S100P - low molecular weight acidic protein has been shown to be involved in processes of proliferation, survival, angiogenesis, multidrug resistance and metastasis in various human malignancies. In breast cancer, S100P expression is associated with immortalization of neoplastic cells and aggressive tumour behaviour, indicating that this protein may have adverse prognostic value. We analyzed nuclear and cytoplasmic expression of S100P in 85 stage II breast cancer patients with a median follow up of 17 years. Immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against S100P. We also studied prognostic value of S100P mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. Moreover, the relationship was examined between expression of S100P in cells of four breast cancer cell lines and their sensitivity to the 11 most frequently applied cytotoxic drugs. Univariate and multivariate analyses showed that higher expression of nuclear S100P (S100Pn) was typical for cases of a shorter overall survival and disease-free time. KM plotter analysis showed that elevated S100P expression was specific for cases of a relapse-free survival and distant metastases-free survival. No relationship could be documented between expression of S100P and sensitivity of breast cancer cells to cytostatic drugs. We demonstrated that a high S100Pn expression level was associated with poor survival in early stage breast cancer patients. Since preliminary data indicated that expression of S100P was up-regulated by activation of glucocorticoid receptor and several agents manifested potential to activate or inhibit S100P promoter activity, this protein might become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value.

  14. Nfix Expression Critically Modulates Early B Lymphopoiesis and Myelopoiesis

    PubMed Central

    O’Connor, Caitríona; Campos, Joana; Osinski, Jason M.; Gronostajski, Richard M.; Michie, Alison M.; Keeshan, Karen

    2015-01-01

    The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification. PMID:25780920

  15. Early changes in corticospinal excitability when seeing fearful body expressions

    PubMed Central

    Borgomaneri, Sara; Vitale, Francesca; Avenanti, Alessio

    2015-01-01

    Quick inhibition of approach tendencies in response to signals of potential threats is thought to promote survival. However, little is known about the effect of viewing fearful expressions on the early dynamics of the human motor system. We used the high temporal resolution of single-pulse and paired-pulse transcranial magnetic stimulation (TMS) over the motor cortex to assess corticospinal excitability (CSE) and intracortical facilitation (ICF) during observation of happy, fearful and neutral body postures. To test motor circuits involved in approach tendencies, CSE and ICF were recorded from the first dorsal interosseous (FDI), a muscle involved in grasping, and the abductor pollicis brevis (APB), which served as a control. To test early motor dynamics, CSE and ICF were measured 70–90 ms after stimulus onset. We found a selective reduction in CSE in the FDI when participants observed fearful body expressions. No changes in ICF or in the excitability of APB were detected. Our study establishes an extremely rapid motor system reaction to observed fearful body expressions. This motor modulation involves corticospinal downstream projections but not cortical excitatory mechanisms, and appears to reflect an inhibition of hand grasping. Our results suggest a fast visuo-motor route that may rapidly inhibit inappropriate approaching actions. PMID:26388400

  16. Enhanced expression of immediate-early genes in mouse hippocampus after trimethyltin treatment.

    PubMed

    Lee, Sueun; Kang, Sohi; Kim, Juhwan; Yoon, Seongwook; Kim, Sung-Ho; Moon, Changjong

    2016-09-01

    Immediate-early genes (IEGs) are transiently and rapidly activated in response to various cellular stimuli. IEGs mediate diverse functions during pathophysiologic events by regulating cellular signal transduction. We investigated the temporal expression of several IEGs, including c-fos, early growth response protein-1 (Egr-1), and activity-regulated cytoskeleton-associated protein (Arc), in trimethyltin (TMT)-induced hippocampal neurodegeneration. Mice (7 weeks old, C57BL/6) administered TMT (2.6mg/kg intraperitoneally) presented severe neurodegenerative lesions in the dentate gyrus (DG) and showed behavioral seizure activity on days 1-4 post-treatment, after which the lesions and behavior recovered spontaneously over time. c-fos, Egr-1, and Arc mRNA and protein levels significantly increased in the mouse hippocampus after TMT treatment. Immunohistochemical analysis showed that nuclear c-fos expression increased mainly in the DG, whereas nuclear Egr-1 expression was increased extensively in cornu ammonis (CA) 1, CA3, and the DG after TMT treatment. Increased Arc levels were detected in the cellular somata/dendrites of the hippocampal subregions after TMT treatment. Therefore, we suggest that increased IEGs are associated with TMT-induced pathological events in mouse hippocampus. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. Alteration in Endometrial Proteins during Early- and Mid-Secretory Phases of the Cycle in Women with Unexplained Infertility

    PubMed Central

    Manohar, Murli; Khan, Huma; Sirohi, Vijay Kumar; Das, Vinita; Agarwal, Anjoo; Pandey, Amita; Siddiqui, Waseem Ahmad; Dwivedi, Anila

    2014-01-01

    Background Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility. Methods 2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women. Results Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women. Conclusions De

  18. Alteration in endometrial proteins during early- and mid-secretory phases of the cycle in women with unexplained infertility.

    PubMed

    Manohar, Murli; Khan, Huma; Sirohi, Vijay Kumar; Das, Vinita; Agarwal, Anjoo; Pandey, Amita; Siddiqui, Waseem Ahmad; Dwivedi, Anila

    2014-01-01

    Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility. 2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women. Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women. De-regulation of the expression of Sorcin

  19. Tools for Co-expressing Multiple Proteins in Mammalian Cells

    PubMed Central

    Assur, Zahra; Hendrickson, Wayne A.; Mancia, Filippo

    2013-01-01

    Summary Structural and functional studies of many mammalian systems are critically dependent on abundant supplies of recombinant multi-protein complexes. Mammalian cells are often the most ideal, if not the only suitable host for such experiments. This is due to their intrinsic capability to generate functional mammalian proteins. This advantage is frequently countered by problems with yields in expression, time required to generate over-expressing lines, and elevated costs. Co-expression of multiple proteins adds another level of complexity to these experiments, as cells need to be screened and selected for expression of suitable levels of each component. Here we present an efficient fluorescence marking procedure for establishing stable cell lines that over-express two proteins in co-ordination, and we validate the method in the production of recombinant monoclonal antibody Fab fragments. This procedure may readily be expanded to systems of greater complexity, comprising more then two components. PMID:21987254

  20. Expression of Sox family genes in early lamprey development

    PubMed Central

    UY, BENJAMIN R.; SIMOES-COSTA, MARCOS; SAUKA-SPENGLER, TATJANA; BRONNER, MARIANNE E.

    2014-01-01

    Members of the Sox (Sry-related high mobility group box) family of transcription factors play a variety of roles during development of both vertebrates and invertebrates. A marked expansion in gene number occurred during emergence of vertebrates, apparently via gene duplication events that are thought to have facilitated new functions. By screening a macroarrayed library as well as the lamprey genome, we have isolated genes of the Sox B, D, E and F subfamilies in the basal jawless vertebrate, lamprey. The expression patterns of all identified Sox genes were examined from gastrulation through early organogenesis (embryonic day 4–14), with particular emphasis on the neural crest, a vertebrate innovation. Coupled with phylogenetic analysis of these Sox genes, the results provide insight into gene duplication and divergence in paralog deployment occurring during early vertebrate evolution. PMID:22811271

  1. Follow-up of children with early expressive phonology disorders.

    PubMed

    Lewis, B A; Freebairn, L A; Taylor, H G

    2000-01-01

    Fifty-two children identified at age 4 to 6 years as demonstrating a moderate to severe expressive phonology disorder were followed to the third and fourth grades. Children were classified into two groups based on the presence of an early phonology disorder in isolation (P) or the presence of a phonology disorder with other language problems (PL). At follow-up, articulation measures failed to differentiate the groups; however, the PL group performed more poorly than the P group on measures of phoneme awareness, language, reading decoding, reading comprehension, and spelling. The P group demonstrated poor spelling skills relative to their reading and language abilities, suggesting residual spelling weaknesses in these children. The PL group reported more nuclear family members with speech-language disorders and with reading disorders than the P group. Findings support previous research linking early language disorders with later reading difficulties.

  2. Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

    PubMed

    Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

    2016-01-01

    BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

  3. Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis

    PubMed Central

    1987-01-01

    A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpole parvalbumin is highly similar to previously characterized beta- parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos. PMID:3558484

  4. Xenopus homeobox-containing cDNAs expressed in early development.

    PubMed Central

    Fritz, A; De Robertis, E M

    1988-01-01

    We report the isolation of six different homeobox-containing genes in Xenopus laevis which are expressed during early embryogenesis. cDNA clones of all of them were partially sequenced including two from previously isolated Xenopus genomic loci and found to contain homeodomains which share a high degree of homology with the Antennapedia protein (50 to 59 out of 60 amino acids). We find a short region of homology (consensus Ile Tyr Pro Trp Met) in four of the cDNAs, which is also present in Antennapedia and that may correspond to a bend region preceding the homeodomain. Northern blots have been performed to show the transcriptional pattern through early frog embryogenesis. Three of the genes are expressed only during a very narrow period of embryogenesis, reinforcing the view that homeobox genes are developmentally regulated. Images PMID:2894634

  5. DPEP1, expressed in the early stages of colon carcinogenesis, affects cancer cell invasiveness.

    PubMed

    Toiyama, Yuji; Inoue, Yasuhiro; Yasuda, Hiromi; Saigusa, Susumu; Yokoe, Takeshi; Okugawa, Yoshinaga; Tanaka, Koji; Miki, Chikao; Kusunoki, Masato

    2011-02-01

    We investigated changes in the gene expression profile in colon cancer in order to identify gene markers that may be useful in the management of this disease. The Cancer Genome Anatomy Project was used to detect differences in gene expression between normal and cancer tissue. The overexpression of dipeptidase-1 (DPEP1) in cancer tissue was confirmed in a sample of 76 patients by real-time PCR. To identify the function of DPEP1, RNA interference (RNAi) was used to inactivate this gene in the colon cancer cell line. Immunohistochemical analysis was performed to characterize the pattern of DPEP1 expression in colon cancer. DPEP1 expression in cancer was significantly higher than that in normal tissue. However, DPEP1 expression decreased with pathological differentiation, lymph-node and distant metastasis. Patients with tumors with decreased DPEP1 expression showed a poorer prognosis, and this was also true of patients with tumors who are treated with curative intent. RNAi-mediated DPEP1 reduction in the colon cancer cell line did not result in cell proliferation or apoptosis, but was associated with an increased invasive ability. DPEP1 protein was observed on the apical side of the cancer cells, and is expressed in the early stages of carcinogenesis, even in adenomas of both sporadic colorectal cancer and familial adenomatous polyposis patients. DPEP1 expression in normal colonic mucosa is very low, but it is highly expressed in colorectal adenoma and cancer specimens and is negatively correlated with parameters of pathological aggressiveness and poor prognosis. DPEP1 is expressed in the early stages of colon carcinogenesis and affects cancer cell invasiveness.

  6. Transient protein expression in three Pisum sativum (green pea) varieties.

    PubMed

    Green, Brian J; Fujiki, Masaaki; Mett, Valentina; Kaczmarczyk, Jon; Shamloul, Moneim; Musiychuk, Konstantin; Underkoffler, Susan; Yusibov, Vidadi; Mett, Vadim

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

  7. Temporal and spatial expression of AIF in the mouse uterus during early pregnancy.

    PubMed

    Zhang, Li-Ying; Hua, Yu-Ping; An, Tie-Zhu; Zuo, Ru-Juan; Nie, Wei-Tian; Wang, Lian-Bang; Shan, Chun-Hua

    2012-01-01

    Apoptosis-inducing factor (AIF) is a phylogenetically old, bifunctional protein with a pro-apoptotic function and redox activity. AIF regulates apoptosis and also plays a role in the defense against stress depending on its subcellular localization. Embryo implantation is a complicated process, in which an activated blastocyst interacts with a receptive uterus. The expression and regulation of AIF were investigated in this study in the mouse uterus during early pregnancy, pseudopregnancy, delayed implantation, artificial decidualization and under hormonal treatment using in situ hybridization, immunohistochemistry and real-time PCR. During early pregnancy, temporally and spatially regulated patterns of AIF expression were found in the mouse uterus; AIF expression in the luminal epithelium and glandular epithelium is regulated by steroid hormones; AIF mRNA expression in the stroma is influenced by the active blastocyst; and AIF protein was found to be located in the cytoplasm rather than the nucleus through confocal microscope. Our data suggest that AIF might play an important role during mouse embryo implantation and that the role of AIF might be implemented through its physiological activity rather than through its pro-apoptotic function in the mouse uterus during this period.

  8. Characterization of sea urchin unconventional myosins and analysis of their patterns of expression during early embryogenesis.

    PubMed

    Sirotkin, V; Seipel, S; Krendel, M; Bonder, E M

    2000-10-01

    Early sea urchin development requires a dynamic reorganization of both the actin cytoskeleton and cytoskeletal interactions with cellular membranes. These events may involve the activities of multiple members of the superfamily of myosin motor proteins. Using RT-PCR with degenerate myosin primers, we identified 11 myosin mRNAs expressed in unfertilized eggs and coelomocytes of the sea urchin Strongylocentrotus purpuratus. Seven of these sea urchin myosins belonged to myosin classes Igamma, II, V, VI, VII, IX, and amoeboid-type I, and the remaining four may be from novel classes. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I were either completely or partially cloned and their molecular structures characterized. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I shared a high degree of sequence identity with their respective family members from vertebrates and they retained their class-specific structure and domain organization. Analysis of expression of myosin-V, -VI, -VII, and amoeboid-type-I mRNAs during development revealed that each myosin mRNA displayed a distinct temporal pattern of expression, suggesting that myosins might be involved in specific events of early embryogenesis. Interestingly, the onset of gastrulation appeared to be a pivotal point in modulation of myosin mRNA expression. The presence of multiple myosin mRNAs in eggs and embryos provides insight into the potential involvement of multiple specific motor proteins in the actin-dependent events of embryo development.

  9. Translational Control of Protein Synthesis: The Early Years

    PubMed Central

    Lodish, Harvey F.

    2012-01-01

    For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of α- and β-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments. PMID:22988251

  10. Signatures of unfolding in the early stages of protein denaturation

    NASA Astrophysics Data System (ADS)

    Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

    2012-04-01

    A comparative study of the early stages of unfolding of five proteins: cyt c, c-b 562, cyt c‧, azurin, and lysozyme is reported. From crystallographic data, helical regions and intervening non-helical (or 'turning') regions are identified in each. Exploiting a previously introduced geometrical model, the paper describes quantitatively the stepwise extension of a polypeptide chain subject to the geometrical constraint that the spatial relationship among the residues of each triplet is fixed by native-state crystallographic data. Despite differences among the above-cited proteins, remarkable universality of behavior is found in the early stages of unfolding. At the very earliest stages, internal residues in each helical region have a common unfolding history; the terminal residues, however, are extraordinarily sensitive to structural perturbations. Residues in non-helical sections of the polypeptide unfold after residues in the internal helical regions, but with increasing steric perturbation playing a dominant role in advancing denaturation.

  11. Translational control of protein synthesis: the early years.

    PubMed

    Lodish, Harvey F

    2012-10-19

    For the past fifty-five years, much of my research has focused on the function and biogenesis of red blood cells, including the cloning and study of many membrane proteins such as glucose and anion transporters and the erythropoietin receptor. We have also elucidated the mechanisms of membrane insertion, folding, and maturation of many plasma membrane and secreted proteins. Despite all of this work and more, I remain extremely proud of our very early work on the regulation of mRNA translation: work on bacteriophage f2 RNA in the 1960s and on translation of α- and β-globin mRNAs in the early 1970s. Using techniques hopelessly antiquated by today's standards, we correctly elucidated many important aspects of translational control, and I thought readers would be interested in learning how we did these experiments.

  12. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  13. Computational Simulations of the Early Steps of Protein Aggregation

    PubMed Central

    Wei, Guanghong; Mousseau, Normand

    2007-01-01

    There is strong evidence that the oligomers of key proteins, formed during the early steps of aggregation, could be the primary toxic species associated with human neurodegenerative diseases, such as Alzheimer's and prion diseases. Here, we review recent progress in the development of computational approaches in order to understand the structures, dynamics and free energy surfaces of oligomers. We also discuss possible research directions for the coming years. PMID:19164927

  14. Temporal dynamics of immediate early gene expression during cellular consolidation of spatial memory.

    PubMed

    Barry, Daniel N; Commins, Sean

    2017-06-01

    The consolidation of newly acquired memories on a cellular level is thought to take place in the first few hours following learning. This process is dependent on de novo protein synthesis during this time, which ultimately leads to long-term structural and functional neuronal changes and the stabilisation of a memory trace. Immediate early genes (IEGs) are rapidly expressed in neurons following learning, and previous research has suggested more than one wave of IEG expression facilitates consolidation in the hours following learning. We analysed the expression of Zif268, c-Fos and Arc protein in a number of brain regions involved in spatial learning either 90min, 4h or 8h following training in the Morris water maze task. Consistent with the role of IEGs in the earliest stages of consolidation, a single wave of expression was observed in most brain regions at 90min, however a subsequent wave of expression was not observed at 8h. In fact, Zif268 expression was observed to fall below the levels of naïve controls at this time-point in the medial prefrontal and perirhinal cortices. This may be indicative of synaptic downscaling in these regions in the hours following learning, and an important marker of the consolidation of spatial memory. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Social isolation stress down-regulates cortical early growth response 1 (Egr-1) expression in mice.

    PubMed

    Matsumoto, Kinzo; Ono, Kazuya; Ouchi, Hirofumi; Tsushima, Ryohei; Murakami, Yukihisa

    2012-07-01

    Social isolation stress induces behavioral disturbances such as aggression, cognitive impairments, and deficits in prepulse inhibition in mice. Social isolation mice have, therefore, been studied as an animal model of neuropsychiatric disorders such as schizophrenia. Recently, the decrease in early growth response (Egr) gene expression levels were reported in the post-mortem brains of schizophrenia patients. In this study, we investigate the effects of social isolation stress on the expression levels of Egr mRNA and protein in the frontal cortex. Social isolation stress exposure significantly down-regulated the expression of Egr-1 protein and Egr-1 gene transcript in nucleus of cortical neurons in a manner dependent on a social isolation period. This stress had no effect on the expression level of Egr-1 in the striatum or the expression levels of other Egr family members (Egr-2, -3, and -4) in the frontal cortex. These results suggest that the decrease in Egr-1 expression in the frontal cortex may be involved in social isolation stress-induced behavioral abnormalities.

  16. Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice.

    PubMed

    Grisolano, J L; Sclar, G M; Ley, T J

    1994-09-13

    The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.

  17. Regulation of chick early B-cell factor-1 gene expression in feather development.

    PubMed

    El-Magd, Mohammed Abu; Sayed-Ahmed, Ahmed; Awad, Ashraf; Shukry, Mustafa

    2014-05-01

    The chick Ebf1 (early B-cell factor-1) gene is a member of a novel family of helix loop helix transcription factors. The expression profile, regulation and significance of this gene have been extensively studied in lymphatic, nervous, adipose and muscular tissues. However, cEbf1 expression, regulation and function in the feather of chick embryo have not yet been investigated. cEbf1 expression was first detected throughout the mesenchymal core of some few feather placodes (D7-D7.5). After feathers became mature and grew distally (D9 and D10), the mesenchymal expression of cEbf1 became confined to the caudal margin of the proximal half of all formed feather buds. Because this dynamic pattern of expression resembles that of Sonic Hedgehog (Shh) protein and bone morphogenetic protein (Bmp4) plus the crucial role of these two major signals in feather development, we hypothesized that cEbf1 expression in the feather may be regulated by Shh and Bmp4. In a feather explant culture system, Shh signals are necessary to initiate and maintain cEbf1 expression in the posterior half of the feather bud, while Bmp4 is crucial for the initial cEbf1 expression in the anterior half of the feather bud. Inhibition of Shh, not only down-regulates cEbf1, but also changes the morphology of feather buds, which become irregular and fused. This is the first study to demonstrate that cEbf1 expression in the feather bud is under the control of Shh and Bmp4 signals and that expression may play a role in the normal development of feathers.

  18. Neuroendocrine secretory protein 7B2: structure, expression and functions.

    PubMed Central

    Mbikay, M; Seidah, N G; Chrétien, M

    2001-01-01

    7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation. PMID:11439082

  19. Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

    PubMed Central

    Fogle, Rachel L.; Lynch, Christopher J.; Palopoli, Mary; Deiter, Gina; Stanley, Bruce A.; Vary, Thomas C.

    2014-01-01

    Background Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. Methods The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT™). Following the reaction with the ICAT™ reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Results Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Conclusions Based on the changes in proteins, we speculate modulation of

  20. Ttyh1 protein is expressed in glia in vitro and shows elevated expression in activated astrocytes following status epilepticus.

    PubMed

    Wiernasz, Elzbieta; Kaliszewska, Aleksandra; Brutkowski, Wojciech; Bednarczyk, Joanna; Gorniak, Malgorzata; Kaza, Beata; Lukasiuk, Katarzyna

    2014-12-01

    In a previous study, we showed that Ttyh1 protein is expressed in neurons in vitro and in vivo in the form of punctuate structures, which are localized to neuropil and neuronal somata. Herein, we provide the first description of Ttyh1 protein expression in astrocytes, oligodendrocytes and microglia in vitro. Moreover, using double immunofluorescence, we show Ttyh1 protein expression in activated astrocytes in the hippocampus following amygdala stimulation-induced status epilepticus. We demonstrate that in migrating astrocytes in in vitro wound model Ttyh1 concentrates at the edges of extending processes. These data suggest that Ttyh1 not only participates in shaping neuronal morphology, as previously described, but may also play a role in the function of activated glia in brain pathology. To localize Ttyh1 expression in the cellular compartments of neurons and astrocytes, we performed in vitro double immunofluorescent staining using markers for the following subcellular structures: endoplasmic reticulum (GRP78), Golgi apparatus (GM130), clathrin-coated vehicles (clathrin), early endosomes (Rab5 and APPL2), recycling endosomes (Rab11), trans-Golgi network (TGN46), endoplasmic reticulum membrane (calnexin), late endosomes and lysosomes (LAMP1) and synaptic vesicles (synaptoporin and synaptotagmin 1). We found that Ttyh1 is present in the endoplasmic reticulum, Golgi apparatus and clathrin-coated vesicles (clathrin) in both neurons and astrocytes and also in late endosomes or lysosomes in astrocytes. The presence of Ttyh1 was negligible in early endosomes, recycling endosomes, trans-Golgi network, endoplasmic reticulum membrane and synaptic vesicles.

  1. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

    Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

    2014-01-01

    Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

  2. Exocyst complex protein expression in the human placenta.

    PubMed

    Gonzalez, I M; Ackerman, W E; Vandre, D D; Robinson, J M

    2014-07-01

    Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. Copyright 2010 Elsevier Inc. All rights reserved.

  4. Improving membrane protein expression by optimizing integration efficiency.

    PubMed

    Niesen, Michiel J M; Marshall, Stephen S; Miller, Thomas F; Clemons, William M

    2017-09-16

    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were four-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effect of double mutations, on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  5. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

    PubMed

    Silva, Haroldo Cid da; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-08-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

  6. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

    PubMed Central

    da Silva, Haroldo Cid; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-01-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  7. BAFF and TACI gene expression are increased in patients with untreated very early rheumatoid arthritis.

    PubMed

    Moura, Rita A; Canhão, Helena; Polido-Pereira, Joaquim; Rodrigues, Ana M; Navalho, Márcio; Mourão, Ana F; Resende, Catarina; Campanilho-Marques, Raquel; Madruga Dias, João; da Silva, José Alberto Pereira; Graca, Luis; Fonseca, João E

    2013-08-01

    B cells play important roles in rheumatoid arthritis (RA). Given the beneficial effect of B cell depletion therapy in RA as well as the observed alterations in B cell subpopulations in this disease, we evaluated whether changes in the expression of genes related to B cell survival and activation were already present in patients with untreated very early RA (VERA; < 6 weeks of disease duration). The expression of a group of B cell-related activation and survival genes was quantified in peripheral blood mononuclear cells from patients with VERA by real-time PCR and compared with untreated early RA (< 1 year), established treated RA, and other untreated early arthritis conditions. Serum B cell-activating factor belonging to the tumor necrosis factor family (BAFF) was quantified by ELISA. BAFF gene expression and serum levels were highest in patients with VERA. The expression of BAFF receptor (BAFF-R) increased with disease progression, while transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) was elevated since the first weeks of RA onset. Paired box 5 gene expression was also increased at all RA stages. Chemokine (C-X-C motif) receptor 5 was elevated only in established RA. No differences were observed in B cell maturation antigen, activation-induced cytidine deaminase, B lymphocyte-induced maturation protein, and B cell lymphoma 2 expression. Disturbances in the expression of B cell-related activation and survival genes, particularly BAFF and TACI, occur from the onset of RA and precede changes in BAFF-R. These alterations can lead to the development of autoreactive B cells from the first weeks of RA onset.

  8. Presence of early stage cancer does not impair the early protein metabolic response to major surgery.

    PubMed

    Engelen, Mariëlle P K J; Klimberg, V Suzanne; Allasia, Arianna; Deutz, Nicolaas Ep

    2017-06-01

    Combined bilateral mastectomy and reconstruction is a common major surgical procedure in women with breast cancer and in those with a family history of breast cancer. As this large surgical procedure induces muscle protein loss, a preserved anabolic response to nutrition is warranted for optimal recovery. It is unclear whether the presence of early stage cancer negatively affects the protein metabolic response to major surgery as this would mandate perioperative nutritional support. In nine women with early stage (Stage II) breast malignancy and nine healthy women with a genetic predisposition to breast cancer undergoing the same large surgical procedure, we examined whether surgery influences the catabolic response to overnight fasting and the anabolic response to nutrition differently. Prior to and within 24 h after combined bilateral mastectomy and reconstruction surgery, whole body protein synthesis and breakdown rates were assessed after overnight fasting and after meal intake by stable isotope methodology to enable the calculation of net protein catabolism in the post-absorptive state and net protein anabolic response to a meal. Major surgery resulted in an up-regulation of post-absorptive protein synthesis and breakdown rates (P < 0.001) and lower net protein catabolism (P < 0.05) and was associated with insulin resistance and increased systemic inflammation (P < 0.01). Net anabolic response to the meal was reduced after surgery (P < 0.05) but higher in cancer (P < 0.05) indicative of a more preserved meal efficiency. The significant relationship between net protein anabolism and the amount of amino acids available in the circulation (R(2)  = 0.85, P < 0.001) was independent of the presence of non-cachectic early stage breast cancer or surgery. The presence of early stage breast cancer does not enhance the normal catabolic response to major surgery or further attenuates the anabolic response to meal intake within 24 h after

  9. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  10. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  11. A gene expression atlas of early craniofacial development.

    PubMed

    Brunskill, Eric W; Potter, Andrew S; Distasio, Andrew; Dexheimer, Phillip; Plassard, Andrew; Aronow, Bruce J; Potter, S Steven

    2014-07-15

    We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical microregions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the cranial mesenchyme, composed of mixed neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. At E9.5 cells from the cranial mesenchyme, overlying olfactory placode/epidermal ectoderm, and underlying neuroepithelium, as well as the emerging mandibular and maxillary arches were sampled. At E10.5, as the facial prominences form, cells from the medial and lateral prominences, the olfactory pit, multiple discrete regions of underlying neuroepithelium, the mandibular and maxillary arches, including both their mesenchymal and ectodermal components, as well as Rathke's pouch, were similarly sampled and profiled using both microarray and RNA-seq technologies. Further, we performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm. Taken together, and analyzable by a variety of biological network approaches, these data provide a complementing and cross validating resource capable of fueling discovery of novel compartment specific markers and signatures whose combinatorial interactions of transcription factors and growth factors/receptors are responsible for providing the master genetic blueprint for craniofacial development. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

    PubMed

    Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

    2010-12-01

    Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

  13. Protein Production for Structural Genomics Using E. coli Expression

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Li, Hui; Zhou, Min; Joachimiak, Grazyna; Babnigg, Gyorgy; Joachimiak, Andrzej

    2014-01-01

    The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail. PMID:24590711

  14. Regulation of pseudorabies virus gG glycoprotein gene promoter independently of pseudorabies immediate early IE180 protein.

    PubMed

    Muñoz, A L; Torres, M; Martín, B; Lerma, L; Tabarés, E

    2010-04-01

    The pseudorabies virus (PRV) glycoprotein known as gG is generally regarded as an early protein, and the immediate early IE180 protein regulates its expression during infection. This study, however, provides evidence that although induction by IE180 is observed, the expression of a marker protein (EGFP), or gG itself, under the control of the gG promoter, can also occur independently of the expression of IE180. This result was demonstrated both with transient transfection assays using plasmids and with viral infections. In transient transfections, the expression under control of the gG promoter depends on the cell type and surprisingly, can be 1.3-fold higher than the expression under the control of the IE180 promoter in Hela Tet-Off cells. Recombinant PRV S3 was constructed by replacing gE in the PRV genome with a chimeric transgene, expressing EGFP under the control of the gG promoter. In PK15 cells infected with NIA-3 wild-type virus or with S3 recombinant virus, expression of gG PRV mRNA (or EGFP mRNA) under the control of the gG promoter in the presence of cycloheximide was detected by RT-PCR. This again indicates that some basal expression was produced in infected cells independently of IE180. This expression was augmented by IE180 protein in both plasmid transfections and viral infections.

  15. The expression level of gC1qR is down regulated at the early time of infection with porcine circovirus of type 2 (PCV-2) and gC1qR interacts differently with the Cap proteins of porcine circoviruses.

    PubMed

    Kouokam Fotso, Guy Baudry; Bernard, Cécilia; Bigault, Lionel; de Boisséson, Claire; Mankertz, Annette; Jestin, André; Grasland, Béatrice

    2016-07-15

    Porcine circoviruses (PCV) are small, non-enveloped single-stranded DNA-viruses. Porcine circovirus type 2 (PCV-2) is the causal agent of post-weaning multisystemic wasting syndrome (PMWS) whereas porcine circovirus of type 1 (PCV-1) is non- pathogenic. gC1qR is a membrane-located receptor of the complement protein subunit C1q and interacts with PCV capsid proteins. The mechanisms associated with the triggering of PMWS are not well known and gC1qR may have a role in the life cycle and eventually in the pathogenicity of PCV. The objectives of this study were to determine the level of expression of gC1qR during early PCV-2 infection, to determine the region of PCV-2 capsid protein (Cap) required for the interaction with gC1qR and to evaluate the interaction of gC1qR with Cap proteins of different PCV strains. The results indicate that gC1qR transcripts are downregulated in the tonsils and the tracheo-bronchial lymph nodes of piglets infected by PCV-2 at the early time of the infection. The N-terminal amino acids (a.a. 1-59) of PCV-2b Cap, an arginine rich region, are involved in the interaction with gC1qR. Porcine gC1qR interacts with Cap proteins of two pathogenic viral strains, PCV-2a and PCV-2b, while interaction has been observed with only one Cap protein of two investigated strains of PCV-1. The amino acids 30 and 49 of PCV-1Cap, solely, were not responsible of the difference of interaction observed. We have also shown that gC1qR interacts strongly with PCV-2Caps and PCV-1 GER Cap. This result suggests that the different interaction of gC1qR with PCV Cap proteins may have an impact on the pathogenicity of the PCV. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Expression of turtle riboflavin-binding protein represses mitochondrial electron transport gene expression and promotes flowering in Arabidopsis.

    PubMed

    Li, Liang; Hu, Li; Han, Li-Ping; Ji, Hongtao; Zhu, Yueyue; Wang, Xiaobing; Ge, Jun; Xu, Manyu; Shen, Dan; Dong, Hansong

    2014-12-30

    Recently we showed that de novo expression of a turtle riboflavin-binding protein (RfBP) in transgenic Arabidopsis increased H2O2 concentrations inside leaf cells, enhanced the expression of floral regulatory gene FD and floral meristem identity gene AP1 at the shoot apex, and induced early flowering. Here we report that RfBP-induced H2O2 presumably results from electron leakage at the mitochondrial electron transport chain (METC) and this source of H2O2 contributes to the early flowering phenotype. While enhanced expression of FD and AP1 at the shoot apex was correlated with early flowering, the foliar expression of 13 of 19 METC genes was repressed in RfBP-expressing (RfBP+) plants. Inside RfBP+ leaf cells, cytosolic H2O2 concentrations were increased possibly through electron leakage because similar responses were also induced by a known inducer of electron leakage from METC. Early flowering no longer occurred when the repression on METC genes was eliminated by RfBP gene silencing, which restored RfBP+ to wild type in levels of FD and AP1 expression, H2O2, and flavins. Flowering was delayed by the external riboflavin application, which brought gene expression and flavins back to the steady-state levels but only caused 55% reduction of H2O2 concentrations in RfBP+ plants. RfBP-repressed METC gene expression remedied the cytosolic H2O2 diminution by genetic disruption of transcription factor NFXLl and compensated for compromises in FD and AP1 expression and flowering time. By contrast, RfBP resembled a peroxisomal catalase mutation, which augments the cytosolic H2O2, to enhance FD and AP1 expression and induce early flowering. RfBP-repressed METC gene expression potentially causes electron leakage as one of cellular sources for the generation of H2O2 with the promoting effect on flowering. The repressive effect on METC gene expression is not the only way by which RfBP induces H2O2 and currently unappreciated factors may also function under RfBP+ background.

  17. Postnatal developmental expression of regulator of G protein signaling 14 (RGS14) in the mouse brain.

    PubMed

    Evans, Paul R; Lee, Sarah E; Smith, Yoland; Hepler, John R

    2014-01-01

    Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and mitogen-activated protein kinase (MAPK) signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal-dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti-RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0), with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7, with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain.

  18. The early embryonic expression of TFII-I during mouse preimplantation development.

    PubMed

    Enkhmandakh, Badam; Bitchevaia, Natalia; Ruddle, Frank; Bayarsaihan, Dashzeveg

    2004-01-01

    We studied the developmentally regulated expression of mouse TFII-I, a founding member of a family of transcription factors characterized by the presence of multiple helix-loop-helix repeat domains. TFII-I and BEN, a second member of this family, are involved in histone modification and SUMOylation. The genes, GTF2I and GTF2IRD1, encoding these proteins in human are located at chromosomal band 7q11.23, within the Williams syndrome critical region. Our immunohistochemical analysis revealed extensive expression of TFII-I at early stages of embryogenesis. Like BEN, TFII-I is detected in the cytoplasm and nuclei of postfertilization through first cleavage stage embryos. However, in E4.5 blastocysts, at the time of implantation, TFII-I is localized in the nucleus and cytoplasm of the inner cell mass (ICM) and trophectoderm. BEN, at this stage, is expressed only in the cytoplasm of trophoblast cells, but not in the ICM [Gene Expr. Patterns, 2003; 3, 577-587]. Using RT-PCR, we detected Gtf2i and Gtf2ird1 mRNA transcripts in unfertilized oocytes, which indicates the maternal expression of these genes. Thus, the early embryonic expression of TFII-I implicates this family of transcription factors in preimplantation development.

  19. Differential expression of renal proteins in a rodent model of Meckel syndrome.

    PubMed

    Mason, Stephen B; Lai, Xianyin; Ringham, Heather N; Bacallao, Robert L; Harris, Peter C; Witzmann, Frank A; Gattone, Vincent H

    2011-01-01

    Meckel syndrome (MKS) is a fatal autosomal recessive condition with prominent renal cystic pathology. Renal protein misexpression was evaluated in the Wpk rat model of human MKS3 gene disease to identify biomarkers for the staging of renal cystic progression. Misexpressed proteins were compared between early and late stages of MKS renal cystic disease using proteomic analysis (two-dimensional gel electrophoresis with LC-MS/MS identification) followed by Western blot analysis. A proteomic analysis identified 76 proteins with statistically different, normalized abundance in at least one group. Subsequently, Western blot was used to confirm differential expression in several of these and polycystic kidney disease (PKD)-associated proteins. Galectin-1 and vimentin were identified as overexpressed proteins, which have been previously found in the jck mouse model of nephronophthisis 9. Ciliopathic PKD proteins, polycystins 1 & 2, and fibrocystin were also differentially expressed in Wpk kidney. In the Wpk rat, misexpressed proteins were identified that were also implicated in other forms of cystic disease. Numerous proteins were either over- or underexpressed in late-stage disease. Differences in protein expression may serve as biomarkers of cystic disease and its progression. Copyright © 2010 S. Karger AG, Basel.

  20. Analysis of incomplete gene expression dataset through protein-protein interaction information.

    PubMed

    Massanet-Vila, Raimon; Padró, Teresa; Cardús, Anna; Badimon, Lina; Caminal, Pere; Perera, Alexandre

    2011-01-01

    This paper shows a graph based method to analyze proteomic expression data. The method allows the prediction of the expression of genes not measured by the gene expression technology based on the local connectivity properties of the measured differentially expressed gene set. The prediction of the expression jointly with the stability of this prediction as a function of the variation of the initial expressed set is computed. The method is able to correctly predict one third of the proteins with independence of variations on the selection of the initial set. The algorithm is validated through a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF) protein expression experiment aiming the study of the protein expression patterns and post-translational modifications in human endothelial vascular cells exposed to atherosclerotic levels of Low Density Lipoproteins (LDL).

  1. Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

    PubMed

    Fernández-Bussy, Rodrigo; Mouguelar, Valeria; Banchio, Claudia; Coux, Gabriela

    2015-04-01

    In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.

  2. Two novel transcripts encoding two Ankyrin repeat containing proteins have preponderant expression during the mouse spermatogenesis.

    PubMed

    Wang, Fei; Hu, Jiarui; Song, Ping; Gong, Wuming

    2007-12-01

    The clone 4921537P18 expressed preponderantly in mouse testis was identified by screening the Riken cDNA database, and two new full-length isoforms of this clone, which were named gsarp1 (Gonad Specific Ankyrin Repeat (ANK) Protein 1) and gsarp2, were found and isolated from mouse testis in the course of the research. Both of the GSARP1 and GSARP2 contain an ANK region circular composed by seven ANKs, and their structural feature is very similar to that of the IkappaB family proteins, while IkappaB proteins associate with the transcription factor NF-kappaB via their ANKs in the NF-kappaB pathway. We investigated the expression pattern at the mRNA level by Reverse transcription PCR. The gsarp1 has high expression level in mouse testis, while has low expression level in the ovary, and the gsarp2 is only expressed in mouse testis. The gsarp1 and gsarp2 begin to be detected at the early and later pachytene stage of meiosis separately, while both have high-expression level at the stage of MI and MII. The result of in situ hybridization reveals that the gsarp1 is primarily expressed in spermatocytes, while gsarp2 is expressed in spermatocytes and spermatids. In view of the structural feature and expression pattern of the GSARP1 and GSARP2, we speculate that they may play a certain role in a signal pathway of meiosis.

  3. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.

    PubMed

    Dhore, C R; Cleutjens, J P; Lutgens, E; Cleutjens, K B; Geusens, P P; Kitslaar, P J; Tordoir, J H; Spronk, H M; Vermeer, C; Daemen, M J

    2001-12-01

    In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification.

  4. Proteins and an Inflammatory Network Expressed in Colon Tumors

    PubMed Central

    Zhu, Wenhong; Fang, Changming; Gramatikoff, Kosi; Niemeyer, Christina C.; Smith, Jeffrey W.

    2011-01-01

    The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of Multidimensional Protein Identification Technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from ApcMin/+ mice. Twenty-seven proteins were found to be up-regulated in colon tumors and twenty-five down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as “seeds” to search for co-expressed genes. This approach revealed a co-expression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The co-expression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer. PMID:21366352

  5. Plasma protein insudation as an index of early coronary atherogenesis.

    PubMed Central

    Zhang, Y.; Cliff, W. J.; Schoefl, G. I.; Higgins, G.

    1993-01-01

    Two hundred ninety-nine paraffin-embedded coronary artery blocks from 68 autopsy cases were serially sectioned. The blocks were selected to provide a range from normal through various stages of atherosclerosis, and sections were examined with the indirect immunofluorescence technique for intramural distribution of plasma albumin, fibrinogen, and immunoglobulin gamma (IgG). Cryostat-sections of 44 blocks from 22 of the same cases were examined with the same technique for distribution of apolipoprotein B. Alteration of protein insudation in the artery wall was a sensitive index of coronary atherogenesis. The sequence in which these proteins were involved in the initiation and development of early atherosclerotic lesions was analyzed by determining the average relative intimal thickness and relative lumen size that was associated with the first occurrence of altered insudation of each of these proteins. Results indicate that changed plasma albumin insudation is the earliest sign of a focal intimal lesion, and increasing albumin insudation shows the strongest association with intimal plaque growth. The other proteins tested showed altered insudation, in the order IgG, fibrinogen, apolipoprotein B. The results indicate that a progressive increase in permeability of the coronary artery endothelium occurs in the early stages of atherogenesis. Patterns of IgG localization provide evidence of both early systemic and subsequent local immune reactions being involved in atherogenesis. Altered albumin and apolipoprotein B insudation levels have stronger correlation coefficients with relative intimal thickness and relative lumen size than do those IgG and fibrinogen. The extremely high correlation coefficients shown by albumin emphasizes the importance of edema in determining plaque size and lumen stenosis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8342598

  6. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses

    PubMed Central

    Chudotvorova, Ilona; Ivanov, Anton; Rama, Sylvain; Hübner, Christian A; Pellegrino, Christophe; Ben-Ari, Yehezkel; Medina, Igor

    2005-01-01

    The development of GABAergic synapses is associated with an excitatory to inhibitory shift of the actions of GABA because of a reduction of [Cl−]i. This is due to a delayed postnatal expression of the K+–Cl− cotransporter KCC2, which has low levels at birth and peaks during the first few postnatal weeks. Whether the expression of the cotransporter and the excitatory to inhibitory shift have other consequences on the operation of GABAA receptors and synapses is not yet known. We have now expressed KCC2 in immature neurones at an early developmental stage and determined the consequences on the formation of GABA and glutamate synapses. We report that early expression of the cotransporter selectively enhances GABAergic synapses: there is a significant increase of the density of GABAA receptors and synapses and an increase of the frequency of GABAergic miniature postsynaptic currents. The density of glutamate synapses and frequency of AMPA miniature postsynaptic currents are not affected. We conclude that the expression of KCC2 and the reduction of [Cl−]i play a critical role in the construction of GABAergic networks that extends beyond the excitatory to inhibitory shift of the actions of GABA. PMID:15961425

  8. Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses.

    PubMed

    Chudotvorova, Ilona; Ivanov, Anton; Rama, Sylvain; Hübner, Christian A; Pellegrino, Christophe; Ben-Ari, Yehezkel; Medina, Igor

    2005-08-01

    The development of GABAergic synapses is associated with an excitatory to inhibitory shift of the actions of GABA because of a reduction of [Cl-]i. This is due to a delayed postnatal expression of the K+ -Cl- cotransporter KCC2, which has low levels at birth and peaks during the first few postnatal weeks. Whether the expression of the cotransporter and the excitatory to inhibitory shift have other consequences on the operation of GABA(A) receptors and synapses is not yet known. We have now expressed KCC2 in immature neurones at an early developmental stage and determined the consequences on the formation of GABA and glutamate synapses. We report that early expression of the cotransporter selectively enhances GABAergic synapses: there is a significant increase of the density of GABA(A) receptors and synapses and an increase of the frequency of GABAergic miniature postsynaptic currents. The density of glutamate synapses and frequency of AMPA miniature postsynaptic currents are not affected. We conclude that the expression of KCC2 and the reduction of [Cl-]i play a critical role in the construction of GABAergic networks that extends beyond the excitatory to inhibitory shift of the actions of GABA.

  9. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  10. Fungal and algal gene expression in early developmental stages of lichen-symbiosis.

    PubMed

    Joneson, Suzanne; Armaleo, Daniele; Lutzoni, François

    2011-01-01

    How plants and microbes recognize each other and interact to form long-lasting relationships remains one of the central questions in cellular communication. The symbiosis between the filamentous fungus Cladonia grayi and the single-celled green alga Asterochloris sp. was used to determine fungal and algal genes upregulated in vitro in early lichen development. cDNA libraries of upregulated genes were created with suppression subtractive hybridization in the first two stages of lichen development. Quantitative PCR subsequently was used to verify the expression level of 41 and 33 candidate fungal and algal genes respectively. Induced fungal genes showed significant matches to genes putatively encoding proteins involved in self and non-self recognition, lipid metabolism, and negative regulation of glucose repressible genes, as well as to a putative d-arabitol reductase and two dioxygenases. Upregulated algal genes included a chitinase-like protein, an amino acid metabolism protein, a dynein-related protein and a protein arginine methyltransferase. These results also provided the first evidence that extracellular communication without cellular contact can occur between lichen symbionts. Many genes showing slight variation in expression appear to direct the development of the lichen symbiosis. The results of this study highlight future avenues of investigation into the molecular biology of lichen symbiosis.

  11. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  12. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  13. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  14. A Statistical Study on Oscillatory Protein Expression

    NASA Astrophysics Data System (ADS)

    Yan, Shiwei

    Motivated by the experiments on the dynamics of a common network motif, p53 and Mdm2 feedback loop, by Lahav et al. [Nat. Genet 36, 147(2004)] in individual cells and Lev Bar-or et al. [Proc. Natl. Acad. Sci. USA 97, 11250(2000)] at the population of cells, we propose a statistical signal-response model with aiming to describe the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and in population of cells in a unified way. At the cellular level, the activities of p53 and Mdm2 proteins are described by a group of nonlinear dynamical equations where the damage-derived signal is assumed to have the form with abrupt transition (”on” leftrightarrow ”off”) as soon as signal strength passes forth and back across a threshold. Each cell responses to the damage with different time duration within which the oscillations persist. For the case of population of cells, the activities of p53 and Mdm2 proteins will be the population average of the individual cells, which results damped oscillations, due to the averaging over the cell population with the different response time.

  15. Early expressed genes showing a dichotomous developing pattern in the lancelet embryo.

    PubMed

    Yasui, K; Saiga, H; Wang, Y; Zhang, P J; Semba, I

    2001-04-01

    Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3beta class genes. The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region, respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling. The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.

  16. Identification of genes differentially expressed in dorsal and ventral chick midbrain during early Development

    PubMed Central

    Chittka, A; Volff, JN; Wizenmann, A

    2009-01-01

    Background During the development of the central nervous system (CNS), patterning processes along the dorsoventral (DV) axis of the neural tube generate different neuronal subtypes. As development progresses these neurons are arranged into functional units with varying cytoarchitecture, such as laminae or nuclei for efficient relaying of information. Early in development ventral and dorsal regions are similar in size and structure. Different proliferation rates and cell migration patterns are likely to result in the formation of laminae or nuclei, eventually. However, the underlying molecular mechanisms that establish these different structural arrangements are not well understood. We undertook a differential display polymerase chain reaction (DD-PCR) screen to identify genes with distinct expression patterns between dorsal and ventral regions of the chick midbrain in order to identify genes which regulate the sculpturing of such divergent neuronal organisation. We focused on the DV axis of the early chick midbrain since mesencephalic alar plate and basal plate develop into laminae and nuclei, respectively. Results We identified 53 differentially expressed bands in our initial screen. Twenty-six of these could be assigned to specific genes and we could unambiguously show the differential expression of five of the isolated cDNAs in vivo by in situ mRNA expression analysis. Additionally, we verified differential levels of expression of a selected number of genes by using reverse transcriptase (RT) PCR method with gene-specific primers. One of these genes, QR1, has been previously cloned and we present here a detailed study of its early developmental time course and pattern of expression providing some insights into its possible function. Our phylogenetic analysis of QR1 shows that it is the chick orthologue of Sparc-like 1/Hevin/Mast9 gene in mice, rats, dogs and humans, a protein involved in cell adhesion. Conclusion This study reveals some possible networks, which

  17. Expression and Purification of Mini G Proteins from Escherichia coli.

    PubMed

    Carpenter, Byron; Tate, Christopher G

    2017-04-20

    Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

  18. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  19. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

    PubMed

    Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

  20. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  1. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    PubMed

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  2. Analysis of the Expression of Tachykinins and Tachykinin Receptors in the Rat Uterus During Early Pregnancy.

    PubMed

    Pinto, Francisco M; Bello, Aixa R; Gallardo-Castro, Manuel; Valladares, Francisco; Almeida, Teresa A; Tena-Sempere, Manuel; Candenas, Luz

    2015-08-01

    The peptides of the tachykinin family participate in the regulation of reproductive function acting at both central and peripheral levels. Our previous data showed that treatment of rats with a tachykinin NK3R antagonist caused a reduction of litter size. In the present study, we analyzed the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy. Uterine samples were obtained from early pregnant rats (Days 1-9 of pregnancy) and from nonpregnant rats during the proestrus stage of the ovarian cycle, and real-time quantitative RT-PCR, immunohistochemistry, and Western blot studies were used to investigate the pattern of expression of tachykinins and tachykinin receptors. We found that all tachykinins and tachykinin receptors were locally synthesized in the uterus of early pregnant rats. The expression of substance P, neurokinin B, and the tachykinin receptors NK1R and NK3R mRNAs and proteins underwent major changes during the days around implantation and they were widely distributed in implantation sites, being particularly abundant in decidual cells. These findings support the involvement of the tachykinin system in the series of uterine events that occur around embryo implantation in the rat. © 2015 by the Society for the Study of Reproduction, Inc.

  3. Expression Analysis of Cathepsin F during Embryogenesis and Early Developmental Stage in Olive Flounder (Paralichthys olivaceus)

    PubMed Central

    Lee, Jang-Wook; Lee, Young Mee; Yang, Hyun; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Hwang, In Joon; Kim, Sung Yeon; Lee, Jeong-Ho

    2013-01-01

    Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87-98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder. PMID:25949137

  4. Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy

    PubMed Central

    2014-01-01

    Background MDC1A is a congenital neuromuscular disorder with developmentally complex and progressive pathologies that results from a deficiency in the protein laminin α2. MDC1A is associated with a multitude of pathologies, including increased apoptosis, inflammation and fibrosis. In order to assess and treat a complicated disease such as MDC1A, we must understand the natural history of the disease so that we can identify early disease drivers and pinpoint critical time periods for implementing potential therapies. Results We found that DyW mice show significantly impaired myogenesis and high levels of apoptosis as early as postnatal week 1. We also saw a surge of inflammatory response at the first week, marked by high levels of infiltrating macrophages, nuclear factor κB activation, osteopontin expression and overexpression of inflammatory cytokines. Fibrosis markers and related pathways were also observed to be elevated throughout early postnatal development in these mice, including periostin, collagen and fibronectin gene expression, as well as transforming growth factor β signaling. Interestingly, fibronectin was found to be the predominant fibrous protein of the extracellular matrix in early postnatal development. Lastly, we observed upregulation in various genes related to angiotensin signaling. Methods We sought out to examine the dysregulation of various pathways throughout early development (postnatal weeks 1-4) in the DyW mouse, the most commonly used mouse model of laminin-deficient muscular dystrophy. Muscle function tests (stand-ups and retractions) as well as gene (qRT-PCR) and protein levels (western blot, ELISA), histology (H&E, picrosirius red staining) and immunohistochemistry (fibronectin, TUNEL assay) were used to assess dysregulation of matricelluar protieins. Conclusions Our results implicate the involvement of multiple signaling pathways in driving the earliest stages of pathology in DyW mice. As opposed to classical dystrophies, such as

  5. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  6. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  7. Smad3 Deficiency Ameliorates Hepatic Fibrogenesis through the Expression of Senescence Marker Protein-30, an Antioxidant-Related Protein

    PubMed Central

    Jeong, Da-Hee; Hwang, Meeyul; Park, Jin-Kyu; Goo, Moon-Jung; Hong, Il-Hwa; Ki, Mi-Ran; Ishigami, Akihito; Kim, Ah-Young; Lee, Eun-Mi; Lee, Eun-Joo; Jeong, Kyu-Shik

    2013-01-01

    Smad3 is a key mediator of the transforming growth factor (TGF)-β1 signaling pathway that plays central role in inflammation and fibrosis. In present study, we evaluated the effect of Smad3 deficiency in Smad3−/− mice with carbon tetrachloride (CCl4)-induced liver fibrosis. The animals were received CCl4 or olive oil three times a week for 4 weeks. Histopathological analyses were performed to evaluate the fibrosis development in the mice. Alteration of protein expression controlled by Smad3 was examined using a proteomic analysis. CCl4-induced liver fibrosis was rarely detected in Smad3−/− mice compared to Smad3+/+. Proteomic analysis revealed that proteins related to antioxidant activities such as senescence marker protein-30 (SMP30), selenium-binding proteins (SP56) and glutathione S-transferases (GSTs) were up-regulated in Smad3−/− mice. Western blot analysis confirmed that SMP30 protein expression was increased in Smad3−/− mice. And SMP30 levels were decreased in CCl4-treated Smad3+/+ and Smad3−/− mice. These results indicate that Smad3 deficiency influences the proteins level related to antioxidant activities during early liver fibrosis. Thus, we suggest that Smad3 deteriorate hepatic injury by inhibitor of antioxidant proteins as well as mediator of TGF-β1 signaling. PMID:24304543

  8. Protein dynamics modulated electron transfer kinetics in early stage photosynthesis.

    PubMed

    Kundu, Prasanta; Dua, Arti

    2013-01-28

    A recent experiment has probed the electron transfer kinetics in the early stage of photosynthesis in Rhodobacter sphaeroides for the reaction center of wild type and different mutants [Science 316, 747 (2007)]. By monitoring the changes in the transient absorption of the donor-acceptor pair at 280 and 930 nm, both of which show non-exponential temporal decay, the experiment has provided a strong evidence that the initial electron transfer kinetics is modulated by the dynamics of protein backbone. In this work, we present a model where the electron transfer kinetics of the donor-acceptor pair is described along the reaction coordinate associated with the distance fluctuations in a protein backbone. The stochastic evolution of the reaction coordinate is described in terms of a non-Markovian generalized Langevin equation with a memory kernel and Gaussian colored noise, both of which are completely described in terms of the microscopics of the protein normal modes. This model provides excellent fits to the transient absorption signals at 280 and 930 nm associated with protein distance fluctuations and protein dynamics modulated electron transfer reaction, respectively. In contrast to previous models, the present work explains the microscopic origins of the non-exponential decay of the transient absorption curve at 280 nm in terms of multiple time scales of relaxation of the protein normal modes. Dynamic disorder in the reaction pathway due to protein conformational fluctuations which occur on time scales slower than or comparable to the electron transfer kinetics explains the microscopic origin of the non-exponential nature of the transient absorption decay at 930 nm. The theoretical estimates for the relative driving force for five different mutants are in close agreement with the experimental estimates obtained using electrochemical measurements.

  9. Protein dynamics modulated electron transfer kinetics in early stage photosynthesis

    NASA Astrophysics Data System (ADS)

    Kundu, Prasanta; Dua, Arti

    2013-01-01

    A recent experiment has probed the electron transfer kinetics in the early stage of photosynthesis in Rhodobacter sphaeroides for the reaction center of wild type and different mutants [Science 316, 747 (2007)]. By monitoring the changes in the transient absorption of the donor-acceptor pair at 280 and 930 nm, both of which show non-exponential temporal decay, the experiment has provided a strong evidence that the initial electron transfer kinetics is modulated by the dynamics of protein backbone. In this work, we present a model where the electron transfer kinetics of the donor-acceptor pair is described along the reaction coordinate associated with the distance fluctuations in a protein backbone. The stochastic evolution of the reaction coordinate is described in terms of a non-Markovian generalized Langevin equation with a memory kernel and Gaussian colored noise, both of which are completely described in terms of the microscopics of the protein normal modes. This model provides excellent fits to the transient absorption signals at 280 and 930 nm associated with protein distance fluctuations and protein dynamics modulated electron transfer reaction, respectively. In contrast to previous models, the present work explains the microscopic origins of the non-exponential decay of the transient absorption curve at 280 nm in terms of multiple time scales of relaxation of the protein normal modes. Dynamic disorder in the reaction pathway due to protein conformational fluctuations which occur on time scales slower than or comparable to the electron transfer kinetics explains the microscopic origin of the non-exponential nature of the transient absorption decay at 930 nm. The theoretical estimates for the relative driving force for five different mutants are in close agreement with the experimental estimates obtained using electrochemical measurements.

  10. [Bax protein expression in the carcinogenesis of human oral mucosa].

    PubMed

    Zeng, X; Chen, Q; Li, B; Tan, N

    2000-04-01

    . Blank controls were fabricated for each specimen by the omission of the primary antibody, which was replaced with PBS. In the process of oral carcinogenesis, each stage had Bax expression. The positive staining appeared in cytoplasm. In the normal oral mucosal specimens Bax expression was evident in the prickle layer, but not in the basal cell layers. Various degrees of Bax expression were seen in the diseased tissues. The staining pattern of hyperkeratotic lesions was similar to the normal oral mucosa, but Bax expression were also seen in the basal cell layer. In the mild, moderate, severe dysplasia and squamous cell carcinomas, Bax expression were seen in all layers, however, the intensity of staining were greater in mild and moderate dysplasia. The number of positive cells tended to increase gradually with the development of cell malignancy in the tissues of hyperkeratosis, mild and moderate dysplasia (P < 0.05). In the tissue of squamous cell carcinomas the number of positive cells had no marked difference comparing with the normal oral mucosa. The expression of Bax is involved in oral carcinogenesis and the compensative increase of Bax protein expression may be an early response.

  11. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  12. Vectors for the expression of tagged proteins in Drosophila.

    PubMed

    Parker, L; Gross, S; Alphey, L

    2001-12-01

    Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

  13. Variation in Protein Intake Induces Variation in Spider Silk Expression

    PubMed Central

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  14. Differential Protein Expression in Congenital and Acquired Cholesteatomas

    PubMed Central

    Kim, Sung Huhn; Choi, Jae Young

    2015-01-01

    Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D) electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5–3), plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5–3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5–3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins may differ

  15. Expression of methylation-modulated tumor-related genes in endoscopically resected early esophageal squamous neoplasia

    PubMed Central

    Hosoda, Kohei; Yashima, Kazuo; Tamoto, Akihiro; Yamamoto, Sohei; Kawata, Soichiro; Ikebuchi, Yuichiro; Matsumoto, Kazuya; Kawaguchi, Koichiro; Harada, Kenichi; Murawaki, Yoshikazu; Isomoto, Hajime

    2017-01-01

    Smoking and alcohol consumption are major risk factors for the development of esophageal squamous cell carcinoma (ESCC). Recent studies have demonstrated that smoking and alcohol consumption may be associated with altered DNA methylation in human cancer development. The aim of the present study was to evaluate methylation-modulated protein expression of tumor-related genes (TRGs) in the early stages of esophageal squamous neoplasia (ESN). ESN tissue samples (n=141) comprising 19 cases of low-grade intraepithelial neoplasia (LGIN), 70 of high-grade intraepithelial neoplasia/carcinoma in situ (HGIN/CIS) and 52 of invasive cancer, were endoscopically resected. The methylation-modulated protein expression of 5 TRGs [fragile histidine triad (FHIT), E-cadherin, MutL homolog 1 (MLH1) /MutS homolog 2 (MSH2) and cyclooxygenase-2 (COX-2)] as well as p53 was examined with immunohistochemistry, and their expression was compared with patient clinicopathological characteristics. Reduced or loss of FHIT, E-cadherin, MLH1/MSH2 and COX-2 expression was detected in 26.3 (5/19), 5.3 (1/19), 0 (0/19) and 63.2% (12/19) of LGIN cases, 61.4 (43/70), 18.6 (13/70), 7.1 (5/70) and 65.7% (46/70) of HGIN/CIS cases, and 78.8 (41/52), 50.0 (26/52), 11.5 (6/52) and 59.6% (31/52) of invasive cancer cases, respectively. Reduced or absent expression of FHIT and E-cadherin was significantly associated with neoplastic progression (FHIT, P=0.0007; E-cadherin, P=0.00014). The mean number of TRGs (FHIT, E-cadherin, MLH1/MSH2, and COX-2) that exhibited reduced or absent expression in LGIN, HGIN/CIS and invasive cancer specimens was 1.12±0.61, 1.66±0.93 and 2.09±0.96, respectively, demonstrating a significant stepwise increment from LGIN to HGIN/CIS and then to invasive cancer (P<0.05). p53 overexpression was frequently detected in ESN with head and neck carcinomas. However p53 overexpression was not significantly associated with ESN progression. An increase in the number of the 5 TRG proteins with

  16. Androgen receptor is expressed in mouse cardiomyocytes at prenatal and early postnatal developmental stages.

    PubMed

    Pedernera, Enrique; Gómora, María José; Meneses, Iván; De Ita, Marlon; Méndez, Carmen

    2017-08-14

    Previous studies show that androgens are involved in hypertrophy and excitability of cardiomyocytes and that their effects are mediated through their receptor. The aim of this study was to evaluate the presence of androgen receptor (AR) in mouse heart during prenatal and early postnatal stages. The expression of AR and related genes, alpha myosin heavy chain -Myh6-, beta myosin heavy chain -Myh7- and atrial natriuretic factor -Nppa- was simultaneously evaluated by semiquantitative RT-PCR. AR was also detected by immunohistochemistry. Androgen receptor mRNA was detected in hearts from 10.5 days post coitum to 16 postnatal days. A higher expression of AR mRNA in atria compared to ventricles was observed in neonatal mouse. A positive correlation between mRNA levels of AR and Nppa was observed in mouse heart at early postnatal development. Androgen receptor expression is similar in males and females during cardiac development. Finally, androgen receptor protein was observed by immunohistochemistry in myocardial cells of atria and ventricles from 12.5 days onwards and restricted after 16.5 days post-coitum to nuclei of cardiomyocytes. Present results provide evidence that androgen receptor is expressed from prenatal stages in mouse heart, supporting the proposition that androgens could be involved in mammalian heart development.

  17. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  18. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  19. Prolonged morphine administration alters protein expression in the rat myocardium

    PubMed Central

    2011-01-01

    Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart. PMID:22129148

  20. Protein microarrays and quantum dot probes for early cancer detection.

    PubMed

    Zajac, Aleksandra; Song, Dansheng; Qian, Wei; Zhukov, Tatyana

    2007-08-01

    We describe here a novel approach for detection of cancer markers using quantum dot protein microarrays. Both relatively new technologies; quantum dots and protein microarrays, offer very unique features that together allow detection of cancer markers in biological specimens (serum, plasma, body fluids) at pg/ml concentration. Quantum dots offer remarkable photostability and brightness. They do not exhibit photobleaching common to organic fluorophores. Moreover, the high emission amplitude for QDs results in a marked improvement in the signal to noise ratio of the final image. Protein microarrays allow highly parallel quantitation of specific proteins in a rapid, low-cost and low sample volume format. Furthermore the multiplexed assay enables detection of many proteins at once in one sample, making it a powerful tool for biomarker analysis and early cancer diagnostics. In a series of multiplexing experiments we investigated ability of the platform to detect six different cytokines in protein solution. We were able to detect TNF-alpha, IL-8, IL-6, MIP-1beta, IL-13 and IL-1beta down to picomolar concentration, demonstrating high sensitivity of the investigated detection system. We have also constructed and investigated two different models of quantum dot probes. One by conjugation of nanocrystals to antibody specific to the selected marker--IL-10, and the second by use of streptavidin coated quantum dots and biotinylated detector antibody. Comparison of those two models showed better performance of streptavidin QD-biotinylated detector antibody model. Data quantitated using custom designed computer program (CDAS) show that proposed methodology allows monitoring of changes in biomarker concentration in physiological range.

  1. Extracellular Stiffness Modulates the Expression of Functional Proteins and Growth Factors in Endothelial Cells.

    PubMed

    Santos, Lívia; Fuhrmann, Gregor; Juenet, Maya; Amdursky, Nadav; Horejs, Christine-Maria; Campagnolo, Paola; Stevens, Molly M

    2015-08-13

    Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.

  2. Changes in gap junction protein (connexin 32) gene expression during rat liver carcinogenesis.

    PubMed

    Fitzgerald, D J; Mesnil, M; Oyamada, M; Tsuda, H; Ito, N; Yamasaki, H

    1989-10-01

    A rat liver gap junction (GJ) cDNA probe that detects mRNA encoding the 32 Kd GJ-protein (connexin 32) was employed to study GJ-protein gene expression in rat liver tumors induced by a single exposure to diethylnitrosamine (DEN) followed by exposure to 2-acetylaminofluorene (AAF)/CCl4/AAF or induced by systemic administration of N-ethyl-N-hydroxyethylnitrosamine (EHEN). All carcinomas generated by these carcinogens showed markedly reduced levels of GJ-protein mRNA. This may indicate that GJ-protein levels and gap-junctional intercellular communication (GJIC) capacity are also severely compromised. Moreover, all hyperplastic nodules also showed a reduced level of GJ-protein mRNA. Taken together with our earlier finding that the liver tumor promoter phenobarbital inhibits GJ-protein gene expression, these results suggest that deranged GJIC is a relatively early event in liver multistage carcinogenesis. A range of other cDNA probes was also used to characterize gene expression in the DEN-induced tumors. Induction of expression was seen for glutathione S-transferase (placental form) (GST-P), gamma-glutamyltranspeptidase (GGT), and c-raf but not for c-Ha-ras or c-myc.

  3. Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions.

    PubMed

    McReynolds, Jayme R; Donowho, Kyle; Abdi, Amin; McGaugh, James L; Roozendaal, Benno; McIntyre, Christa K

    2010-03-01

    Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a beta-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of beta-adrenoceptors with propranolol (0.5 microg/0.2 microL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA beta-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.

  4. Expression analysis of a novel p75(NTR) signaling protein, which regulates cell cycle progression and apoptosis.

    PubMed

    Kendall, Stephen E; Goldhawk, Donna E; Kubu, Chris; Barker, Philip A; Verdi, Joseph M

    2002-09-01

    Neurotrophin receptor-interacting MAGE (NRAGE) is the most recently identified p75 neurotrophin receptor (p75(NTR)) intracellular binding protein. Previously, NRAGE over-expression was shown to mediate cell cycle arrest and facilitate nerve growth factor (NGF) dependent apoptosis of sympathetic neuroblasts in a p75(NTR) specific manner. Here we have examined the temporal and spatial expression patterns of NRAGE over the course of murine embryogenesis to determine whether NRAGE's expression is consistent with its proposed functions. We demonstrate that NRAGE mRNA and protein are expressed throughout embryonic and adult tissues. The mRNA is constitutively expressed within each tissue across development. However, expression of NRAGE protein displays a tight temporal tissue specific regulation. During early CNS development, NRAGE protein is expressed throughout the neural tube, but by later stages of neurogenesis, NRAGE protein is restricted within the ventricular zone, subplate and cortical plate. Moreover, NRAGE protein expression is limited to proliferative neural subpopulations as we fail to detect NRAGE expression co-localized with mature/differentiation associated neuronal markers. Interestingly, NRAGE's expression is not restricted solely to areas of p75(NTR) expression suggesting that NRAGE may mediate proliferation and/or apoptosis from other environmental signals in addition to NGF within the CNS. Our data support previously characterized roles for NRAGE as a mediator of precursor apoptosis and a repressor of cell cycle progression in neural development.

  5. Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia.

    PubMed

    Ihnatko, Robert; Edén, Ulla; Lagali, Neil; Dellby, Anette; Fagerholm, Per

    2013-12-06

    Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P<0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly α-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-α2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients' tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition. This study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears. © 2013 Elsevier B.V. All rights reserved.

  6. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  7. Analysis of protein expression profile of oral squamous cell carcinoma by MALDI-TOF-MS.

    PubMed

    Roman, Eric; Lunde, Mai Lill Suhr; Miron, Talia; Warnakulasauriya, Saman; Johannessen, Anne Christine; Vasstrand, Endre Normann; Ibrahim, Salah Osman

    2013-03-01

    In this study, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF-MS) technology was used to examine differentially expressed proteins in oral squamous cell carcinoma (OSCC) tissues from Norway (n=15) and the UK (n=45). Twenty-nine proteins were found to be significantly overexpressed in the OSCCs examined compared to the normal controls. Identified proteins included, family of annexin proteins that play important roles in signal transduction pathways and regulation of cellular growth, keratin-1, heat-shock proteins (HSP), squamous cell carcinoma antigen (SCC-Ag), cytoskeleton proteins, and proteins involved in mitochondrial and intracellular signalling pathways. The expression of four selected proteins (annexin II and V, HSP-27, and SCC-Ag) was verified using western blot analysis of 76 fresh tissue biopsy specimens in total, from Norway (n=53) and the UK (n=23). Proteomic analysis of OSCCs examined here demonstrated involvement of several proteins that might function as potential biomarkers and molecular targets for early cancer diagnostics, and may contribute to a novel approach to therapeutics and for predicting prognosis of OSCC.

  8. Recent patents on alphavirus protein expression and vector production.

    PubMed

    Aranda, Alejandro; Ruiz-Guillen, Marta; Quetglas, Jose I; Bezunartea, Jaione; Casales, Erkuden; Smerdou, Cristian

    2011-12-01

    Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

  9. Expression and purification of GST-FHL2 fusion protein.

    PubMed

    Yu, H; Ma, Q; Lin, J; Sun, Y F; Zheng, F

    2013-12-06

    Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.

  10. Enhanced Expression of Hedgehog Pathway Proteins in Oral Epithelial Dysplasia.

    PubMed

    Dias, Rosane Borges; Valverde, Ludmila de Faro; Sales, Caroline Brandi Schlaepfer; Guimarães, Vanessa Sousa Nazaré; Cabral, Márcia Grillo; de Aquino Xavier, Flávia Caló; Dos Santos, Jean Nunes; Ramos, Eduardo Antônio Gonçalves; Gurgel Rocha, Clarissa Araújo

    2016-09-01

    The aim of this study was to characterize the profile of the proteins involved in the Hedgehog signaling pathway to aid in the understanding of the pathogenesis of oral epithelial dysplasia (OED). The proteins SHH, PTCH1, HHIP, SUFU, GLI1, and cyclin D1 were evaluated by immunohistochemistry in 25 cases of OED, 4 of non-neoplasic oral mucosa, 8 of inflammatory fibrous hyperplasia and 5 of hyperkeratosis. SHH proteins were predominant in OED cases. Although PTCH1 protein was observed in all cases, this molecule was more highly expressed in OED. The inhibitor protein SUFU was present in OED and HHIP protein was overexpressed in OED. GLI1 proteins were predominantly found in the nuclei of epithelial cells in OED. Basal and suprabasal cells in the epithelial lining were positive for cyclin D1 only in OED. In conclusion, comparative analysis of the proteins involved in the Hedgehog pathway suggests that enhanced expression of these proteins can play an important role in the biological behavior of OED.

  11. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    PubMed Central

    Chakrabarti, Sanjukta; Barrow, Colin J.; Kanwar, Rupinder K.; Ramana, Venkata; Kanwar, Jagat R.

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

  12. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  13. Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.

    PubMed

    Adler, M J; Coronel, C; Shelton, E; Seegmiller, J E; Dewji, N N

    1991-01-01

    The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4- to 5-kDa amyloid beta-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of beta-amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyclonal antibodies to a synthetic peptide of the beta-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. Regulated expression of beta-amyloid precursor protein may be an important determinant of growth and metabolic responses to serum and growth factors under physiological as well as pathological conditions.

  14. NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons.

    PubMed

    Vaes, N; Lentjes, M H F M; Gijbels, M J; Rademakers, G; Daenen, K L; Boesmans, W; Wouters, K A D; Geuzens, A; Qu, X; Steinbusch, H P J; Rutten, B P F; Baldwin, S H; Sharkey, K A; Hofstra, R M W; van Engeland, M; Vanden Berghe, P; Melotte, V

    2017-09-01

    Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract. We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy. NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase. NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression. © 2017 John Wiley & Sons Ltd.

  15. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

    Gibbs, David L.; Baratt, Arie; Baric, Ralph; Kawaoka, Yoshihiro; Smith, Richard D.; Orwoll, Eric S.; Katze, Michael G.; Mcweeney, Shannon K.

    2013-06-01

    Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology. We have evaluated the construction of network models using methods derived from weighted gene co-expression network analysis (WGCNA). We show that approximately scale-free peptide networks, composed of statistically significant modules, are feasible and biologically meaningful using two mouse lung experiments and one human plasma experiment. Within each network, peptides derived from the same protein are shown to have a statistically higher topological overlap and concordance in abundance, which is potentially important for inferring protein abundance. The module representatives, called eigenpeptides, correlate significantly with biological phenotypes. Furthermore, within modules, we find significant enrichment for biological function and known interactions (gene ontology and protein-protein interactions). Biological networks are important tools in the analysis of complex systems. In this paper we evaluate the application of weighted co-expression network analysis to quantitative proteomics data. Protein co-expression networks allow novel approaches for biological interpretation, quality control, inference of protein abundance, a framework for potentially resolving degenerate peptide-protein mappings, and a biomarker signature discovery.

  16. Thyroid-Related Protein Expression in the Human Thymus

    PubMed Central

    Park, Do Joon; Jung, Kyeong Cheon

    2017-01-01

    Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS. PMID:28386277

  17. Expression of Escherichia coli cspA during early exponential growth at 37 °C.

    PubMed

    Brandi, Anna; Pon, Cynthia L

    2012-01-25

    CspA is a small (7.4 kDa) nucleic acid binding protein of Escherichia coli whose expression is stimulated after cold-stress but whose level is also extraordinarily high during the early phase of growth of non-stressed cells. In this study the relationship existing between cspA transcription/translation on the one hand and the acquisition of critical mass for cell division and chromosome replication, on the other, in stationary phase cells subjected to a nutritional up-shift at 37 °C has been analyzed. Measurements of optical density and viable counts, pulse-chase, real-time PCR and immunodetection experiments, as well as cytofluorimetric and DNA duplication analyses show that synthesis of new CspA molecules at 37 °C is not only restricted to the lag phase ensuing the nutritional up-shift, but continues also during the first stages of logarithmic growth, when cells have already started dividing; although the early synthesized molecules are diluted by the following cell divisions and new synthesis occurs at an extremely low level, cspA mRNA and CspA continue to be present. A possible explanation for the apparent paradox that cspA is activated not only following cold stress, but also under non-stress and other stress conditions which entail a down-regulation of bulk gene expression and protein synthesis is presented.

  18. Expression dynamics and protein localization of rhabdomeric opsins in Platynereis larvae.

    PubMed

    Randel, Nadine; Bezares-Calderón, Luis A; Gühmann, Martin; Shahidi, Réza; Jékely, Gáspár

    2013-07-01

    The larval stages of polychaete annelids are often responsive to light and can possess one to six eyes. The early trochophore larvae of the errant annelid Platynereis dumerilii have a single pair of ventral eyespots, whereas older nectochaete larvae have an additional two pairs of dorsal eyes that will develop into the adult eyes. Early Platynereis trochophores show robust positive phototaxis starting on the first day of development. Even though the mechanism of phototaxis in Platynereis early trochophore larvae is well understood, no photopigment (opsin) expression has yet been described in this stage. In late trochophore larvae, a rhabdomeric-type opsin, r-opsin1, expressed in both the eyespots and the adult eyes has already been reported. Here, we identify another Platynereis rhabdomeric opsin, r-opsin3, that is expressed in a single photoreceptor in the eyespots in early trochophores, suggesting that it mediates early larval phototaxis. We also show that r-opsin1 and r-opsin3 are expressed in adjacent photoreceptor cells in the eyespots in later stages, indicating that a second eyespot-photoreceptor differentiates in late trochophore larvae. Using serial transmission electron microscopy (TEM), we identified and reconstructed both photoreceptors and a pigment cell in the late larval eyespot. We also characterized opsin expression in the adult eyes and found that the two opsins co-express there in several photoreceptor cells. Using antibodies recognizing r-opsin1 and r-opsin3 proteins, we demonstrate that both opsins localize to the rhabdomere in all six eyes. In addition, we found that r-opsin1 mRNA is localized to, and translated in, the projections of the adult eyes. The specific changes we describe in opsin transcription and translation and in the cellular complement suggest that the six larval eyes undergo spectral and functional maturation during the early planktonic phase of the Platynereis life cycle.

  19. TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion.

    PubMed

    Rostasy, Kevin; Augood, Sarah J; Hewett, Jeffrey W; Leung, Joanne Chung-on; Sasaki, Hikaru; Ozelius, Laurie J; Ramesh, Vijaya; Standaert, David G; Breakefield, Xandra O; Hedreen, John C

    2003-02-01

    Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. Most cases are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which is widely expressed in human brain and encodes the protein torsinA. This study compares neuropathology and torsinA expression in the normal human brain with that in dystonia cases with and without the GAG deletion. TorsinA-like protein was expressed in neuronal cytoplasm throughout the human brain, including cerebellum, substantia nigra, hippocampus, and neostriatum, with higher levels in specific neurons. This immunostaining pattern was not discernibly different in dystonia and normal brains in midbrain and neostriatal regions. However, nigral dopaminergic neurons appeared to be larger in both GAG-deletion and non-GAG-deletion dystonia brains compared to normal, and may be more closely spaced in GAG-deletion brains. Beyond these apparent changes in neuronal size and spacing in dystonia brains, there was no indication of neuron loss, inflammation, DNA strand breaks, or altered distribution of torsin-like immunoreactivity, supporting a functional rather than degenerative etiology of early onset torsion dystonia.

  20. Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

    PubMed

    Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

    2017-04-01

    The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.

  1. Protein Z: A putative novel biomarker for early detection of ovarian cancer.

    PubMed

    Russell, Matthew R; Walker, Michael J; Williamson, Andrew J K; Gentry-Maharaj, Aleksandra; Ryan, Andy; Kalsi, Jatinderpal; Skates, Steven; D'Amato, Alfonsina; Dive, Caroline; Pernemalm, Maria; Humphryes, Phillip C; Fourkala, Evangelia-Ourania; Whetton, Anthony D; Menon, Usha; Jacobs, Ian; Graham, Robert L J

    2016-06-15

    Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded-evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down-regulated up to 2 years pre-diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up-regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA-125 and CA-125 combined with Protein Z showed a statistically significant (p = 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA-125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes.

  2. Protein Z: A putative novel biomarker for early detection of ovarian cancer

    PubMed Central

    Russell, Matthew R.; Walker, Michael J.; Williamson, Andrew J. K.; Gentry‐Maharaj, Aleksandra; Ryan, Andy; Kalsi, Jatinderpal; Skates, Steven; D'Amato, Alfonsina; Dive, Caroline; Pernemalm, Maria; Humphryes, Phillip C.; Fourkala, Evangelia‐Ourania; Whetton, Anthony D.; Menon, Usha

    2016-01-01

    Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded‐evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down‐regulated up to 2 years pre‐diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up‐regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA‐125 and CA‐125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA‐125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes. PMID:26815306

  3. In vivo expression and regulation of genes associated with vascularization during early response of sutures to tensile force.

    PubMed

    Takeshita, Nobuo; Hasegawa, Masakazu; Sasaki, Kiyo; Seki, Daisuke; Seiryu, Masahiro; Miyashita, Shunro; Takano, Ikuko; Oyanagi, Toshihito; Miyajima, Yuki; Takano-Yamamoto, Teruko

    2017-01-01

    Sutures are fibrous tissues that connect bones in craniofacial skeletal complexes. Cranio- and dentofacial skeletal deformities in infant and adolescent patients can be treated by applying tensile force to sutures to induce sutural bone formation. The early gene expression induced by mechanical stress is essential for bone formation in long bones; however, early gene expression during sutural bone formation induced by tensile force is poorly characterized. In vivo studies are essential to evaluate molecular responses to mechanical stresses in heterogeneous cell populations, such as sutures. In this paper we examined in vivo early gene expression and the underlying regulatory mechanism for this expression in tensile-force-applied cranial sutures, focusing on genes involved in vascularization. Tensile force upregulated expression of vascular factors, such as vascular endothelial growth factor (Vegf) and endothelial cell markers, in sutures within 3 h. The expression of connective tissue growth factor (Ctgf) and Rho-associated coiled-coil containing protein kinase 2 (Rock2) was also upregulated by tensile force. A CTGF-neutralizing antibody and the ROCK inhibitor, Y-27632, abolished tensile-force-induced Vegf expression. Moreover, tensile force activated extracellular signal-related kinase 1/2 (ERK1/2) signaling in sagittal sutures, and the ERK1/2 inhibitor, U0126, partially inhibited tensile-force-induced Ctgf expression. These results indicate that tensile force induces in vivo gene expression associated with vascularization early in tensile-force-induced sutural bone formation. Moreover, the early induction of Vegf gene expression is regulated by CTGF and ROCK2.

  4. Comprehensive analysis of TCP transcription factors and their expression during cotton (Gossypium arboreum) fiber early development.

    PubMed

    Ma, Jun; Liu, Fang; Wang, Qinglian; Wang, Kunbo; Jones, Don C; Zhang, Baohong

    2016-02-09

    TCP proteins are plant-specific transcription factors implicated to perform a variety of physiological functions during plant growth and development. In the current study, we performed for the first time the comprehensive analysis of TCP gene family in a diploid cotton species, Gossypium arboreum, including phylogenetic analysis, chromosome location, gene duplication status, gene structure and conserved motif analysis, as well as expression profiles in fiber at different developmental stages. Our results showed that G. arboreum contains 36 TCP genes, distributing across all of the thirteen chromosomes. GaTCPs within the same subclade of the phylogenetic tree shared similar exon/intron organization and motif composition. In addition, both segmental duplication and whole-genome duplication contributed significantly to the expansion of GaTCPs. Many these TCP transcription factor genes are specifically expressed in cotton fiber during different developmental stages, including cotton fiber initiation and early development. This suggests that TCP genes may play important roles in cotton fiber development.

  5. Expression of growth hormone gene during early development of Siberian sturgeon (Acipenser baerii)

    PubMed Central

    Abdolahnejad, Zeinab; Pourkazemi, Mohammad; Khoshkholgh, Majid Reza; Yarmohammadi, Mahtab

    2015-01-01

    The mRNA expression of growth hormone (GH) gene in early development stages of Siberian sturgeon was investigated using RT-PCR method. Samples were collected from unfertilized eggs up to 50 days post hatched (dph) larvae in 11 different times. Ribosomal protein L6 (RPL6) transcripts were used as the internal standard during quantification of GH mRNA expression. The results showed that the GH mRNA could be observed in the eyed eggs and even at unfertilized eggs of Siberian sturgeon. The highest amounts of GH mRNA were found at 25 and 50 dph larvae, while the lowest levels were detected at 1 and 3 dph larvae stage. These findings suggest that, the GH mRNA play a key role during developmental stages of Siberian sturgeon. PMID:27844010

  6. Expression of growth hormone gene during early development of Siberian sturgeon (Acipenserbaerii).

    PubMed

    Abdolahnejad, Zeinab; Pourkazemi, Mohammad; Khoshkholgh, Majid Reza; Yarmohammadi, Mahtab

    2015-12-01

    The mRNA expression of growth hormone (GH) gene in early development stages of Siberian sturgeon was investigated using RT-PCR method. Samples were collected from unfertilized eggs up to 50 days post hatched (dph) larvae in 11 different times. Ribosomal protein L6 (RPL6) transcripts were used as the internal standard during quantification of GH mRNA expression. The results showed that the GH mRNA could be observed in the eyed eggs and even at unfertilized eggs of Siberian sturgeon. The highest amounts of GH mRNA were found at 25 and 50 dph larvae, while the lowest levels were detected at 1 and 3 dph larvae stage. These findings suggest that, the GH mRNA play a key role during developmental stages of Siberian sturgeon.

  7. Temporal expression and immunogold localization of Plodia interpunctella granulosis virus structural proteins

    NASA Technical Reports Server (NTRS)

    Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Monospecific antisera were produced against four structural proteins (VP12, VP17, VP31, and granulin) of the Plodia interpunctella granulosis virus using polypeptides derived by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or acid extraction. The antisera were shown to be specific on immunoblots of SDS-PAGE separated granulosis virus and were further used to detect structural proteins in infected fat body lysates. Immunoblots of fat body lysates from early stages of infection indicated that VP12, VP17, VP31, and granulin were expressed by 2.5 days post-infection. Immunogold labeling of the virus using the monospecific antisera and electron microscopy confirmed earlier reports that granulin is located in the protein matrix, V17 is an envelope protein, and VP31 is a capsid protein.

  8. Temporal expression and immunogold localization of Plodia interpunctella granulosis virus structural proteins

    NASA Technical Reports Server (NTRS)

    Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Monospecific antisera were produced against four structural proteins (VP12, VP17, VP31, and granulin) of the Plodia interpunctella granulosis virus using polypeptides derived by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or acid extraction. The antisera were shown to be specific on immunoblots of SDS-PAGE separated granulosis virus and were further used to detect structural proteins in infected fat body lysates. Immunoblots of fat body lysates from early stages of infection indicated that VP12, VP17, VP31, and granulin were expressed by 2.5 days post-infection. Immunogold labeling of the virus using the monospecific antisera and electron microscopy confirmed earlier reports that granulin is located in the protein matrix, V17 is an envelope protein, and VP31 is a capsid protein.

  9. Protein-directed ribosomal frameshifting temporally regulates gene expression

    PubMed Central

    Napthine, Sawsan; Ling, Roger; Finch, Leanne K.; Jones, Joshua D.; Bell, Susanne; Brierley, Ian; Firth, Andrew E.

    2017-01-01

    Programmed −1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins. PMID:28593994

  10. Using ion exchange chromatography to purify a recombinantly expressed protein.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  11. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    PubMed Central

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2013-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease. PMID:23012103

  12. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  13. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

    PubMed

    Smits, Arne H; Lindeboom, Rik G H; Perino, Matteo; van Heeringen, Simon J; Veenstra, Gert Jan C; Vermeulen, Michiel

    2014-09-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

  14. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

    PubMed Central

    Smits, Arne H.; Lindeboom, Rik G.H.; Perino, Matteo; van Heeringen, Simon J.; Veenstra, Gert Jan C.; Vermeulen, Michiel

    2014-01-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs. PMID:25056316

  15. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  16. Screening and validation of serum protein biomarkers for early postmenopausal osteoporosis diagnosis.

    PubMed

    Wang, Long; Hu, Ya-Qian; Zhao, Zhuo-Jie; Zhang, Hong-Yang; Gao, Bo; Lu, Wei-Guang; Xu, Xiao-Long; Lin, Xi-Sheng; Wang, Jin-Peng; Jie, Qiang; Luo, Zhuo-Jing; Yang, Liu

    2017-09-26

    Postmenopausal osteoporosis is one of the most prominent worldwide public health problems and the morbidity is increasing with the aging population. It has been demonstrated that early diagnosis and intervention delay the disease progression and improve the outcome. Therefore, searching for biomarkers that are able to identify postmenopausal women at high risk for developing osteoporosis is an effective way to improve the quality of life of patients, and alleviate social and economic burdens. In the present study, a protein array was used to identify potential biomarkers. The bone mineral densities of 10 rats were dynamically measured in an ovariectomized model by micro‑computed tomography assessment, and the early stage of osteoporosis was defined. Through the protein array‑based screening, the expression levels of six serum protein biomarkers in ovariectomized rats were observed to alter at the initiation stage of the postmenopausal osteoporosis. Fractalkine, tissue inhibitor of metalloproteinases‑1 and monocyte chemotactic protein‑1 were finally demonstrated to be increased in the serum of eight enrolled postmenopausal osteoporosis patients using ELISA assay and were correlated with the severity of progressive bone loss. These biomarkers may be explored as potential early biomarkers to readily evaluate and diagnose postmenopausal osteoporosis in the clinic.

  17. Cell-type-specific expression pattern of ceramide synthase 2 protein in mouse tissues.

    PubMed

    Kremser, Christiane; Klemm, Anna-Lena; van Uelft, Martina; Imgrund, Silke; Ginkel, Christina; Hartmann, Dieter; Willecke, Klaus

    2013-11-01

    Ceramide synthase 2 (CerS2) catalyzes the synthesis of dihydroceramides from dihydrosphingosine and very long fatty acyl (C22-C24)-CoAs. CerS2-deficient (gene trap) mice were reported to exhibit myelin and behavioral abnormalities, associated with the expression of CerS2 in oligodendrocytes and neurons based on expression of lacZ reporter cDNA instead of the cers2 gene in these mice. In order to clarify the cell-type-specific expression of CerS2 protein, we have raised antibodies that specifically recognize the glycosylated and non-glycosylated CerS2 protein in wild-type but not in CerS2-deficient mouse tissues. In early postnatal, juvenile and adult mouse brain, the new antibodies detect CerS2 protein only in oligodendrocytes but not in neurons, suggesting that the gene trap vector in CerS2-deficient mice led to ectopic expression of the lacZ reporter gene in neurons. In liver, the CerS2 protein is expressed in hepatocytes but not in Ito cells or Kupffer cells. We conclude that the behavioral abnormalities observed in CerS2-deficient mice originate primarily in oligodendrocytes and not in neurons. The identification of specific cell types in which CerS2 protein is expressed is prerequisite to further mechanistic characterization of phenotypic abnormalities exhibited by CerS2-deficient mice. The amount of CerS2 protein detected in different tissues by immunoblot analyses does not strictly correspond to the activity of the CerS2 enzyme. Disproportional results are likely due to post-translational regulation of the CerS2 protein.

  18. Serum Protein Markers for the Early Detection of Lung Cancer: A Focus on Autoantibodies.

    PubMed

    Broodman, Ingrid; Lindemans, Jan; van Sten, Jenny; Bischoff, Rainer; Luider, Theo

    2017-01-06

    Lung cancer has the highest mortality rate among cancer patients in the world, in particular because most patients are only diagnosed at an advanced and noncurable stage. Computed tomography (CT) screening on high-risk individuals has shown that early detection could reduce the mortality rate. However, the still high false-positive rate of CT screening may harm healthy individuals because of unnecessary follow-up scans and invasive follow-up procedures. Alternatively, false-negative and indeterminate results may harm patients due to the delayed diagnosis and treatment of lung cancer. Noninvasive biomarkers, complementary to CT screening, could lower the false-positive and false-negative rate of CT screening at baseline and thereby reduce the number of patients that need follow-up and diagnose patients at an earlier stage of lung cancer. Lung cancer tissue generates lung cancer-associated proteins to which the immune system might produce high-affinity autoantibodies. This autoantibody response to tumor-associated antigens starts during early stage lung cancer and may endure over years. Identification of tumor-associated antigens or the corresponding autoantibodies in body fluids as potential noninvasive biomarkers could thus be an effective approach for early detection and monitoring of lung cancer. We provide an overview of differentially expressed protein, antigen, and autoantibody biomarkers that combined with CT imaging might be of clinical use for early detection of lung cancer.

  19. Global Analysis of Protein Expression of Inner Ear Hair Cells

    PubMed Central

    Wong, Ann C.Y.; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel

    2017-01-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that

  20. SPINK 1 Protein Expression and Prostate Cancer Progression

    PubMed Central

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  1. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  2. Expression of protein kinase C genes during ontogenic development of the central nervous system

    SciTech Connect

    Sposi, N.M.; Bottero, L.; Testa, U.; Peschle, C.; Russo, G.

    1989-05-01

    The authors have analyzed the RNA expression of three protein kinase C (PKC) genes in (/alpha/, /beta/, and /gamma/) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)/sup +/ RNA indicates that expression of PKC /alpha/ and /beta/ genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage,and that the PKC /gamma/ gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)/sup +/ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenic patterns were similar to those observed for human brain. Furthermore, they observed that the expression of PKC /gamma/ is induced in the peri- and postnatal phases. These results suggest that expression of PKC /alpha/, /beta/, and /gamma/ genes possibly mediates the development of central neuronal functions, and expression of PKC /gamma/ in particular may be involved in the development of peri- and postnatal functions.

  3. Early Days of Pressure Denaturation Studies of Proteins.

    PubMed

    Suzuki, Keizo

    2015-01-01

    The denaturation of protein by pressure has been generally well known since the findings of the perfect coagulation of egg white by a pressure of 7,000 atm within 30 min by Bridgman (J Biol Chem 19:511-512, 1914), and Kiyama and Yanagimoto (Rev Phys Chem Jpn 21:41-43, 1951) confirmed that the coagulation occurs above 3,880 kg cm(-2). Grant et al. (Science 94:616, 1941) and Suzuki and Kitamura (Abstracts of 30th annual meeting of Japanese Biochemical Society, 1957) found that SH groups are detected at the compressed sample of ovalbumin. On the other hand, Johnson and Campbell (J Cell Comp Physiol 26:43-49, 1945), Tongur (Kolloid Zhur 11:274-279, 1949; Biokhimiya 17:495-503, 1952) and Suzuki et al. (Mem Res Inst Sci Eng Ritsumeikan Univ 3:1-4, 1958) reported that the thermal denaturation of proteins is retarded in a few examples by the low pressure of about 1,000 atm. Before 1960, the studies of denaturation under high pressure were, however, rare and almost qualitative compared with those by heat, acid, urea and so on, so that there was no theory for the influence of hydrostatic pressure on the mechanism of denaturation. Here I review how I started experiments and analysis on pressure denaturation of proteins in early days of 1950s and 1960s in my laboratory and others.

  4. Early Fluid and Protein Shifts in Men During Water Immersion

    NASA Technical Reports Server (NTRS)

    Hinghofer-Szalkay, H.; Harrison, M. H.; Greenleaf, J. E.

    1987-01-01

    High precision blood and plasma densitometry was used to measure transvascular fluid shifts during water immersion to the neck. Six men (28-49 years) undertook 30 min of standing immersion in water at 35.0 +/- 0.2 C; immersion was preceded by 30 min control standing in air at 28 +/- 1 C. Blood was sampled from an antecubital catheter for determination of Blood Density (BD), Plasma Density (PD), Haematocrit (Ht), total Plasma Protein Concentration (PPC), and Plasma Albumin Concentration (PAC). Compared to control, significant decreases (p less than 0.01) in all these measures were observed after 20 min immersion. At 30 min, plasma volume had increased by 11.0 +/- 2.8%; the average density of the fluid shifted from extravascular fluid into the vascular compartment was 1006.3 g/l; albumin moved with the fluid and its albumin concentration was about one-third of the plasma protein concentration during early immersion. These calculations are based on the assumption that the F-cell ratio remained unchanged. No changes in erythrocyte water content during immersion were found. Thus, immersion-induced haemodilution is probably accompanied by protein (mainly albumin) augmentation which accompanies the intra-vascular fluid shift.

  5. Enhancement of G Protein-Coupled Receptor Surface Expression

    PubMed Central

    Dunham, Jill H.; Hall, Randy A.

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

  6. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    USDA-ARS?s Scientific Manuscript database

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  7. Atrial expression of the CCN1 and CCN2 proteins in chronic heart failure.

    PubMed

    Bonda, Tomasz A; Kamiński, Karol A; Dziemidowicz, Magdalena; Litvinovich, Sergey; Kożuch, Marcin; Hirnle, Tomasz; Dmitruk, Iwona; Chyczewski, Lech; Winnicka, Maria M

    2012-04-24

    Previous studies have reported the upregulation of CCN proteins early after acute heart injury. The aim of the present work was to evaluate the expression of the CCN1 and CCN2 proteins and their regulation by angiotensin II in the atrial myocardium of a chronically failing heart. Male adult mice were subjected to ligation of the left coronary artery to produce myocardial infarction (the MI group), and 16 of them were treated for 12 weeks with the AT1 receptor antagonist telmisartan (the MI-Tel group). Sham-operated mice served as controls. The expression of proteins was evaluated by immunohistochemistry 12 weeks after the operation. In shamoperated mice, stainings for CCN1 and CCN2 proteins were positive within atrial cardiomyocytes. CCN1-positive reaction revealed diffused cytoplasmic localization, while CCN2 was present mainly within the perinuclear cytoplasm. CCN1 was upregulated in the MI group, while CCN2 remained at basal level. Telmisartan prevented the upregulation of CCN1 and decreased CCN2 level. We compared the experimental data with the expression of CCN1 and CCN2 proteins in human right atrial appendages. We found an inverse, but not significant, relation between the level of either protein and the left ventricular ejection fraction. This suggests a similar atrial regulation of CCN1 and CCN2 expression also in humans. We conclude that in the murine atria, CCN1 and CCN2 proteins are expressed constitutively. In chronic heart failure, CCN proteins tend to be upregulated, which may be related to the action of angiotensin II.

  8. Expression of an early Epstein-Barr virus antigen (EA-D) in E. coli. Brief report.

    PubMed

    Roeckel, D; Boos, H; Mueller-Lantzsch, N

    1987-01-01

    The 1.34 kb BcII-BgIII-fragment of the BamHI-M region of Epstein-Barr virus genome, comprising the complete BMRF1 open reading frame, was cloned into the tryptophan regulated E. coli expression vector pATH1. The resulting fusion protein, having a molecular weight of 80 kd, is recognized not only by anti-early antigen (EA)-positive human sera but also by the monoclonal antibody R3 directed against the diffuse component of EA (EA-D). A possible use for this fusion protein as an indicator protein in diagnosis of IgA antibodies against EA-D is presented.

  9. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  10. Argonaute Family Protein Expression in Normal Tissue and Cancer Entities

    PubMed Central

    Bruckmann, Astrid; Hauptmann, Judith; Deutzmann, Rainer; Meister, Gunter; Bosserhoff, Anja Katrin

    2016-01-01

    The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease. PMID:27518285

  11. Expression analysis of BRUCE protein in esophageal squamous cell carcinoma.

    PubMed

    Salehi, Somayeh; Jafarian, Amir Hossein; Forghanifard, Mohammad Mahdi

    2016-10-01

    Apoptosis is a form of cell death in response to diverse stressful physiological or pathological stimuli. One of the most important gene families involved in apoptosis is inhibitors of apoptosis. As a member of inhibitors of apoptosis, BRUCE can suppress apoptosis and promote cell division. Because esophageal squamous cell carcinoma (ESCC) cells, as well as other cancer cells, are immortal, our aim in this study was to analyze BRUCE protein expression in ESCC and evaluate its correlation with tumoral clinicopathologic features. Fifty ESCC specimens were examined for BRUCE protein expression using immunohistochemistry. A defined scoring method was applied. BRUCE protein was detected in 82% of tumors. Tumor progression stage and invasion depth correlated significantly with BRUCE protein expression (P=.019 and .005, respectively). Furthermore, association of BRUCE expression with tumor location was near significant (P=.058). The correlation of BRUCE overexpression in ESCC and disease aggressiveness may confirm the importance of BRUCE in ESCC progression and invasiveness. Therefore, BRUCE protein may be a molecular marker for aggressive ESCC and, thus, a potential therapeutic target to inhibit tumor cell progression and invasion. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

    PubMed Central

    Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

  13. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  14. AB223. Expression of tight junction proteins in rat vagina

    PubMed Central

    Oh, Kyung Jin; Lee, Hyun-Suk; Chung, Ho Suck; Ahn, Kyu Youn; Park, Kwangsung

    2014-01-01

    Aim Tight junction plays a role in apical cell-to-cell adhesion and epithelial polarity. In this study, we investigated the expression of tight junction proteins, such as Claudin-1, zonula occludens (ZO)-1, junction adhesion molecule (JAM)-A, and occludin in rat vagina. Methods Female Sprague-dawley rats (230-240 g, n=20) were divided into two groups: control (n=10) and bilateral ovariectomy (n=10). The expression and cellular localization of claudin-1, ZO-1, JAM-A, and occludin were determined in each group by immunohistochemistry and Western blot. Results Immunolabeling of ZO-1 was mainly expressed in the capillaries and venules of the vagina. Claudin-1, JAM-A, and occludin were expressed in the epithelium of the vagina. The immunoreactivity and protein expression of claudin-1 was significantly decreased in the ovariectomy group compared with the control group. Conclusions Our results suggest that tight junction proteins may have an important role in the vagina. Further studies are needed to clarify the role of each tight junction protein on vaginal lubrication.

  15. Functional interaction between co-expressed MAGE-A proteins

    PubMed Central

    Laiseca, Julieta E.; Ladelfa, María F.; Cotignola, Javier; Peche, Leticia Y.; Pascucci, Franco A.; Castaño, Bryan A.; Galigniana, Mario D.; Schneider, Claudio

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior. PMID:28542476

  16. Functional interaction between co-expressed MAGE-A proteins.

    PubMed

    Laiseca, Julieta E; Ladelfa, María F; Cotignola, Javier; Peche, Leticia Y; Pascucci, Franco A; Castaño, Bryan A; Galigniana, Mario D; Schneider, Claudio; Monte, Martin

    2017-01-01

    MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior.

  17. Protein synthesis rate is the predominant regulator of protein expression during differentiation

    PubMed Central

    Kristensen, Anders R; Gsponer, Joerg; Foster, Leonard J

    2013-01-01

    External perturbations, by forcing cells to adapt to a new environment, often elicit large-scale changes in gene expression resulting in an altered proteome that improves the cell's fitness in the new conditions. Steady-state levels of a proteome depend on transcription, the levels of transcripts, translation and protein degradation but system-level contribution that each of these processes make to the final protein expression change has yet to be explored. We therefore applied a systems biology approach to characterize the regulation of protein expression during cellular differentiation using quantitative proteomics. As a general rule, it seems that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins stays constant. In these data, we also observe that the proteins in defined sub-structures of larger protein complexes tend to have highly correlated synthesis and degradation rates but that this does not necessarily extend to the holo-complex. Finally, we provide strong evidence that the generally poor correlation observed between transcript and protein levels can fully be explained once the protein synthesis and degradation rates are taken into account. PMID:24045637

  18. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  19. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  20. Nanog suppresses the expression of vasa by directly regulating nlk1 in the early zebrafish embryo.

    PubMed

    Liu, Yanhua; Xue, Weiwei; Zhu, Lin; Ye, Ding; Zhu, Xiaoqin; Wang, Huannan; Sun, Yonghua; Deng, Fengjiao

    2017-07-28

    Nanog is a homeodomain transcription factor that is essential for maintenance of pluripotency and self-renewal of embryonic stem cells (ESCs). In the present study, we demonstrate that zebrafish Nanog (zNanog) directly binds to the promoter region of zebrafish nlk1 (znlk1) by ChIP-Seq analysis and that it up-regulates the expression of znlk1 in fibroblast-like embryonic cells of Danio rerio (ZEM-2S cells) and in zebrafish embryos at 30% epiboly both at the mRNA and protein levels. In addition, compared with control (MO-C) embryos at 30% epiboly, the mRNA and protein expression of vasa and the numbers of vasa-positive cells were increased in embryos injected with zNanog morpholino (MO-zNanog). Further, injection of znlk1 mRNA into zNanog-depleted embryos restored the expression of vasa and the number of vasa-positive cells. These data indicated that zNanog up-regulates the expression of znlk1 through directly binding to the znlk1 promoter, thereby suppressing the expression of vasa. Vasa is a marker gene for PGCs. Our results suggest that zNanog plays a role in restraint of PGC cell number through regulating the expression of znlk1 in the early embryonic development. The current results provide fundamental information to support further investigation regarding the regulatory mechanism of zNanog during the development of PGCs. Copyright © 2017. Published by Elsevier B.V.

  1. Gene Expression Profiles Deciphering Leaf Senescence Variation between Early- and Late-Senescence Cotton Lines

    PubMed Central

    Kong, Xiangqiang; Luo, Zhen; Dong, Hezhong; Eneji, A. Egrinya; Li, Weijiang; Lu, Hequan

    2013-01-01

    Leaf senescence varies greatly among genotypes of cotton (Gossypium hirsutium L), possibly due to the different expression of senescence-related genes. To determine genes involved in leaf senescence, we performed genome-wide transcriptional profiling of the main-stem leaves of an early- (K1) and a late-senescence (K2) cotton line at 110 day after planting (DAP) using the Solexa technology. The profiling analysis indicated that 1132 genes were up-regulated and 455 genes down-regulated in K1 compared with K2 at 110 DAP. The Solexa data were highly consistent with, and thus were validated by those from real-time quantitative PCR (RT-PCR). Most of the genes related to photosynthesis, anabolism of carbohydrates