Science.gov

Sample records for early protein expression

  1. 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.

  2. 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

  3. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis.

    PubMed Central

    Goulding, M D; Chalepakis, G; Deutsch, U; Erselius, J R; Gruss, P

    1991-01-01

    We describe the isolation and characterization of Pax-3, a novel murine paired box gene expressed exclusively during embryogenesis. Pax-3 encodes a 479 amino acid protein with an Mr of 56 kd containing both a paired domain and a paired-type homeodomain. The Pax-3 protein is a DNA binding protein that specifically recognizes the e5 sequence present upstream of the Drosophila even-skipped gene. Pax-3 transcripts are first detected in 8.5 day mouse embryos where they are restricted to the dorsal part of the neuroepithelium and to the adjacent segmented dermomyotome. During early neurogenesis, Pax-3 expression is limited to mitotic cells in the ventricular zone of the developing spinal cord and to distinct regions in the hindbrain, midbrain and diencephalon. In 10-12 day embryos, expression of Pax-3 is also seen in neural crest cells of the developing spinal ganglia, the craniofacial mesectoderm and in limb mesenchyme of 10 and 11 day embryos. Images PMID:2022185

  4. Alteration of podocyte protein expression and localization in the early stage of various hemodynamic conditions.

    PubMed

    Li, Kai; Wang, Juan; Yin, Xiaohui; Zhai, Xiaoyue; Li, Zilong

    2013-01-01

    Given that podocalyxin (PCX) and nestin play important roles in podocyte morphogenesis and the maintenance of structural integrity, we examined whether the expression and localization of these two podocyte proteins were influenced in the early stage of various hemodynamic conditions. Mice kidney tissues were prepared by in vivo cryotechnique (IVCT). The distribution of glomeruli and podocyte proteins was visualized with DAB staining, confocal laser scanning microscopy and immunoelectron microscopy. The mRNA levels were examined by real-time quantitative PCR. The results showed the following: Under the normal condition, PCX stained intensely along glomerular epithelial cells, whereas nestin was clearly staining in the endothelial cells and appeared only weakly in the podocytes. Under the acute hypertensive and cardiac arrest conditions, PCX and nestin staining was not clear, with a disarranged distribution, but the colocalization of PCX and nestin was apparent under this condition. In addition, under the acute hypertensive and cardiac arrest conditions, the mRNA levels of PCX and nestin were significantly decreased. Collectively, the abnormal redistribution and decreased mRNA expressions of PCX and nestin are important molecular events at the early stage of podocyte injury during hemodynamic disorders. IVCT may have more advantages for morphological analysis when researching renal diseases. PMID:23502465

  5. 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. PMID:25313365

  6. 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…

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

    PubMed

    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 AND 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

  8. 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

  9. Stress protein expression in early phase spinal cord ischemia/reperfusion injury.

    PubMed

    Zhang, Shanyong; Wu, Dankai; Wang, Jincheng; Wang, Yongming; Wang, Guoxiang; Yang, Maoguang; Yang, Xiaoyu

    2013-08-25

    Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins (n > 2) in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initially improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradually decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6-12 hours, showing a characterization of induction-inhibition-induction. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons. PMID:25206532

  10. The expression of N-terminal deletion DNA pilot proteins inhibits the early stages of phiX174 replication.

    PubMed

    Ruboyianes, Mark V; Chen, Min; Dubrava, Mathew S; Cherwa, James E; Fane, Bentley A

    2009-10-01

    The phiX174 DNA pilot protein H contains four predicted C-terminal coiled-coil domains. The region of the gene encoding these structures was cloned, expressed in vivo, and found to strongly inhibit wild-type replication. DNA and protein synthesis was investigated in the absence of de novo H protein synthesis and in wild-type-infected cells expressing the inhibitory proteins (DeltaH). The expression of the DeltaH proteins interfered with early stages of DNA replication, which did not require de novo H protein synthesis, suggesting that the inhibitory proteins interfere with the wild-type H protein that enters the cell with the penetrating DNA. As transcription and protein synthesis are dependent on DNA replication in positive single-stranded DNA life cycles, viral protein synthesis was also reduced. However, unlike DNA synthesis, efficient viral protein synthesis required de novo H protein synthesis, a novel function for this protein. A single amino acid change in the C terminus of protein H was both necessary and sufficient to confer resistance to the inhibitory DeltaH proteins, restoring both DNA and protein synthesis to wild-type levels. DeltaH proteins derived from the resistant mutant did not inhibit wild-type or resistant mutant replication. The inhibitory effects of the DeltaH proteins were lessened by the coexpression of the internal scaffolding protein, which may suppress H-H protein interactions. While coexpression relieved the block in DNA biosynthesis, viral protein synthesis remained suppressed. These data indicate that protein H's role in DNA replication and stimulating viral protein synthesis can be uncoupled. PMID:19640994

  11. 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...

  12. 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

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

    PubMed Central

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

    2013-01-01

    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. PMID:23838326

  14. Histone Deacetylase Inhibitors Activate Tristetraprolin Expression through Induction of Early Growth Response Protein 1 (EGR1) in Colorectal Cancer Cells

    PubMed Central

    Sobolewski, Cyril; Sanduja, Sandhya; Blanco, Fernando F.; Hu, Liangyan; Dixon, Dan A.

    2015-01-01

    The RNA-binding protein tristetraprolin (TTP) promotes rapid decay of mRNAs bearing 3' UTR AU-rich elements (ARE). In many cancer types, loss of TTP expression is observed allowing for stabilization of ARE-mRNAs and their pathologic overexpression. Here we demonstrate that histone deacetylase (HDAC) inhibitors (Trichostatin A, SAHA and sodium butyrate) promote TTP expression in colorectal cancer cells (HCA-7, HCT-116, Moser and SW480 cells) and cervix carcinoma cells (HeLa). We found that HDAC inhibitors-induced TTP expression, promote the decay of COX-2 mRNA, and inhibit cancer cell proliferation. HDAC inhibitors were found to promote TTP transcription through activation of the transcription factor Early Growth Response protein 1 (EGR1). Altogether, our findings indicate that loss of TTP in tumors occurs through silencing of EGR1 and suggests a therapeutic approach to rescue TTP expression in colorectal cancer. PMID:26343742

  15. 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. PMID:27122561

  16. Expression of early growth response protein 1 in vasopressin neurones of the rat anterior olfactory nucleus following social odour exposure

    PubMed Central

    Wacker, Douglas W; Tobin, Vicky A; Noack, Julia; Bishop, Valerie R; Duszkiewicz, Adrian J; Engelmann, Mario; Meddle, Simone L; Ludwig, Mike

    2010-01-01

    The anterior olfactory nucleus (AON), a component of the main olfactory system, is a cortical region that processes olfactory information and acts as a relay between the main olfactory bulbs and higher brain regions such as the piriform cortex. Utilizing a transgenic rat in which an enhanced green fluorescent protein reporter gene is expressed in vasopressin neurones (eGFP-vasopressin), we have discovered a population of vasopressin neurones in the AON. These vasopressin neurones co-express vasopressin V1 receptors. They also co-express GABA and calbinin-D28k indicating that they are neurochemically different from the newly described vasopressin neurons in the main olfactory bulb. We utilized the immediate early gene product, early growth response protein 1 (Egr-1), to examine the functional role of these vasopressin neurons in processing social and non-social odours in the AON. Exposure of adult rats to a conspecific juvenile or a heterospecific predator odour leads to increases in Egr-1 expression in the AON in a subregion specific manner. However, only exposure to a juvenile increases Egr-1 expression in AON vasopressin neurons. These data suggest that vasopressin neurones in the AON may be selectively involved in the coding of social odour information. PMID:20921194

  17. The ICP0 protein of equine herpesvirus 1 is an early protein that independently transactivates expression of all classes of viral promoters.

    PubMed Central

    Bowles, D E; Holden, V R; Zhao, Y; O'Callaghan, D J

    1997-01-01

    To assess the role of the equine herpesvirus type 1 (EHV-1) ICP0 protein (EICP0) in gene regulation, a variety of molecular studies on the EICP0 gene and gene products of both the attenuated cell culture-adapted Kentucky A (KyA) strain and the Ab4p strain were conducted. These investigations revealed that (i) the ICP0 open reading frame (ORF) of the KyA virus strain is 1,257 bp in size and would encode a protein of 419 amino acids, and in comparison to the ICP0 gene (ORF63) of the Ab4p strain of 1,596 bp (E. A. Telford, M. S. Watson, K. McBride, and A. J. Davison, Virology 189:304-316, 1992), it has an internal in-frame deletion of 339 bp; (ii) one early transcript of 1.4 kb predicted to encode the EICP0 protein and a late transcript of 1.8 kb are detected in Northern blot analyses using probes containing the EICP0 ORF; (iii) the KyA EICP0 protein (50 kDa) and the Ab4p EICP0 protein (80 kDa) are expressed as several species of early proteins that are first detected at 3 to 4 h postinfection by Western blot analyses of infected-cell polypeptides, using an antiserum generated to a TrpE fusion protein that harbors amino acids 46 to 153 of the EICP0 protein; and (iv) the EICP0 protein of both EHV-1 strains is a potent transactivator of EHV-1 genes. Transient expression assays using a simian virus 40 expression construct of the EICP0 protein of the KyA strain showed that the EICP0 protein independently transactivated chloramphenicol acetyltransferase reporter constructs under the control of the immediate-early promoter (3.9-fold), the early thymidine kinase promoter (95-fold), the late (gamma1) IR5 promoter (85-fold), and the late (gamma2) glycoprotein K promoter (21-fold). The finding that the EICP0 protein of the KyA virus can function as an activator of gene expression indicates that amino acids corresponding to residues 319 to 431 of the Ab4p EICP0 protein are not essential for EICP0 transactivation of EHV-1 promoters. PMID:9188552

  18. Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis

    PubMed Central

    Malminen, Maria; Peltonen, Sirkku; Koivunen, Jussi; Peltonen, Juha

    2002-01-01

    Background NF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes. Methods The expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization. Results The expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins. Conclusions These results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes. PMID:12199909

  19. 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. PMID:25756589

  20. 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

    2009-01-01

    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. PMID:18706452

  1. Regulation of the expression of the sea urchin mitochondrial D-loop binding protein during early development.

    PubMed

    Musicco, C; Roberti, M; Polosa, P L; Milella, F; Sagliano, A; Gadaleta, M N; Cantatore, P

    2000-10-22

    The Paracentrotus lividus mitochondrial D-loop binding protein (mtDBP) is a DNA-binding protein which is involved in the regulation of sea urchin mtDNA transcription. Immunoblots of Heparin Sepharose-bound proteins at selected early developmental stages, as well as electrophoretic mobility shift assay, show that mtDBP is present in the egg at a concentration of about 1 x 10(6) molecules/egg. Its level increases after fertilization of about twofold, remaining substantially unchanged between 16-h blastula stage and early pluteus stage and declines thereafter. The content of mtDBP mRNA, determined by RNase protection experiments, increases about sevenfold at the 16-h blastula stage compared to the egg. A considerable decrease occurs at the 40-h pluteus stage, which precedes that of the protein. These results suggest that the expression of mtDBP is regulated at transcriptional level up to blastula stage, while other factors, in addition to the level of the RNA, may control the content of this protein in the following stages of embryogenesis. PMID:11032721

  2. Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

    PubMed Central

    Nourizadeh-Lillabadi, Rasoul; Seilø Torgersen, Jacob; Vestrheim, Olav; König, Melanie; Aleström, Peter; Syed, Mohasina

    2010-01-01

    Background The Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. Methodology/Principal Findings The zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO) knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf) as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. Conclusions/Significance The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development. PMID:21042590

  3. 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.

  4. Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

    PubMed Central

    Wheaton, Keith; Riabowol, Karl

    2004-01-01

    Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKCδ) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKCδ activity as cells age, production of the PKCδ catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKCδ in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKCδ inhibitor rottlerin and ectopic expression of a dominant negative form of PKCδ independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKCδ activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKCδ contributes to the senescent phenotype. PMID:15282327

  5. CMF608-a novel mechanical strain-induced bone-specific protein expressed in early osteochondroprogenitor cells.

    PubMed

    Segev, Orit; Samach, Aviva; Faerman, Alexander; Kalinski, Hagar; Beiman, Merav; Gelfand, Anna; Turam, Hagit; Boguslavsky, Shlomit; Moshayov, Anat; Gottlieb, Helen; Kazanov, Eugeniy; Nevo, Zvi; Robinson, Dror; Skaliter, Rami; Einat, Paz; Binderman, Itzhak; Feinstein, Elena

    2004-02-01

    Microarray gene expression analysis was utilized to identify genes upregulated in primary rat calvaria cultures in response to mechanical force. One of the identified genes designated CMF608 appeared to be novel. The corresponding full-length cDNA was cloned and characterized in more details. It encodes a putative 2597 amino acid protein containing N-terminal signal peptide, six leucine-rich repeats (LRRs), and 12 immunoglobulin-like repeats, 10 of which are clustered within the C-terminus. Expression of CMF608 is bone-specific and the main type of CMF608-positive cells is mesenchymal osteochondroprogenitors with fibroblast-like morphology. These cells reside in the perichondral fibrous ring of La Croix, periosteum, endosteum of normal bone as well as in the activated periosteum and early fibrous callus generated postfracture. Expression of CMF608 is notably absent from the regions of endochondral ossification. Mature bone cell types do not produce CMF608 with the exception of chondrocytes of the tangential layer of the articular cartilage, which are thought to be under constant mechanical loading. Ectopic expression of CMF608 in HEK293T cells shows that the protein is subjected to post-translational processing and its N-terminal approximately 90 kDa polypeptide can be found in the conditioned medium. Ectopic expression of either the full-length cDNA of CMF608 or of its N-terminal region in CMF608-negative ROS17/2.8 rat osteosarcoma cells results in transfected clones displaying increased proliferation rate and the characteristics of less-differentiated osteoblasts compared to the control cells. Our data indicate that CMF608 is a unique marker of early osteochondroprogenitor cells. We propose that it could be functionally involved in maintenance of the osteochondroprogenitor cells pool and its down-regulation precedes terminal differentiation. PMID:14962803

  6. Expression of mitogen-activated protein kinase phosphatase-1 in the early phases of human epithelial carcinogenesis.

    PubMed Central

    Loda, M.; Capodieci, P.; Mishra, R.; Yao, H.; Corless, C.; Grigioni, W.; Wang, Y.; Magi-Galluzzi, C.; Stork, P. J.

    1996-01-01

    Many mitogens and human oncogenes activate extracellular regulated kinases (ERKs), which in turn convey proliferation signals. ERKs or mitogen-activated protein (MAP) kinases are inactivated in vitro by MAP kinase phosphatases (MKPs). The gene encoding one of these MKPs, MKP-1, is a serum-inducible gene and is transcriptionally activated by mitogenic signals in cultured cells. As MKP-1 has been shown to block DNA synthesis by inhibiting ERKs when expressed at elevated levels in cultured cells, it has been suggested that it may act as a tumor suppressor. MKP-1 mRNA and MAP kinase (ERK-1 and -2) protein expression was assessed in 164 human epithelial tumors of diverse tissue origin by in situ hybridization and immunohistochemistry. MKP-1 was overexpressed in the early phases of prostate, colon, and bladder carcinogenesis, with progressive loss of expression with higher histological grade and in metastases. In contrast, breast carcinomas showed significant MKP-1 expression even when poorly differentiated or in late stages of the disease. MKP-1, ERK-1, and ERK-2 were co-expressed in most tumors examined. In a subset of 15 tumors, ERK-1 enzymatic activity as well as structural alterations that might be responsible for loss of function of MKP-1 during tumor progression, were examined. ERK-1 enzymatic activity was found to be elevated despite MKP-1 overexpression. No loss of 5q35-ter (containing the MKP-1 locus) was detected by polymerase chain reaction in metastases compared with primary tumors. Finally, no mutations were found in the catalytic domain of MKP-1. These data indicate that MKP-1 is an early marker for a wide range of human epithelial tumors and suggest that MKP-1 does not behave as a tumor suppressor in epithelial tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8909245

  7. 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

  8. Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

    PubMed Central

    Whalen, S G; Marcellus, R C; Whalen, A; Ahn, N G; Ricciardi, R P; Branton, P E

    1997-01-01

    A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter. PMID:9094626

  9. 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}.

  10. 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 PMID:26020220

  11. Environmental manipulations early in development alter seizure activity, Ih and HCN1 protein expression later in life.

    PubMed

    Schridde, Ulrich; Strauss, Ulf; Bräuer, Anja U; van Luijtelaar, Gilles

    2006-06-01

    Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glaxo/Rijswijk; WAG/Rij) suggests that seizures are sensitive to environmental manipulations. Here, we show that manipulations of the early rearing environment (neonatal handling, maternal deprivation) of WAG/Rij rats leads to a pronounced decrease in seizure activity later in life. Recent observations link seizure activity in WAG/Rij rats to the hyperpolarization-activated cation current (Ih) in the somatosensory cortex, the site of seizure generation. Therefore, we investigated whether the alterations in seizure activity between rats reared differently might be correlated with changes in Ih and its channel subunits hyperpolarization-activated cation channel HCN1, 2 and 4. Whole-cell recordings from layer 5 pyramidal neurons, in situ hybridization and Western blot of the somatosensory cortex revealed an increase in Ih and HCN1 in neonatal handled and maternal deprived, compared to control rats. The increase was specific to HCN1 protein expression and did not involve HCN2/4 protein expression, or mRNA expression of any of the subunits (HCN1, 2, 4). Our findings provide the first evidence that relatively mild changes in the neonatal environment have a long-term impact of absence seizures, Ih and HCN1, and suggest that an increase of Ih and HCN1 is associated with absence seizure reduction. Our findings shed new light on the role of Ih and HCN in brain functioning and development and demonstrate that genetically determined absence seizures are quite sensitive for early interventions. PMID:16820024

  12. RING1 proteins contribute to early proximal-distal specification of the forelimb bud by restricting Meis2 expression.

    PubMed

    Yakushiji-Kaminatsui, Nayuta; Kondo, Takashi; Endo, Takaho A; Koseki, Yoko; Kondo, Kaori; Ohara, Osamu; Vidal, Miguel; Koseki, Haruhiko

    2016-01-15

    Polycomb group (PcG) proteins play a pivotal role in silencing developmental genes and help to maintain various stem and precursor cells and regulate their differentiation. PcG factors also regulate dynamic and complex regional specification, particularly in mammals, but this activity is mechanistically not well understood. In this study, we focused on proximal-distal (PD) patterning of the mouse forelimb bud to elucidate how PcG factors contribute to a regional specification process that depends on developmental signals. Depletion of the RING1 proteins RING1A (RING1) and RING1B (RNF2), which are essential components of Polycomb repressive complex 1 (PRC1), led to severe defects in forelimb formation along the PD axis. We show that preferential defects in early distal specification in Ring1A/B-deficient forelimb buds accompany failures in the repression of proximal signal circuitry bound by RING1B, including Meis1/2, and the activation of distal signal circuitry in the prospective distal region. Additional deletion of Meis2 induced partial restoration of the distal gene expression and limb formation seen in the Ring1A/B-deficient mice, suggesting a crucial role for RING1-dependent repression of Meis2 and likely also Meis1 for distal specification. We suggest that the RING1-MEIS1/2 axis is regulated by early PD signals and contributes to the initiation or maintenance of the distal signal circuitry. PMID:26674308

  13. 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

  14. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  15. Expression of proteolipid protein gene in spinal cord stem cells and early oligodendrocyte progenitor cells is dispensable for normal cell migration and myelination.

    PubMed

    Harlow, Danielle E; Saul, Katherine E; Culp, Cecilia M; Vesely, Elisa M; Macklin, Wendy B

    2014-01-22

    Plp1 gene expression occurs very early in development, well before the onset of myelination, creating a conundrum with regard to the function of myelin proteolipid protein (PLP), one of the major proteins in compact myelin. Using PLP-EGFP mice to investigate Plp1 promoter activity, we found that, at very early time points, PLP-EGFP was expressed in Sox2+ undifferentiated precursors in the spinal cord ventricular zone (VZ), as well as in the progenitors of both neuronal and glial lineages. As development progressed, most PLP-EGFP-expressing cells gave rise to oligodendrocyte progenitor cells (OPCs). The expression of PLP-EGFP in the spinal cord was quite dynamic during development. PLP-EGFP was highly expressed as cells delaminated from the VZ. Expression was downregulated as cells moved laterally through the cord, and then robustly upregulated as OPCs differentiated into mature myelinating oligodendrocytes. The presence of PLP-EGFP expression in OPCs raises the question of its role in this migratory population. We crossed PLP-EGFP reporter mice into a Plp1-null background to investigate the role of PLP in early OPC development. In the absence of PLP, normal numbers of OPCs were generated and their distribution throughout the spinal cord was unaffected. However, the orientation and length of OPC processes during migration was abnormal in Plp1-null mice, suggesting that PLP plays a role either in the structural integrity of OPC processes or in their response to extracellular cues that orient process outgrowth. PMID:24453324

  16. Okadaic acid mimics multiple changes in early protein phosphorylation and gene expression induced by tumor necrosis factor or interleukin-1.

    PubMed

    Guy, G R; Cao, X; Chua, S P; Tan, Y H

    1992-01-25

    Okadaic acid, a phosphatase inhibitor from a marine organism, mimics tumor necrosis factor/interleukin-1 (TNF/IL-1) in inducing changes in early cellular protein phosphorylation. A total of approximately 116 proteins exhibit significant and concordant changes in phosphorylation or dephosphorylation within 15 min in human fibroblasts activated by either okadaic acid, TNF, or IL-1. The fidelity of this mimicry by okadaic acid extends to the phosphorylation of the 27 hsp complex, stathmin, eIF-4E, myosin light chain, nucleolin, epidermal growth factor receptor, and other cdc2-kinase substrates (c-abl, RB, and p53). The okadaic acid-induced pattern of protein phosphorylation is distinct from that observed in cells treated with phorbol 12-myristate 13-acetate or with ligands like epidermal growth factor, cyclic AMP agonists, bradykinin, or interferons. Like TNF, okadaic acid also induces the transcription of immediate early response genes like c-jun and Egr-1 as well as the interleukin-6 genes. The overall early effects of okadaic acid uniquely parallel those of TNF/IL-1 and not those of other cytokines or ligands. Regulation of protein phosphatase inhibition is discussed as a mechanism for TNF/IL-1 signal transduction. PMID:1370482

  17. Increased expression of multidrug resistance related proteins Pgp, MRP1, and LRP/MVP occurs early in colorectal carcinogenesis.

    PubMed Central

    Meijer, G A; Schroeijers, A B; Flens, M J; Meuwissen, S G; van der Valk, P; Baak, J P; Scheper, R J

    1999-01-01

    AIM: To analyse the expression of multidrug resistance (MDR) related proteins at different steps in colorectal carcinogenesis. METHODS: The presence of three MDR related proteins (Pgp, MRP1, and LRP/MVP) was studied by means of immunohistochemistry in normal, adenomatous, and malignant colorectal epithelium. Formaldehyde fixed, paraffin embedded tissue sections of 17 samples of colorectal tissue were used (normal mucosa, n = 4; adjacent mucosa, n = 5; adenoma, n = 5; carcinoma, n = 3). RESULTS: For all three proteins, expression was found in the surface epithelium and the upper parts of the crypts in normal colon. In the adenomas, staining was seen along the complete length of the crypts. In the carcinomas analysed, all epithelium showed positive staining. Mucosa adjacent to either carcinoma or adenoma showed staining patterns mostly resembling those of normal mucosa, but sometimes some extension of staining was seen along the crypt. CONCLUSIONS: These proteins already show increased expression in the adenoma stage. In the absence of adequate mucin production in adenomas, MDR related proteins could be an important factor in protecting the epithelium against further environmentally induced genetic damage. This could be one of the reasons why only about 5% of colorectal adenomas will actually progress to carcinomas. Images PMID:10562814

  18. Time Course of Immediate Early Gene Protein Expression in the Spinal Cord following Conditioning Stimulation of the Sciatic Nerve in Rats

    PubMed Central

    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. PMID:25860146

  19. The HOXC13-controlled expression of early hair keratin genes in the human hair follicle does not involve TALE proteins MEIS and PREP as cofactors.

    PubMed

    Jave-Suárez, Luis Felipe; Schweizer, Jürgen

    2006-02-01

    We previously showed that the homeodomain protein HOXC13 is involved in the expression control of the early human hair keratin genes hHa5 and hHa2, which contain specific HOXC13 binding sites in their proximal promoters. Hox specificity is generally thought to be enhanced by the interaction with members of the TALE superclass of homeodomain proteins Pbx, Meis, and Prep. Using reverse transcription PCR with total human hair follicle RNA, we demonstrated transcripts of the major TALE proteins PBX1-4, MEIS1, 2 and PREP1, 2 in the human hair follicle. In view of the presence of MEIS/PREP responsive elements in close vicinity to the HOXC13 binding sites of the hHa5 and hHa2 promoters, we determined the expression sites of these TALE proteins in the human hair follicle. We found that MEIS1, MEIS2, PREP1 and PREP2 were differentially expressed in the three layers of the inner root sheath. In addition, MEIS2 and PREP1 exhibited expression in the mid-to upper hair cortex, with PREP1 being also expressed in the dermal papilla and the connective tissue sheath of the hair follicle. In virtually all cases, the expression of these TALE proteins was exclusively cytoplasmic. Considering that in contrast, HOXC13 is expressed in the nuclei of matrix, precortex and lower cuticle cells of the hair follicle, our data suggest that despite the presence of MEIS/PREP binding sites in the hHa5 and hHa2 promoters, the HOXC13-controlled activation of these genes in the hair follicle does not seem to involve these TALE proteins as cofactors. PMID:16292560

  20. 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

    2016-02-25

    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. PMID:26923026

  1. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

    PubMed

    Alwine, J C

    1985-05-01

    The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed. PMID:2987671

  2. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

    PubMed Central

    Alwine, J C

    1985-01-01

    The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed. Images PMID:2987671

  3. Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells

    PubMed Central

    Neelam, Sudha; Brooks, Morgan M.

    2015-01-01

    Purpose The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. Methods HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression. Results SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed

  4. Clinical significance and expression of the PRSS3 and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 for the early detection of epithelial ovarian cancer.

    PubMed

    Azizmohammadi, Sima; Safari, Aghdas; Seifoleslami, Mehri; Rabati, Rahman Ghaffarzadegan; Mohammadi, Mohsen; Yahaghi, Hamid; Azizmohammadi, Susan

    2016-05-01

    In this study, we evaluate the clinical significance of the PRSS3 and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) in patients with epithelial ovarian cancer (EOC) by immunohistochemistry.In current study, all adjacent non-cancerous tissues showed absent or low expression of PRSS3. The expression of PRSS3 was significantly increased in the EOCs than adjacent non-cancerous tissues. Moreover, the expression of WAVE1 was significantly observed in all EOC tissues when compared with normal tissues. Furthermore, WAVE1 expression was absent in 35 (89.74 %) adjacent non-cancerous tissues.Our findings showed that high expression of PRSS3 was markedly linked to FIGO stage (P = 0.02), advanced grade (P = 0.017), and lymph node metastases (P = 0.001), but no relationship was determined with other clinicopathological parameters. Furthermore, high expression of WAVE1 was significantly correlated with FIGO stage (P = 0.001), grade of tumor (P = 0.011), and residual tumor size (P = 0.041), but no significant associations were found between WAVE1 expression and age, lymph node metastasis, and histological subtypes (all P > 0.05). In conclusion, our study showed that increased expression of PRSS3 and WAVE1 may be involved in development of EOC. PMID:26662304

  5. Oocyte-expressed yes-associated protein is a key activator of the early zygotic genome in mouse

    PubMed Central

    Yu, Chao; Ji, Shu-Yan; Dang, Yu-Jiao; Sha, Qian-Qian; Yuan, Yi-Feng; Zhou, Jian-Jie; Yan, Li-Ying; Qiao, Jie; Tang, Fuchou; Fan, Heng-Yu

    2016-01-01

    In early mammalian embryos, the genome is transcriptionally quiescent until the zygotic genome activation (ZGA) which occurs 2-3 days after fertilization. Despite a long-standing effort, maternal transcription factors regulating this crucial developmental event remain largely elusive. Here, using maternal and paternal mouse models of Yap1 deletion, we show that maternally accumulated yes-associated protein (YAP) in oocyte is essential for ZGA. Maternal Yap1-knockout embryos exhibit a prolonged two-cell stage and develop into the four-cell stage at a much slower pace than the wild-type controls. Transcriptome analyses identify YAP target genes in early blastomeres; two of which, Rpl13 and Rrm2, are required to mediate maternal YAP's effect in conferring developmental competence on preimplantation embryos. Furthermore, the physiological YAP activator, lysophosphatidic acid, can substantially improve early development of wild-type, but not maternal Yap1-knockout embryos in both oviduct and culture. These observations provide insights into the mechanisms of ZGA, and suggest potentials of YAP activators in improving the developmental competence of cultured embryos in assisted human reproduction and animal biotechnology. PMID:26902285

  6. Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle.

    PubMed

    Okumu, L A; Fair, T; Szekeres-Bartho, J; O'Doherty, A M; Crowe, M A; Roche, J F; Lonergan, P; Forde, N

    2011-07-27

    Progesterone-induced blocking factor (PIBF) and galectins modulate the maternal immune response during pregnancy. We hypothesized that the relative transcript abundance of the above genes would be different during the luteal phase/early pregnancy and would be affected by progesterone supplementation. To further test this, hypothesis protein expression analyses were carried out to evaluate the abundance and localization of LGALS9 and PIBF. Following estrus synchronization, heifers were inseminated (n = 140) or not (n = 70). Half the heifers in each status (cyclic or potentially pregnant) were randomly assigned to receive a progesterone-releasing intravaginal device (PRID) on day 3 after estrus, which elevated progesterone concentrations from day 3.5 to 8 (P < 0.05), resulting in four treatment groups: cyclic and pregnant heifers, each with normal and high progesterone. After confirmation of pregnancy status in inseminated animals, uterine tissue was collected on days 5, 7, 13, or 16 of the luteal phase of the cycle/pregnancy. Gene and protein expression was determined using Q-RT-PCR and IHC, respectively, on 5 heifers per treatment per time point (i.e., 80 in total). Progesterone concentrations did not affect expression of any of the genes (P > 0.05). LGALS9 and LGALS3BP were expressed at low levels in both cyclic and pregnant endometria until day 13. On day 16, expression increased only in the pregnant heifers (P < 0.0001). LGALS1 and LGALS3 decreased on day 7 (P < 0.0001) and remained low until day 16. Pregnancy had no effect on the expression of LGALS1, LGALS3, and PIBF. Additionally, LGALS9 and PIBF proteins were expressed in distinct uterine cell types. These results indicate that the galectins may be involved in uterine receptivity and/or implantation in heifers. PMID:21610087

  7. Expression of the cell adhesion proteins BEN/SC1/DM-GRASP and TAG-1 defines early steps of axonogenesis in the human spinal cord.

    PubMed

    Karagogeos, D; Pourquié, C; Kyriakopoulou, K; Tavian, M; Stallcup, W; Péault, B; Pourquié, O

    1997-03-17

    We have studied the expression pattern of two cell adhesion proteins of the immunoglobin (Ig) superfamily, BEN/SC1/DM-GRASP (BEN) and the transient axonal glycoprotein TAG-1, during the development of the human nervous system. This study was performed by immunocytochemistry on sections of human embryos ranging from 4 to 13 weeks postconception. The overall distribution of the two proteins during development is very similar to that reported in other vertebrate species, but several important differences have been observed. Both proteins exhibit a transient expression on selected neuronal populations, which include the motor and the sensory neurons. In addition, BEN was also detected on virtually all neurons derived from the neural crest as well as in nonneuronal tissues. A major difference of expression with the chick embryo is that, in the motor neurons, BEN expression was not observed at early stages of development, thus arguing against a role of this molecule in pathfinding and fasciculation. BEN was observed to be restricted to subsets of motor neurons, such as the medial column at the upper limb level. Expression was also detected in a laterodorsal population of the ventral horn cells, which are likely to correspond to migrating preganglionic neurons that originate from the motor pool at the thoracic level. TAG-1 was found on commissural neurons and weakly on the sympathetic neurons; it was also detected on restricted nonneuronal populations. In addition, we observed TAG-1 expression in fibers that could correspond either to subsets of dorsal root ganglia (DRGs) central afferences (including the Ia fibers) or to the axons of association interneurons and in scattered motoneurons likely to correspond either to preganglionic neurons, to gamma-motoneurons, or to late-born motoneurons. Therefore, our results indicate that the molecular strategies used to establish the axonal scaffolding of the nervous system in humans are extremely conserved among the different

  8. Possible role of the 72,000 dalton DNA-binding protein in regulation of adenovirus type 5 early gene expression.

    PubMed Central

    Carter, T H; Blanton, R A

    1978-01-01

    Relative abundances of early virus RNA species in the cytoplasm of cells infected with wild-type adenovirus type 5 (WT Ad5) and a temperature-sensitive "early" mutant, H5ts125 (ts125), were compared by hybridization kinetics using separated strands of HindIII restriction endonuclease fragments of Ad5 DNA. 1-beta-D-Arabinofuranosylcytosine (ara-C) was used to limit transcription to early virus genes in cells infected by WT virus. At 40.5 degrees C, a restrictive temperature for ts125, three to seven times as much virus RNA from all four early regions of the genome accumulated in the cytoplasm of cells infected by the mutant as accumulated in cells infected by WT. At 32 degrees C, no such difference in the relative abundances of cytoplasmic virus RNA was observed. The capacity to synthesize a 72,000-dalton (72K) virus polypeptide, presumably the single-stranded DNA-binding protein that is defective in ts125 at restrictive temperatures, was compared in cells infected at 40.5 degrees C in the presence of ara-C with the mutant or WT Ad5. The rate of 72K polypeptide synthesis, measured by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of [35S]methionine-labeled polypeptides and autoradiography, was greater at 15 h after infection in ts125-infected cells than in cells infected by WT. A time course experiment showed that the rate of synthesis of the 72K polypeptide increased continuously in ts125-infected cells during the first 15 h of infection, relative to the rate in WT-infected cells. These data are consistent with the hypothesis that Ad5 early gene expression is modulated by the product of an early gene, the 72K DNA-binding protein. Images PMID:203722

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

    PubMed Central

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

    2013-01-01

    Early life adversity and stress in humans has 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. PMID:22982514

  10. 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.

  11. 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

  12. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Regulation of Early Light-Inducible Protein Gene Expression by Blue and Red Light in Etiolated Seedlings Involves Nuclear and Plastid Factors.

    PubMed Central

    Adamska, I.

    1995-01-01

    Early light-inducible proteins (ELIPs) are nuclear-encoded chloroplast proteins whose genes are transiently transcribed during the greening process of etiolated plants. In the present work the regulation of ELIP gene expression by blue and red light has been investigated in plumulas of etiolated pea plants (Pisum sativum). The results show that the steady-state level of ELIP transcripts is controlled by a combined action of phytochrome and blue light receptor systems and, in addition, depends on the age of the seedlings. Both a low-light fluence system of blue and a very-low-fluence system of red light are involved in ELIP induction. The threshold for accumulation of ELIP transcripts was as low as 10-5 [mu]E m-2 s-1 for both light qualities but a different pattern of accumulation was obtained in blue and in red light. Blue light not only acts at the level of transcription but also regulates the stability of the ELIP transcripts in a light intensity-dependent manner. Moreover, it is shown that product(s) of nuclear gene(s) negatively regulate the steady-state level of ELIP transcripts during the 1st h of illumination with red light. Preillumination of seedlings with white light abolishes this repression. Accumulation of ELIP transcripts requires "plastid factors" in both blue and red light qualities. PMID:12228423

  14. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.

    PubMed Central

    Bruening, W; Giasson, B; Mushynski, W; Durham, H D

    1998-01-01

    The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-activated protein kinases [SAPKs, also called Jun N-terminal kinases (JNKs)] and p38 kinases. We have found that the same conditions that lead to activation of SAPK/JNKs and p38 kinases can also dramatically increase expression from the CMV promoter. Inhibitors of p38 kinases abolished basal transcription from the CMV promoter and completely blocked stress-induced up-regulation of the CMV promoter. Overexpression of a dominant negative JNK kinase had no effect on basal transcription, but significantly reduced up-regulation caused by stress. These results have grave implications for use of the CMV promoter. If the CMV promoter can be up-regulated by cellular stresses, inadvertent activation of the stress kinase pathways may complicate, if not invalidate, the interpretation of a wide range of experiments. PMID:9421504

  15. 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.

  16. Enhanced expression of adenovirus transforming proteins.

    PubMed Central

    Gaynor, R B; Tsukamoto, A; Montell, C; Berk, A J

    1982-01-01

    Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated. Images PMID:7143568

  17. 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

  18. 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. PMID:26287569

  19. Diosmectite-zinc oxide composite improves intestinal barrier function, modulates expression of pro-inflammatory cytokines and tight junction protein in early weaned pigs.

    PubMed

    Hu, Caihong; Song, Juan; Li, Yali; Luan, Zhaoshuang; Zhu, Kang

    2013-08-01

    The study evaluated whether feeding diosmectite-ZnO composite (DS-ZnO) at 500 mg Zn/kg to early weaned pigs would alleviate the weaning-related intestinal disorders as a substitute for high concentration of ZnO (2250 mg Zn/kg). The pigs weaned at an age of 21 ± 1 d were allotted to four treatments groups as follows: (1) control; (2) DS-ZnO, 500 mg Zn/kg diet; (3) ZnO, 2250 mg Zn/kg diet; and (4) mixture of 2·0 g DS/kg and 500 mg Zn/kg from ZnO (equal amount of DS and ZnO in the DS-ZnO treatment group). The results showed that, compared with the control on days 7 and 14 post-weaning, addition of DS-ZnO at 500 mg Zn/kg improved (P<0·05) daily gain and feed intake, decreased (P<0·05) post-weaning scour scores, increased (P<0·05) jejunal villus height and the ratio of villus height and crypt depth, decreased (P<0·05) jejunal paracellular permeability of fluorescein isothiocyanate dextran 4 kDa and up-regulated (P<0·05) tight junction protein expression of occludin, claudin-1 and zonula occludens-1 in jejunal mucosa. The mRNA levels of TNF-α, IL-6 and interferon-γ (IFN-γ) on day 7 post-weaning were also decreased (P<0·05). The piglets fed DS-ZnO at 500 mg Zn/kg did not differ in the above parameters from those fed ZnO at 2250 mg Zn/kg, while they had better performance than those fed the mixture of DS and ZnO. Supplementation with DS-ZnO at 500 mg Zn/kg was effective in alleviating diarrhoea, barrier dysfunction and inflammatory cytokine expression and up-regulating tight junction protein expression. PMID:23308387

  20. Early secreted antigenic target of 6 kDa (ESAT-6) protein of Mycobacterium tuberculosis induces interleukin-8 (IL-8) expression in lung epithelial cells via protein kinase signaling and reactive oxygen species.

    PubMed

    Boggaram, Vijay; Gottipati, Koteswara R; Wang, Xisheng; Samten, Buka

    2013-08-30

    Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis is critical for the virulence and pathogenicity of M. tuberculosis. IL-8, a major chemotactic cytokine for neutrophils and T lymphocytes, plays important roles in the development of lung injury. To further understand the role of ESAT-6 in lung pathology associated with tuberculosis development, we studied the effects of ESAT-6 on the regulation of IL-8 expression in lung epithelial cells. ESAT-6 induced IL-8 expression by increasing IL-8 gene transcription and mRNA stability. ESAT-6 induction of IL-8 promoter activity was dependent on nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding and sensitive to pharmacological inhibition of PKC and ERK and p38 MAPK pathways. ESAT-6 activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. Dimethylthiourea but not mannitol inhibited IL-8 induction by ESAT-6, further supporting the involvement of ROS in the induction of IL-8 expression. Exposure of mice to ESAT-6 induced localized inflammatory cell aggregate formation with characteristics of early granuloma concomitant with increased keratinocyte chemoattractant CXCL1 staining in bronchiolar and alveolar type II epithelial cells and alveolar macrophages. Our studies have identified a signal transduction pathway involving ROS, PKC, ERK, and p38 MAPKs and NF-κB and AP-1 in the ESAT-6 induction of IL-8 expression in lung epithelial cells. This has important implications for the understanding of lung innate immune responses to tuberculosis and the pathogenesis of lung injury in tuberculosis. PMID:23867456

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Involvement of the major capsid protein and two early-expressed phage genes in the activity of the lactococcal abortive infection mechanism AbiT.

    PubMed

    Labrie, Simon J; Tremblay, Denise M; Moisan, Maxim; Villion, Manuela; Magadán, Alfonso H; Campanacci, Valérie; Cambillau, Christian; Moineau, Sylvain

    2012-10-01

    The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14(bIL170)]), orf41 (P008 [orf41(P008)]), and orf6 (p2 [orf6(p2)] and P008 [orf6(P008)]). The genes e14(bIL170) and orf41(P008) are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6(p2) and ORF5(p2), a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes. PMID:22820334

  3. Motor-Coordination-Dependent Learning, More than Others, Is Impaired in Transgenic Mice Expressing Pseudorabies Virus Immediate-Early Protein IE180

    PubMed Central

    López-Ramos, Juan C.; Tomioka, Yukiko; Morimatsu, Masami; Yamamoto, Sayo; Ozaki, Kinuyo; Ono, Etsuro; Delgado-García, José M.

    2010-01-01

    The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96) was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning (classical eyeblink conditioning), object recognition, spatial orientation (water maze), startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests. PMID:20711341

  4. A novel ARID DNA-binding protein interacts with SymRK and is expressed during early nodule development in Lotus japonicus.

    PubMed

    Zhu, Hui; Chen, Tao; Zhu, Maosheng; Fang, Qing; Kang, Heng; Hong, Zonglie; Zhang, Zhongming

    2008-09-01

    During the establishment of symbiosis in legume roots, the rhizobial Nod factor signal is perceived by the host cells via receptor-like kinases, including SymRK. The NODULE INCEPTION (NIN) gene in Lotus japonicus is required for rhizobial entry into root cells and for nodule organogenesis. We describe here a novel DNA-binding protein from L. japonicus, referred to as SIP1, because it was identified as a SymRK-interacting protein. SIP1 contains a conserved AT-rich interaction domain (ARID) and represents a unique member of the ARID-containing proteins in plants. The C terminus of SIP1 was found to be responsible for its interaction with the kinase domain of SymRK and for homodimerization in the absence of DNA. SIP1 specifically binds to the promoter of LjNIN but not to that of LjCBP1 (a calcium-binding protein gene), both of which are known to be inducible by Nod factors. SIP1 recognizes two of the three AT-rich domains present in the NIN gene promoter. Deletion of one of the AT-rich domains at the NIN promoter diminishes the binding of SIP1 to the NIN promoter. The protein is localized to the nuclei when expressed as a red fluorescence fusion protein in the onion (Allium cepa) epidermal cells. The SIP1 gene is expressed constitutively in the uninfected roots, and its expression levels are elevated after infection by Mesorhizobium loti. It is proposed that SIP1 may be required for the expression of NIN and involved in the initial communications between the rhizobia and the host root cells. PMID:18633121

  5. 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

  6. Protein identification and Peptide expression resolver: harmonizing protein identification with protein expression data.

    PubMed

    Kearney, Paul; Butler, Heather; Eng, Kevin; Hugo, Patrice

    2008-01-01

    Proteomic discovery platforms generate both peptide expression information and protein identification information. Peptide expression data are used to determine which peptides are differentially expressed between study cohorts, and then these peptides are targeted for protein identification. In this paper, we demonstrate that peptide expression information is also a powerful tool for enhancing confidence in protein identification results. Specifically, we evaluate the following hypothesis: tryptic peptides originating from the same protein have similar expression profiles across samples in the discovery study. Evidence supporting this hypothesis is provided. This hypothesis is integrated into a protein identification tool, PIPER (Protein Identification and Peptide Expression Resolver), that reduces erroneous protein identifications below 5%. PIPER's utility is illustrated by application to a 72-sample biomarker discovery study where it is demonstrated that false positive protein identifications can be reduced below 5%. Consequently, it is recommended that PIPER methodology be incorporated into proteomic studies where both protein expression and identification data are collected. PMID:18062667

  7. Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity.

    PubMed

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

    2007-12-01

    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

  8. 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

  9. Bovine Herpesvirus 1 Regulatory Proteins bICP0 and VP16 Are Readily Detected in Trigeminal Ganglionic Neurons Expressing the Glucocorticoid Receptor during the Early Stages of Reactivation from Latency

    PubMed Central

    Frizzo da Silva, Leticia; Kook, Insun; Doster, Alan

    2013-01-01

    Bovine herpesvirus 1 (BHV-1) establishes a lifelong latent infection in sensory neurons following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Within minutes, corticosteroids activate the glucocorticoid receptor and transcription of promoters containing a glucocorticoid receptor element. A single intravenous injection of the synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. Lytic cycle viral gene expression is detected within 6 h after dexamethasone treatment of calves latently infected with BHV-1. Cellular transcription factors are induced by dexamethasone in trigeminal ganglionic neurons within 1.5 h after dexamethasone treatment, suggesting they promote viral gene expression during the early phases of reactivation from latency, which we operationally defined as the escape from latency. In this study, immunohistochemistry was utilized to examine viral protein expression during the escape from latency. Within 1.5 h after dexamethasone treatment, bICP0 and a late protein (VP16) were consistently detected in a subset of trigeminal ganglionic neurons. Most neurons expressing bICP0 also expressed VP16. Additional studies revealed that neurons expressing the glucocorticoid receptor also expressed bICP0 or VP16 at 1.5 h after dexamethasone treatment. Two other late proteins, glycoprotein C and D, were not detected until 6 h after dexamethasone treatment and were detected in only a few neurons. These studies provide evidence that VP16 and the promiscuous viral trans-activator (bICP0) are expressed during the escape from latency, suggesting they promote the production of infectious virus in a small subset of latently infected neurons. PMID:23926348

  10. Bovine herpesvirus 1 regulatory proteins bICP0 and VP16 are readily detected in trigeminal ganglionic neurons expressing the glucocorticoid receptor during the early stages of reactivation from latency.

    PubMed

    Frizzo da Silva, Leticia; Kook, Insun; Doster, Alan; Jones, Clinton

    2013-10-01

    Bovine herpesvirus 1 (BHV-1) establishes a lifelong latent infection in sensory neurons following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Within minutes, corticosteroids activate the glucocorticoid receptor and transcription of promoters containing a glucocorticoid receptor element. A single intravenous injection of the synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. Lytic cycle viral gene expression is detected within 6 h after dexamethasone treatment of calves latently infected with BHV-1. Cellular transcription factors are induced by dexamethasone in trigeminal ganglionic neurons within 1.5 h after dexamethasone treatment, suggesting they promote viral gene expression during the early phases of reactivation from latency, which we operationally defined as the escape from latency. In this study, immunohistochemistry was utilized to examine viral protein expression during the escape from latency. Within 1.5 h after dexamethasone treatment, bICP0 and a late protein (VP16) were consistently detected in a subset of trigeminal ganglionic neurons. Most neurons expressing bICP0 also expressed VP16. Additional studies revealed that neurons expressing the glucocorticoid receptor also expressed bICP0 or VP16 at 1.5 h after dexamethasone treatment. Two other late proteins, glycoprotein C and D, were not detected until 6 h after dexamethasone treatment and were detected in only a few neurons. These studies provide evidence that VP16 and the promiscuous viral trans-activator (bICP0) are expressed during the escape from latency, suggesting they promote the production of infectious virus in a small subset of latently infected neurons. PMID:23926348

  11. Assessment of microtubule-associated protein (MAP)-Tau expression as a predictive and prognostic marker in TACT; a trial assessing substitution of sequential docetaxel for FEC as adjuvant chemotherapy for early breast cancer.

    PubMed

    Irshad, S; Gillett, C; Pinder, S E; A'hern, R P; Dowsett, M; Ellis, I O; Bartlett, J M S; Bliss, J M; Hanby, A; Johnston, S; Barrett-Lee, P; Ellis, P; Tutt, A

    2014-04-01

    The TACT trial is the largest study assessing the benefit of taxanes as part of adjuvant therapy for early breast cancer. The goal of this translational study was to clarify the predictive and prognostic value of Tau within the TACT trial. Tissue microarrays (TMA) were available from 3,610 patients. ER, PR, HER2 from the TACT trial and Tau protein expression was determined by immunohistochemistry on duplicate TMAs. Two parallel scoring systems were generated for Tau expression ('dichotomised' vs. 'combined' score). The positivity rate of Tau expression was 50 % in the trial population (n = 2,483). Tau expression correlated positively with ER (p < 0.001) and PR status (p < 0.001); but negatively with histological grade (p < 0.001) and HER2 status (p < 0.001). Analyses with either scoring systems for Tau expression demonstrated no significant interaction between Tau expression and efficacy of docetaxel. Contrary to the hypothesis that taxane benefit would be enriched in Tau negative/low patients, the only groups with a suggestion of a reduced event rate in the taxane group were the HER2-positive, Tau positive subgroups. Tau expression was seen to be a prognostic factor on univariate analysis associated with an improved DFS, independent of the treatment group (p < 0.001). It had no prognostic value in ER-negative tumours and the weak prognostic effect of Tau in ER-positive tumours (p = 0.02) diminished, when considering ER as an ordinal variable. On multivariable analyses, Tau had no prognostic value in either group. In addition, no significant interaction between Tau expression and benefit from docetaxel in patients within the PR-positive and negative subsets was seen. This is now the second large adjuvant study, and the first with quantitative analysis of ER and Tau expression, failing to show an association between Tau and taxane benefit with limited utility as a prognostic marker for Tau in ER-positive early breast cancer patients. PMID:24519386

  12. Protective role of fish oil (Maxepa) on early events of rat mammary carcinogenesis by modulation of DNA-protein crosslinks, cell proliferation and p53 expression

    PubMed Central

    Manna, Sangita; Chakraborty, Tridib; Damodaran, Suresh; Samanta, Kartick; Rana, Basabi; Chatterjee, Malay

    2007-01-01

    Background Fish oil is known to protect from many types of cancers of the colon, liver, breast, prostate and lung [1-3]. The objective of the present study was to evaluate the role of fish oil [Maxepa, supplemented at a dose of 0.5 ml is equivalent to 90 mg eicosapentaenoic acid (EPA) and 60 mg docosahexaenoic acid (DHA)] on cell proliferation, expression of p53 tumor suppressor protein and DNA protein crosslinks (DPCs) in a defined model of chemical rat mammary carcinogenesis. Mammary carcinogenesis was initiated by a single, intravenous (i.v.) tail vein injection of 7,12 dimethylbenz(α)anthracene (DMBA) at a dose of 5 mg DMBA/2 ml corn oil/kg body weight in female Sprague-Dawley rats at 7 weeks of age. Fish oil supplementation was started daily, 2 weeks prior to DMBA injection and continued for 24 (31 weeks of animal age) weeks and 35 (42 weeks of animal age) weeks of post DMBA injection, for histopathological and immunohistochemical and for morphological studies, respectively. Results Our results indicate the chemopreventive effect of fish oil (Maxepa) on DMBA-induced rat mammary carcinogenesis. Administration of fish oil further showed a prominent reduction of cell proliferation (24.34%, P = 0.001); DPCs (25%, P < 0.001) and an increased expression of p53 protein (4.636 ± 0.19, P < 0.001) in preneoplastic mammary tissue when compared to carcinogen control counterpart. Histopathological and morphological analyses were carried out as end-point biomarkers. Conclusion Our study thus provides evidence for the anticarcinogenic effect of fish oil (Maxepa) in limiting mammary preneoplasia in Sprague-Dawley rats. PMID:17470299

  13. 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. PMID:24342663

  14. CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins

    PubMed Central

    Pfannes, Loretta; Chen, Gubin; Shah, Simant; Solomou, Elena E.; Barrett, John; Young, Neal S.

    2007-01-01

    CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) are distinguished from other MDS cells and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We previously demonstrated up-regulation of c-myc and CD1 by microarray analysis. Here, we confirmed these findings by real-time polymerase chain reaction (PCR), demonstrated up-regulation of survivin, c-myc, and CD1 protein expression, and documented comparable colony formation by annexin+ trisomy 8− CD34+ and annexin− CD34 cells. There were low levels of DNA degradation in annexin+ trisomy 8 CD34 cells, which were comparable with annexin− CD34 cells. Trisomy 8 cells were resistant to apoptosis induced by gamma irradiation. Knock-down of survivin by siRNA resulted in preferential loss of trisomy 8 cells. These results suggest that trisomy 8 cells undergo incomplete apoptosis and are nonetheless capable of colony formation and growth. PMID:17090657

  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. Effect of Boschniakia rossica on expression of GST-P, p53 and p21(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats.

    PubMed

    Yin, Zong-Zhu; Jin, Hai-Ling; Yin, Xue-Zhe; Li, Tian-Zhu; Quan, Ji-Shu; Jin, Zeng-Nan

    2000-12-01

    AIM:To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21(ras) proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS:The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21(ras) proteins were investigated by immunohisto-chemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet induced mouse granuloma formation methods.RESULTS:The 500mg/kg of BR-H2O extract frac-tionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, P<0.05) and the expression of mutant p53 and p21(ras) protein was lower than that of hepatic preneoplastic lesions (33% and 22% positive respectively in DEN+AAF group vs negative in DEN+AAF+BR group). Both CH(2)Cl(2) and H(2)O extracts from BR had anti-inflamatory effect in xylene and crotonoil induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR H(2)O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.CONCLUSION:BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis.Both CH(2)Cl(2) and H(2)O extracts from BR exerted anti-inflammatory effect in rats and mice. PMID:11819701

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

    PubMed

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

    2015-07-01

    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

  18. Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

    PubMed Central

    2011-01-01

    Background Herpes simplex viruses (HSVs) rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1), a host cell protein markedly up-regulated by HSV infection. Results INSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP) assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA. Conclusions The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter. PMID:21609490

  19. Surface expression of GABAA receptors is transcriptionally controlled by the interplay of cAMP-response element-binding protein and its binding partner inducible cAMP early repressor.

    PubMed

    Hu, Yinghui; Lund, Ingrid V; Gravielle, Maria C; Farb, David H; Brooks-Kayal, Amy R; Russek, Shelley J

    2008-04-01

    The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers. PMID:18180303

  20. 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.

  1. Predictable tuning of protein expression in bacteria.

    PubMed

    Bonde, Mads T; Pedersen, Margit; Klausen, Michael S; Jensen, Sheila I; Wulff, Tune; Harrison, Scott; Nielsen, Alex T; Herrgård, Markus J; Sommer, Morten O A

    2016-03-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expression level of any Escherichia coli gene by changing only a few bases. Measured protein levels for 91% of our designed sequences were within twofold of the desired target level. PMID:26752768

  2. 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.

  3. 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

  4. Expression and purification of GST fusion proteins.

    PubMed

    Harper, S; Speicher, D W

    2001-05-01

    An increasingly common strategy for expressing proteins and large peptides in prokaryotic systems is to express the protein of interest connected to a "tag" that provides the basis for rapid high-affinity purification. This unit describes the expression and purification of fusion proteins containing the 26-kDa glutathione-S-transferase protein as well as methods for cleaving the affinity tag and repurifying the target protein. Advantages of this popular fusion protein system include high protein yields, high-affinity one-step protein purification of the fusion protein, existence of several alternative protease cleavage sites for removing the affinity tag when required, and ease of removal of the cleaved affinity tag. PMID:18429193

  5. 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

  6. 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

  7. 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. PMID:27512005

  8. 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. PMID:14717545

  9. 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. PMID:23662649

  10. T(lys), a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes.

    PubMed

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta; Contursi, Patrizia

    2013-05-01

    While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (T(lys)) was identified. Transcriptional analysis revealed that T(lys) is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and T(ind)) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA-binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and T(ind) transcripts, as well as of its own promoter. Binding sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the T(ind) promoter. Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1. PMID:23514883

  11. 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

  12. 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.

  13. High expression of Y-box-binding protein 1 correlates with poor prognosis and early recurrence in patients with small invasive lung adenocarcinoma

    PubMed Central

    Zhao, Shilei; Guo, Wei; Li, Jinxiu; Yu, Wendan; Guo, Tao; Deng, Wuguo; Gu, Chundong

    2016-01-01

    Background Prognosis of small (≤2 cm) invasive lung adenocarcinoma remains poor, and identification of high-risk individuals from the patients after complete surgical resection of lung adenocarcinoma has become an urgent problem. YBX1 has been reported to be able to predict prognosis in many cancers (except lung adenocarcinoma) that are independent of TNM (tumor, nodes, metastases) staging, especially small invasive lung adenocarcinoma. Therefore, we examined the significance of YBX1 expression on prognosis and recurrence in patients with small invasive lung adenocarcinoma. Material and methods A total of 75 patients with small invasive lung adenocarcinoma after complete resection were enrolled from January 2008 to December 2010. Immunohistochemical staining was used to detect the expression of YBX1, and receiver operating characteristic curve analysis was performed to precisely assess the overall expression of YBX1. Meanwhile, primary lesions were identified based on the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society’s classification of lung adenocarcinoma. The effect of different clinicopathological factors on patients’ survival was examined. Furthermore, Western blot analysis was used to show the expression of YBX1 in vitro. Results Sensitivity and specificity of YBX1 for detecting small invasive lung adenocarcinoma from normal surrounding tissue were 66.7% and 74.7% (area under the receiver operating characteristic curve =0.731; P<0.001), respectively. High YBX1 expression was detected in 31 (41.3%) patients, and in A549, H322, Hcc827, and H1299 lung adenocarcinoma cells but not in HLF cells. In addition to sex, age, tumor size, TNM staging, pleural invasion, and lymph node metastasis, the expression of YBX1 was associated with the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society pathological grade risk (P

  14. Adenovirus type 2 encoded early 11 kDa protein

    SciTech Connect

    Murthy, S.V.K.N.; Kapoor, Q.S.

    1986-05-01

    Several adenovirus type 2 (Ad2) encoded early proteins have been identified in viral infected human KB cells. These proteins are of great interest as they play key roles in cell transformation, viral DNA synthesis and gene expression. They have partially purified an AD2 encoded early polypeptide of an apparent molecular weight of 11 kilodaltons from the nuclei of viral infected cells labelled with /sup 35/S-methionine. After DNA removal from the nuclear extracts, the polypeptide was isolated using DEAE-Sephacel anion exchange and Biogel P-10 gel filtration columns. This simple two step procedure yielded several fold purification of the polypeptide. Antisera raised in mice against an Ad2 transformed rat cell line 8617 was found to immunoprecipitate the 11 kDa polypeptide from the nuclear extract of Ad2 infected KB cells. After relating this protein to an open reading frame of an Ad2 early gene block by matching the amino acid sequences to the nucleotide sequences of early genes, they plan to functionally characterize this protein by using monoclonal antibodies in in vivo and in vitro experiments.

  15. 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. PMID:26590793

  16. Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.

    PubMed Central

    Sanz-Ezquerro, J J; de la Luna, S; Ortín, J; Nieto, A

    1995-01-01

    In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit. PMID:7884889

  17. 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

  18. 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

  19. Expression of developmental genes during early embryogenesis of Hydra.

    PubMed

    Fröbius, Andreas C; Genikhovich, Gregory; Kürn, Ulrich; Anton-Erxleben, Friederike; Bosch, Thomas C G

    2003-09-01

    Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning. PMID:12883882

  20. 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.

  1. 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. PMID:26614281

  2. 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.

  3. 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

  4. Membrane protein expression in Lactococcus lactis.

    PubMed

    King, Martin S; Boes, Christoph; Kunji, Edmund R S

    2015-01-01

    The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels. PMID:25857778

  5. 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

  6. Proteomics for Protein Expression Profiling in Neuroscience*

    PubMed Central

    Freeman, Willard M.; Hemby, Scott E.

    2013-01-01

    As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future. PMID:15176464

  7. 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. PMID:26664034

  8. Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

    PubMed Central

    Lange Consiglio, Anna; Dell'Aquila, Maria Elena; Fiandanese, Nadia; Ambruosi, Barbara; Cho, Yoon S; Bosi, Giampaolo; Arrighi, Silvana; Lacalandra, Giovanni M; Cremonesi, Fausto

    2009-01-01

    Background The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI). Methods Compact and expanded-cumulus horse oocytes were matured in medium containing different concentrations (1, 10, 100, 1000 ng/ml) of recombinant human leptin and the effects on maturation, fertilization and embryo cleavage were evaluated. Furthermore, early developmental expression of Ob and leptin receptor (Ob-R) was investigated by immunocytochemical staining. Results In expanded-cumulus oocytes, the addition of leptin in IVM medium improved maturation (74% vs 44%, for 100 ng/ml leptin-treated and control groups, respectively; P < 0.05) and fertilization after ICSI (56% vs 23% for 10 ng/ml leptin-treated and control groups, respectively; P < 0.05). However, the developmental rate and quality of 8-cell stage embryos derived from leptin-treated oocytes (100 ng/ml) was significantly reduced, in contrast to previous data in other species where leptin increased embryo cleavage. Ob and Ob-R proteins were detected up to the 8-cell stage with cortical and cytoplasmic granule-like distribution pattern in each blastomere. Conclusion Leptin plays a cumulus cell-mediated role in the regulation of oocyte maturation in the mare. Species-specific differences may exist in oocyte sensitivity to leptin. PMID:19835605

  9. Expression Pattern of Id Proteins in Medulloblastoma

    PubMed Central

    Snyder, Andrew D.; Dulin-Smith, Ashley N.; Houston, Ronald H.; Durban, Ashley N.; Brisbin, Bethany J.; Oostra, Tyler D.; Marshall, Jordan T.; Kahwash, Basil M.

    2013-01-01

    Inhibitor of DNA binding or inhibitor of differentiation (Id) proteins are up regulated in a variety of neoplasms, particularly in association with high-grade, poorly differentiated tumors, while differentiated tissues show little or no Id expression. The four Id genes are members of the helix-loop-helix (HLH) family of transcription factors and act as negative regulators of transcription by binding to and sequestering HLH complexes. We tested the hypothesis that Id proteins are overexpressed in medulloblastoma by performing immunohistochemistry using a medulloblastoma tissue microarray with 45 unique medulloblastoma and 11 normal control cerebella, and antibodies specific for Id1, Id2, Id3, and Id4. A semi-quantitative staining score that took staining intensity and the proportion of immunoreactive cells into account was used. Id1 was not detected in normal cerebella or in medulloblastoma cells, but 78 % of tumors showed strong Id1 expression in endothelial nuclei of tumor vessels. Id2 expression was scant in normal cerebella and increased in medulloblastoma (median staining score: 4). Id3 expression was noted in some neurons of the developing cerebellar cortex, but it was markedly up regulated in medulloblastoma (median staining score: 12) and in tumor endothelial cells. Id4 was not expressed in normal cerebella or in tumor cells. Id2 or Id3 overexpression drove proliferation in medulloblastoma cell lines by altering the expression of critical cell cycle regulatory proteins in favor of cell proliferation. This study shows that Id1 expression in endothelial cells may contribute to angiogenic processes and that increased expression of Id2 and Id3 in medulloblastoma is potentially involved in tumor cell proliferation and survival. PMID:23397264

  10. Early light-induced proteins protect Arabidopsis from photooxidative stress.

    PubMed

    Hutin, Claire; Nussaume, Laurent; Moise, Nicolae; Moya, Ismaël; Kloppstech, Klaus; Havaux, Michel

    2003-04-15

    The early light-induced proteins (ELIPs) belong to the multigenic family of light-harvesting complexes, which bind chlorophyll and absorb solar energy in green plants. ELIPs accumulate transiently in plants exposed to high light intensities. By using an Arabidopsis thaliana mutant (chaos) affected in the posttranslational targeting of light-harvesting complex-type proteins to the thylakoids, we succeeded in suppressing the rapid accumulation of ELIPs during high-light stress, resulting in leaf bleaching and extensive photooxidative damage. Constitutive expression of ELIP genes in chaos before light stress resulted in ELIP accumulation and restored the phototolerance of the plants to the wild-type level. Free chlorophyll, a generator of singlet oxygen in the light, was detected by chlorophyll fluorescence lifetime measurements in chaos leaves before the symptoms of oxidative stress appeared. Our findings indicate that ELIPs fulfill a photoprotective function that could involve either the binding of chlorophylls released during turnover of pigment-binding proteins or the stabilization of the proper assembly of those proteins during high-light stress. PMID:12676998

  11. Microgravity alters the expression of salivary proteins.

    PubMed

    Mednieks, Maija; Khatri, Aditi; Rubenstein, Renee; Burleson, Joseph A; Hand, Arthur R

    2014-06-01

    Spaceflight provides a unique opportunity to study how physiologic responses are influenced by the external environment. Microgravity has been shown to alter the function of a number of tissues and organ systems. Very little, however, is known about how microgravity affects the oral cavity. The rodent model is useful for study in that their salivary gland morphology and physiology is similar to that of humans. Useful also is the fact that saliva, a product of the salivary glands with a major role in maintaining oral health, can be easily collected in humans whereas the glands can be studied in experimental animals. Our working hypothesis is that expression of secretory proteins in saliva will respond to microgravity and will be indicative of the nature of physiologic reactions to travel in space. This study was designed to determine which components of the salivary proteome are altered in mice flown on the US space shuttle missions and to determine if a subset with predictive value can be identified using microscopy and biochemistry methods. The results showed that the expression of secretory proteins associated with beta-adrenergic hormone regulated responses and mediated via the cyclic AMP pathway was significantly altered, whereas that of a number of unrelated proteins was not. The findings are potentially applicable to designing a biochemical test system whereby specific salivary proteins can be biomarkers for stress associated with travel in space and eventually for monitoring responses to conditions on earth. PMID:24984624

  12. Differential Expression of Borrelia burgdorferi Proteins during Growth In Vitro

    PubMed Central

    Ramamoorthy, Ramesh; Philipp, Mario T.

    1998-01-01

    In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165–1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host. PMID:9784512

  13. Early pregnancy peripheral blood gene expression and risk of preterm delivery: a nested case control study

    PubMed Central

    2009-01-01

    Background Preterm delivery (PTD) is a significant public health problem associated with greater risk of mortality and morbidity in infants and mothers. Pathophysiologic processes that may lead to PTD start early in pregnancy. We investigated early pregnancy peripheral blood global gene expression and PTD risk. Methods As part of a prospective study, ribonucleic acid was extracted from blood samples (collected at 16 weeks gestational age) from 14 women who had PTD (cases) and 16 women who delivered at term (controls). Gene expressions were measured using the GeneChip® Human Genome U133 Plus 2.0 Array. Student's T-test and fold change analysis were used to identify differentially expressed genes. We used hierarchical clustering and principle components analysis to characterize signature gene expression patterns among cases and controls. Pathway and promoter sequence analyses were used to investigate functions and functional relationships as well as regulatory regions of differentially expressed genes. Results A total of 209 genes, including potential candidate genes (e.g. PTGDS, prostaglandin D2 synthase 21 kDa), were differentially expressed. A set of these genes achieved accurate pre-diagnostic separation of cases and controls. These genes participate in functions related to immune system and inflammation, organ development, metabolism (lipid, carbohydrate and amino acid) and cell signaling. Binding sites of putative transcription factors such as EGR1 (early growth response 1), TFAP2A (transcription factor AP2A), Sp1 (specificity protein 1) and Sp3 (specificity protein 3) were over represented in promoter regions of differentially expressed genes. Real-time PCR confirmed microarray expression measurements of selected genes. Conclusions PTD is associated with maternal early pregnancy peripheral blood gene expression changes. Maternal early pregnancy peripheral blood gene expression patterns may be useful for better understanding of PTD pathophysiology and PTD risk

  14. Development of early PCLP1-expressing haematopoietic cells within the avian dorsal aorta.

    PubMed

    Suonpää, P; Kohonen, P; Koskela, K; Koskiniemi, H; Salminen-Mankonen, H; Lassila, O

    2005-09-01

    The first haematopoietic stem cells (HSC) develop in the dorsal aorta as haematopoietic intra-aortic clusters (HIAC). To evaluate the initial steps of definitive haematopoiesis, we have studied the emergence and the expression profile of podocalyxin-like protein 1 (PCLP1)-expressing cells in early chick embryos. Here we demonstrate that at embryonic day 2 (E2), the PCLP1+ cells are present in the splanchnic mesoderm and in the ventral lining of the paired dorsal aorta. Following aortic fusion at E3, the PCLP1-expressing cells are exclusively found in the aortic floor and as the development proceeds, both the haematopoietic clusters and the aortic endothelial cells express PCLP1. In parallel with the early PCLP1 expression, bone morphogenetic protein 4 (BMP4) expression was detected in the splanchnopleura and thereafter in the densely packed mesenchymal cells beneath the HIAC. The microarray analyses of early E3 PCLP1+ cells revealed elevated expression of genes known to be involved in the stem cell function. These data suggest that splanchnopleura-derived PCLP1-expressing cells give rise to the earliest definitive haematopoietic progenitors. PMID:16179008

  15. Regulation of Mutant p53 Protein Expression

    PubMed Central

    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. PMID:26734569

  16. Transcriptional Factor PU.1 Regulates Decidual C1q Expression in Early Pregnancy in Human.

    PubMed

    Madhukaran, Shanmuga Priyaa; Kishore, Uday; Jamil, Kaiser; Teo, Boon Heng Dennis; Choolani, Mahesh; Lu, Jinhua

    2015-01-01

    C1q is the first recognition subcomponent of the complement classical pathway, which in addition to being synthesized in the liver, is also expressed by macrophages and dendritic cells (DCs). Trophoblast invasion during early placentation results in accumulation of debris that triggers the complement system. Hence, both early and late components of the classical pathway are widely distributed in the placenta and decidua. In addition, C1q has recently been shown to significantly contribute to feto-maternal tolerance, trophoblast migration, and spiral artery remodeling, although the exact mechanism remains unknown. Pregnancy in mice, genetically deficient in C1q, mirrors symptoms similar to that of human preeclampsia. Thus, regulated complement activation has been proposed as an essential requirement for normal successful pregnancy. Little is known about the molecular pathways that regulate C1q expression in pregnancy. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells, and its expression is tissue-specific. Recently, PU.1 has been shown to regulate C1q gene expression in DCs and macrophages. Here, we have examined if PU.1 transcription factor regulates decidual C1q expression. We used immune-histochemical analysis, PCR, and immunostaining to localize and study the gene expression of PU.1 transcription factor in early human decidua. PU.1 was highly expressed at gene and protein level in early human decidual cells including trophoblast and stromal cells. Surprisingly, nuclear as well as cytoplasmic PU.1 expression was observed. Decidual cells with predominantly nuclear PU.1 expression had higher C1q expression. It is likely that nuclear and cytoplasmic PU.1 localization has a role to play in early pregnancy via regulating C1q expression in the decidua during implantation. PMID:25762996

  17. 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.

  18. Gene Expression Analyses of Subchondral Bone in Early Experimental Osteoarthritis by Microarray

    PubMed Central

    Chen, YuXian; Shen, Jun; Lu, HuaDing; Zeng, Chun; Ren, JianHua; Zeng, Hua; Li, ZhiFu; Chen, ShaoMing; Cai, DaoZhang; Zhao, Qing

    2012-01-01

    Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA. PMID:22384228

  19. 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

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

    PubMed

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

    2016-04-01

    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

  1. NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse

    PubMed Central

    PENG, Hui; LIN, Xiujiao; LIU, Fang; WANG, Cheng; ZHANG, Wenchang

    2015-01-01

    Nlrp9a, Nlrp9b and Nlrp9c are preferentially expressed in oocytes and early embryos in the mouse. Simultaneous genetic ablation of Nlrp9a and Nlrp9c does not affect early embryonic development, but the function of Nlrp9b in the process of oocyte maturation and embryonic development has not been elucidated. Here we show that both Nlrp9b mRNA and its protein are expressed in ovaries and the small intestine. Moreover, the NLRP9B protein was restricted to oocytes in the ovary and declined with oocyte aging. After ovulation and fertilization, NLRP9B protein was found in preimplantation embryos. Confocal microscopy demonstrated that it was mainly localized in the cytoplasm in the oocytes and blastomeres. Thus, this protein might play a role in oocyte maturation and early embryonic development. However, knockdown of Nlrp9b expression in GV-stage oocytes using RNA interference did not affect oocyte maturation or subsequent parthenogenetic development after Nlrp9b-deficient oocytes were activated. Furthermore, Nlrp9b knockdown zygotes could reach the blastocyst stage after being cultured for 3.5 days in vitro. These results provide the first evidence that the NLRP9B protein is dispensable for oocyte maturation and early embryonic development in the mouse. PMID:26411641

  2. Expression of KRAS in the endometrium of early pregnant mice and its effect during embryo implantation.

    PubMed

    Long, Xia; Zhang, Min; Chen, Xuemei; He, Junlin; Ding, Yubin; Zhang, Cuizhen; Liu, Xueqing; Wang, Yingxiong

    2015-07-01

    This study investigated the expression pattern of Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) in the endometrium of early-stage pregnant mice and its function during embryo implantation. The expression of KRAS was measured at the mRNA level using real-time polymerase chain reaction (PCR) and at the protein level using immunohistochemistry and western blotting. The expressions of KRAS mRNA and protein were not significantly different in the endometrium of pseudopregnant and early-stage pregnant mice. However, the immunohistochemistry results showed that KRAS was highly expressed in the decidualizing stromal cells on days 5-7 of mouse pregnancy and was enhanced in the epithelial cells as pregnancy progressed. The expression of KRAS protein was higher after the stromal cell was artificially decidualized in vivo and in vitro. Stromal cell proliferation was attenuated after down-regulating KRAS expression. After silencing KRAS in the mouse uterus, the embryo implantation rate was significantly reduced (P < 0.005). We speculate that KRAS may regulate the stromal cell proliferation and differentiation progress and then affect the embryo implantation process. This study reveals that KRAS plays an important role in regulating the embryo implantation process. PMID:25999213

  3. Differences between human and mouse alpha-fetoprotein expression during early development

    PubMed Central

    JONES, ELIZABETH A.; CLEMENT-JONES, MARK; JAMES, OLIVER F. W.; WILSON, DAVID I.

    2001-01-01

    Alpha-fetoprotein (AFP) is the major serum protein during development. AFP is one of the earliest proteins to be synthesised by the embryonic liver. The synthesis of AFP decreases dramatically after birth and only trace amounts are expressed in the adult liver. The tissue distribution of AFP in early human embryogenesis has not been defined. We have studied the expression pattern of AFP mRNA in human and mouse embryos by in situ hybridisation. In humans, AFP is expressed in the hepatic diverticulum at 26 d postovulation as it differentiates from the foregut endoderm (i.e. in the most primitive hepatocytes). It is also expressed in the endoderm of the gastrointestinal tract and in the yolk sac at this age. AFP is subsequently expressed in the mesonephros and transiently in the developing pancreas. In the mouse, no expression of AFP was observed in the mesonephros but other sites of expression were similar. Thus AFP has a distinct temporospatial expression pattern during the embryonic period and this differs between human and mouse species. It is interesting that AFP is expressed by tumours such as primitive gastrointestinal, renal cell and pancreatic tumours as well as those of hepatocyte origin. This distribution reflects the sites of AFP expression during development. PMID:11430694

  4. Arabidopsis thaliana SEPALLATA3 protein prokaryotic expression and purification.

    PubMed

    He, Q; Fu, A Y; Zhang, G C; Li, T J; Zhang, J H

    2015-01-01

    SEPALLATA3 (SEP3) can be attributed to E class gene of the ABCE model of floral organ development. In order to reveal how SEP3 proteins form polymers, and the relationship between the polymers and their biological functions, the experiments of Arabidopsis thaliana AtSEP3 protein soluble expression in vitro were performed to construct a vector of prokaryotic expression, and investigate induced expression of recombinant proteins in Escherichia coli cells. The protein soluble expression was analyzed through the aspects of different protein domains, induction time, induction temperature, etc. Different structural domains and expression conditions were screened, and 0.1% IPTG inducing at 22 oC for 15 h was estimated as an optimal expression strategy. The nickel chelating resin was used to purify the protein in size exclusion chromatography (SEC) and the results indicated that AtSEP3 protein was present in the form of tetramer. PMID:26025404

  5. 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. PMID:18537683

  6. Arabidopsis AMY1 expressions and early flowering mutant phenotype.

    PubMed

    Jie, Wang; Dashi, Yu; XinHong, Guo; Xuanming, Liu

    2009-02-28

    The homozygous T-DNA mutant of the AMY1 gene in Arabidopsis was identified and importantly, shown to cause an early flowering phenotype. We found that the disruption of AMY1 enhanced expression of CO and FT. The expression analyses of genes related to starch metabolism revealed that expression of the AGPase small subunit APS1 in the wild type was higher than in the amy1 mutant. However, there were no significant differences in expression levels of the AGPase large subunit genes ApL1, AMY2, or AMY3 between wild type and the amy1 mutant. Expression profiling showed that AMY1 was highly expressed in leaves, stems, and flowers, and expressed less in leafstalks and roots. Furthermore, the level of AMY1 mRNA was highly elevated with age and in senescing leaves. RT-PCR analyses showed that the expression of AMY1 was induced by heat shock, GA, and ABA, while salt stress had no apparent effect on its expression. PMID:19250611

  7. p53 protein expression in human breast carcinoma: relationship to expression of epidermal growth factor receptor, c-erbB-2 protein overexpression, and oestrogen receptor.

    PubMed Central

    Poller, D. N.; Hutchings, C. E.; Galea, M.; Bell, J. A.; Nicholson, R. A.; Elston, C. W.; Blamey, R. W.; Ellis, I. O.

    1992-01-01

    The expression of p53 protein, oestrogen receptor protein, epidermal growth factor receptor (EGFR) and overexpression of the c-erbB-2 oncoprotein was examined in a series of 149 primary symptomatic breast carcinomas. Expression of p53 was present in 62 of 146 cases (42.5%) of the invasive carcinoma and one of three cases (33.3%) of ductal carcinoma in situ (DCIS) examined. Statistical associations of tumour oestrogen receptor positivity and lack of p53 protein expression, chi 2 = 19.78 (d.f. = 1), P less than 0.001, positive tumour p53 status and poor tumour grade; chi 2 = 14.1 (d.f. = 2), P less than 0.001, EGFR expression chi 2 = 7.07, (d.f. = 1), P less than 0.01 and tumour c-erbB-2 protein overexpression; chi 2 = 4.61 (d.f. = 1), P = 0.032 were identified. Expression of p53 is rare in invasive lobular carcinoma of classical type (8.3% of cases examined) in contrast to other common types of mammary carcinoma. Non-significant trends of p53 protein expression and increased regional tumour recurrence; chi 2 = 3.20 (d.f. = 1), P = 0.074 and also poorer patient survival; chi 2 = 3.76 (d.f. = 1), P = 0.053 were identified. p53 protein expression is a common event in human breast cancer and is present in both DCIS and invasive mammary carcinoma. Abnormal expression of p53 protein is a feature of both in situ and invasive breast carcinoma, implying that the abnormal p53 protein expression may be implicated in the early stages of mammary carcinoma progression. Images Figure 1 PMID:1355662

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Pan-Cancer Analysis for Studying Cancer Stage using Protein Expression Data

    PubMed Central

    Mishra, Sameer; Kaddi, Chanchala D.; Wang, May D.

    2016-01-01

    Pan-cancer analyses attempt to discover similar features among multiple cancers in order to identify fundamental patterns common to cancer development and progression. Pan-cancer analysis at the level of protein expression is particularly important because protein expression is more immediately related to patient phenotype than genomic or transcriptomic data. This study aims to analyze differentially expressed (DE) proteins between early and advanced cases of multiple cancer types through the usage of reverse-phase protein array data. The relevance of these proteins is further investigated by developing predictive models using K-nearest neighbor and linear discriminant analysis classifiers. The results of this study suggest that a pan-cancer analysis may be highly complementary to standard analysis of an individual cancer for identifying biologically relevant DE proteins, and can assist in developing effective predictive models for cancer progression. PMID:26738195

  10. Stochastic Spatio-Temporal Dynamic Model for Gene/Protein Interaction Network in Early Drosophila Development

    PubMed Central

    Li, Cheng-Wei; Chen, Bor-Sen

    2009-01-01

    In order to investigate the possible mechanisms for eve stripe formation of Drosophila embryo, a spatio-temporal gene/protein interaction network model is proposed to mimic dynamic behaviors of protein synthesis, protein decay, mRNA decay, protein diffusion, transcription regulations and autoregulation to analyze the interplay of genes and proteins at different compartments in early embryogenesis. In this study, we use the maximum likelihood (ML) method to identify the stochastic 3-D Embryo Space-Time (3-DEST) dynamic model for gene/protein interaction network via 3-D mRNA and protein expression data and then use the Akaike Information Criterion (AIC) to prune the gene/protein interaction network. The identified gene/protein interaction network allows us not only to analyze the dynamic interplay of genes and proteins on the border of eve stripes but also to infer that eve stripes are established and maintained by network motifs built by the cooperation between transcription regulations and diffusion mechanisms in early embryogenesis. Literature reference with the wet experiments of gene mutations provides a clue for validating the identified network. The proposed spatio-temporal dynamic model can be extended to gene/protein network construction of different biological phenotypes, which depend on compartments, e.g. postnatal stem/progenitor cell differentiation. PMID:20054403

  11. Early postnatal testosterone predicts sex-related differences in early expressive vocabulary.

    PubMed

    Kung, Karson T F; Browne, Wendy V; Constantinescu, Mihaela; Noorderhaven, Rebecca M; Hines, Melissa

    2016-06-01

    During the first few years of life, girls typically have a larger expressive vocabulary than boys. This sex difference is important since a small vocabulary may predict subsequent language difficulties, which are more prevalent in boys than girls. The masculinizing effects of early androgen exposure on neurobehavioral development are well-documented in nonhuman mammals. The present study conducted the first test of whether early postnatal testosterone concentrations influence sex differences in expressive vocabulary in toddlers. It was found that testosterone measured in saliva samples collected at 1-3 months of age, i.e., during the period called mini-puberty, negatively predicted parent-report expressive vocabulary size at 18-30 months of age in boys and in girls. Testosterone concentrations during mini-puberty also accounted for additional variance in expressive vocabulary after other predictors such as sex, child's age at vocabulary assessment, and paternal education, were taken into account. Furthermore, testosterone concentrations during mini-puberty mediated the sex difference in expressive vocabulary. These results suggest that testosterone during the early postnatal period contributes to early language development and neurobehavioral sexual differentiation in humans. PMID:26970201

  12. 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

  13. 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.

  14. 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.

  15. P53 protein expression in human leukemia and lymphoma cells.

    PubMed

    Koníková, E; Kusenda, J

    2001-01-01

    The purpose of this study was to determine the value of p53 protein overexpression in human leukemia and lymphoma cells. We examined PB and/or BM samples on a series of 111 patients with immunophenotypically defined hematological malignancies at diagnosis, in remission and in relapsed disease comparing to 20 control samples of healthy individuals. p53 protein has been studied by flow cytometry using three monoclonal antibodies specific for epitopes on N-terminus (Bp53-12, DO-1) and central region (DO-11) of p53 protein. Our findigs showed, that p53 expression may contribute to phenotype of leukemic cells and that overexpression of this protein is often associated with progression of disease. All samples of early B-ALL patients and samples of patients with immunophenotypically defined T- cell disorders examined at diagnosis of disease were p53 positive. Eleven of 19 patient samples from AML at diagnosis showed also increased expression of p53 protein. The cells of all patients who responded to therapy with complete immunophenotypically defined remission were p53 negative. Relapsed T-, B- ALL and AML develop p53 alteration. We reported positive p53 expression in cells of patients with advanced stages of CLL in comparison to them with initial stage of disease at examination. As well as in the group of B- cell lymphomas only samples of patients with generalized FCC lymphoma at diagnosis were p53 positive. We detected p53 positive cells in immunologically defined myeloid blast crisis of CML opposite to p53 negativity in chronic phase of disease. The finding of p53 positive BM cells without immunophenotypic blast markers in two of followed cases documented the contributing value of p53 detection in their characterization. On the basis of above findings we conclude, that cytofluorometric determination of p53 expression may contribute to the better definition of leukemic phenotype. Loss of the normal p53 function may be important in the genesis of some leukemias

  16. 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

  17. Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

    PubMed Central

    Peeples, M E; Wang, C; Gupta, K C; Coleman, N

    1992-01-01

    A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization. Images PMID:1560547

  18. Autoregulation of Adenovirus Type 5 Early Gene Expression II. Effect of Temperature-Sensitive Early Mutations on Virus RNA Accumulation

    PubMed Central

    Carter, T. H.; Blanton, R. A.

    1978-01-01

    The kinetics of accumulation of early virus RNA in the cytoplasm of KB cells infected at 40.5°C by wild-type (WT) adenovirus type 5 and a temperature-sensitive “early” mutant, H5ts125 (ts125), were compared by hybridization of unlabeled RNA in solution to the 3H-labeled l strand of Ad5 DNA HindIII restriction endonuclease fragment A. In the presence of 1-β-d-arabinofuranosylcytosine, Al RNA accumulated in WT-infected cells for 9 h and then decreased in concentration to 6% of the 9-h concentration by 18 h. In ts125-infected cells, Al RNA accumulated for 12 h and then remained at the same concentration for at least 6 h thereafter. The concentrations of virus RNA from the four early transcription regions of the genome were measured at 15 h in cells infected at 40.5°C in the presence of 1-β-d-arabinofuranosylcytosine by: (i) ts125 and WT; (ii) two other ts early mutants, ts107 and ts149; and (iii) a revertant of ts125. The revertant and ts149, a mutant from a different complementation group than ts125, both accumulated all early virus cytoplasmic RNA species in amounts similar to, or less than, WT. However, both ts125 and ts107, independently isolated mutations in the 72,000-molecular-weight (72K) DNA-binding protein gene, accumulated cytoplasmic early RNA in excess of that found in WT infection. This pattern of RNA accumulation with the mutants and WT virus was the same in the nuclei as in the cytoplasm at 40.5°C. At 32°C, however, the abundance of nuclear virus RNA from all four early regions was the same in cells infected by either ts125 or WT. Differences in the relative abundance of nuclear RNA from the four early regions were observed in cells infected at 40.5 and 32°C, but were not dependent upon the infecting virus genotype. These results are consistent with autoregulation of early gene expression by the 72K protein and support the hypothesis that the 72K protein either decreases the rate of early virus transcription or increases the rate of virus

  19. Purify First: rapid expression and purification of proteins from XMRV.

    PubMed

    Gillette, William K; Esposito, Dominic; Taylor, Troy E; Hopkins, Ralph F; Bagni, Rachel K; Hartley, James L

    2011-04-01

    Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale. PMID:21146612

  20. 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

  1. Differential expression of CaMK-II genes during early zebrafish embryogenesis.

    PubMed

    Rothschild, Sarah C; Lister, James A; Tombes, Robert M

    2007-01-01

    CaMK-II is a highly conserved Ca(2+)/calmodulin-dependent protein kinase expressed throughout the lifespan of all vertebrates. During early development, CaMK-II regulates cell cycle progression and "non-canonical" Wnt-dependent convergent extension. In the zebrafish, Danio rerio, CaMK-II activity rises within 2 hr after fertilization. At the time of somite formation, zygotic expression from six genes (camk2a1, camk2b1, camk2g1, camk2g2, camk2d1, camk2d2) results in a second phase of increased activity. Zebrafish CaMK-II genes are 92-95% identical to their human counterparts in the non-variable regions. During the first three days of development, alternative splicing yields at least 20 splice variants, many of which are unique. Whole-mount in situ hybridization reveals that camk2g1 comprises the majority of maternal expression. All six genes are expressed strongly in ventral regions at the 18-somite stage. Later, camk2a1 is expressed in anterior somites, heart, and then forebrain. Camk2b1 is expressed in somites, mid- and forebrain, gut, retina, and pectoral fins. Camk2g1 appears strongly along the midline and then in brain, gut, and pectoral fins. Camk2g2 is expressed early in the midbrain and trunk and exhibits the earliest retinal expression. Camk2d1 is elevated early at somite boundaries, then epidermal tissue, while camk2d2 is expressed in discrete anterior locations, steadily increasing along either side of the dorsal midline and then throughout the brain, including the retina. These findings reveal a complex pattern of CaMK-II gene expression consistent with pleiotropic roles during development. PMID:17103413

  2. Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells.

    PubMed Central

    Cai, W; Schaffer, P A

    1992-01-01

    The herpes simplex virus type 1 protein, ICP0, can activate expression of all kinetic classes of viral promoters in transient expression assays. To examine the role of ICP0 in the regulation of viral gene expression during productive infection, we characterized the wild-type virus, an ICP0 null mutant (7134), and several ICP0 nonsense mutant viruses with regard to virus replication and protein synthesis in Vero cells. Relative to wild-type virus, 7134 was severely deficient in viral growth and protein synthesis at low multiplicities of infection but exhibited a nearly wild-type phenotype at high multiplicities. The phenotypes of the ICP0 nonsense mutants were intermediate between those of the wild-type virus and 7134 in that the more ICP0-coding sequence expressed by a given nonsense mutant, the more wild type-like was its phenotype. The location of the ICP0 domain responsible for transactivation during productive infection was confirmed to be within the N-terminal portion of the protein, as previously shown in transient expression assays. Immunoprecipitation and immunofluorescence tests were used to detect low-level expression of selected immediate-early (IE), early (E), and late (L) proteins by mutant and wild-type viruses following low-multiplicity infection. The 7134 deletion mutant and several nonsense mutants expressed markedly reduced levels of E and L proteins but wild-type levels of the IE protein, ICP4. Because the latency-associated transcripts (LATs) are specified by the strand opposite that which encodes ICP0, the ICP0 deletion and nonsense mutants are by definition ICP0-LAT double mutants. The ability of a LAT- ICP0+ mutant to replicate as efficiently as wild-type virus at low multiplicities and the ability of ICP0-expressing 0-28 cells to complement the defects of the mutants in E and L protein synthesis indicates that the phenotypes of the mutants are caused by mutations in ICP0 and not the LATs. Thus, we conclude that ICP0 up-regulates E and L but

  3. Proteomics beyond large-scale protein expression analysis.

    PubMed

    Boersema, Paul J; Kahraman, Abdullah; Picotti, Paola

    2015-08-01

    Proteomics is commonly referred to as the application of high-throughput approaches to protein expression analysis. Typical results of proteomics studies are inventories of the protein content of a sample or lists of differentially expressed proteins across multiple conditions. Recently, however, an explosion of novel proteomics workflows has significantly expanded proteomics beyond the analysis of protein expression. Targeted proteomics methods, for example, enable the analysis of the fine dynamics of protein systems, such as a specific pathway or a network of interacting proteins, and the determination of protein complex stoichiometries. Structural proteomics tools allow extraction of restraints for structural modeling and identification of structurally altered proteins on a proteome-wide scale. Other variations of the proteomic workflow can be applied to the large-scale analysis of protein activity, location, degradation and turnover. These exciting developments provide new tools for multi-level 'omics' analysis and for the modeling of biological networks in the context of systems biology studies. PMID:25636126

  4. HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins.

    PubMed

    Yadavalli, Raghavendra; Sam-Yellowe, Tobili

    2015-01-01

    Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed "in vitro human cell free expression systems". The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 µl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer's Cleft - 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 µl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford's assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be

  5. 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.

  6. SUMO fusion technology for difficult-to-express proteins.

    PubMed

    Butt, Tauseef R; Edavettal, Suzanne C; Hall, John P; Mattern, Michael R

    2005-09-01

    The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the field of protein production and purification. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good purification methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, purification, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems. PMID:16084395

  7. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    PubMed

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

  8. Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.

    PubMed

    Song, Hye-Won; Cauffman, Karen; Chan, Agnes P; Zhou, Yi; King, Mary Lou; Etkin, Laurence D; Kloc, Malgorzata

    2007-07-01

    The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs. PMID:17309605

  9. 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. PMID:25788524

  10. Influence of Isoflurane on Immediate-Early Gene Expression

    PubMed Central

    Bunting, Kristopher M.; Nalloor, Rebecca I.; Vazdarjanova, Almira

    2016-01-01

    Background: Anterograde amnesia is a hallmark effect of volatile anesthetics. Isoflurane is known to affect both the translation and transcription of plasticity-associated genes required for normal memory formation in many brain regions. What is not known is whether isoflurane anesthesia prevents the initiation of transcription or whether it halts transcription already in progress. We tested the hypothesis that general anesthesia with isoflurane prevents learning-induced initiation of transcription of several memory-associated immediate-early genes (IEGs) correlated with amnesia; we also assessed whether it stops transcription initiated prior to anesthetic administration. Methods: Using a Tone Fear Conditioning paradigm, rats were trained to associate a tone with foot-shock. Animals received either no anesthesia, anesthesia immediately after training, or anesthesia before, during, and after training. Animals were either sacrificed after training or tested 24 h later for long-term memory. Using Cellular Compartment Analysis of Temporal Activity by Fluorescence in situ Hybridization (catFISH), we examined the percentage of neurons expressing the IEGs Arc/Arg3.1 and Zif268/Egr1/Ngfi-A/Krox-24 in the dorsal hippocampus, primary somatosensory cortex, and primary auditory cortex. Results: On a cellular level, isoflurane administered at high doses (general anesthesia) prevented initiation of transcription, but did not stop transcription of Arc and Zif268 mRNA initiated prior to anesthesia. On a behavioral level, the same level of isoflurane anesthesia produced anterograde amnesia for fear conditioning when administered before and during training, but did not produce retrograde amnesia when administered immediately after training. Conclusion: General anesthesia with isoflurane prevents initiation of learning-related transcription but does not stop ongoing transcription of two plasticity-related IEGs, Arc and Zif268, a pattern of disruption that parallels the effects of

  11. Aberrant expression of signaling proteins in essential thrombocythemia.

    PubMed

    Hui, Wuhan; Ye, Fei; Zhang, Wei; Liu, Congyan; Cui, Miao; Li, Wei; Xu, Juan; Zhang, David Y

    2013-09-01

    Dysregulated expression of signaling proteins may contribute to the pathophysiology of essential thrombocythemia (ET). This study aimed to characterize protein expression in ET and to correlate the dysregulated proteins with phenotypes and prognosis of ET patients. The expression of 128 proteins in peripheral blood neutrophils from 74 ET patients was assessed and compared with those from 29 healthy subjects and 35 polycythemia vera (PV) patients using protein pathway array. Fifteen proteins were differentially expressed between ET patients and normal controls. These dysregulated proteins were involved in the signaling pathways related with apoptosis and inflammation. Our results showed a significant overlap in protein expression between ET patients with JAK2V617F mutation and PV patients. In addition, nine proteins were associated with JAK2V617F mutation status in ET patients. Furthermore, estrogen receptor beta (ERβ) and Stat3 were independent risk factors for subsequent thrombosis during follow-up on multivariable analysis. Our study shows a broad dysregulation of signaling protein in ET patients, suggesting their roles in ET pathogenesis. The expression levels of ERβ and Stat3 could be promising predictors of subsequent thrombosis in ET patients. PMID:23639951

  12. Cloning and expression of special F protein from human liver

    PubMed Central

    Liu, Shu-Ye; Yu, Xin-Da; Song, Chun-Juan; Lu, Wei; Zhang, Jian-Dong; Shi, Xin-Rong; Duan, Ying; Zhang, Ju

    2007-01-01

    AIM: To clone human liver special F protein and to express it in a prokaryotic system. METHODS: Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this, cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein’s cDNA was subcloned into the expression vector pET-15b and transformed into E. coli BL21 (DE3) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein. RESULTS: The cDNA clone of human liver special F protein (1134bp) was successfully produced, with the cDNA sequence being published in Gene-bank: DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted. CONCLUSION: F protein expresses cDNA clone in a prokaryotic system, which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein. PMID:17465469

  13. Relating protein adduction to gene expression changes: a systems approach

    PubMed Central

    Zhang, Bing; Shi, Zhiao; Duncan, Dexter T; Prodduturi, Naresh; Marnett, Lawrence J; Liebler, Daniel C

    2013-01-01

    Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data. PMID:21594272

  14. Pannexin 2 protein expression is not restricted to the CNS

    PubMed Central

    Le Vasseur, Maxence; Lelowski, Jonathan; Bechberger, John F.; Sin, Wun-Chey; Naus, Christian C.

    2014-01-01

    Pannexins (Panx) are proteins homologous to the invertebrate gap junction proteins called innexins (Inx) and are traditionally described as transmembrane channels connecting the intracellular and extracellular compartments. Three distinct Panx paralogs (Panx1, Panx2 and Panx3) have been identified in vertebrates but previous reports on Panx expression and functionality focused primarily on Panx1 and Panx3 proteins. Several gene expression studies reported that Panx2 transcript is largely restricted to the central nervous system (CNS) hence suggesting that Panx2 might serve an important role in the CNS. However, the lack of suitable antibodies prevented the creation of a comprehensive map of Panx2 protein expression and Panx2 protein localization profile is currently mostly inferred from the distribution of its transcript. In this study, we characterized novel commercial monoclonal antibodies and surveyed Panx2 expression and distribution at the mRNA and protein level by real-time qPCR, Western blotting and immunofluorescence. Panx2 protein levels were readily detected in every tissue examined, even when transcriptional analysis predicted very low Panx2 protein expression. Furthermore, our results indicate that Panx2 transcriptional activity is a poor predictor of Panx2 protein abundance and does not correlate with Panx2 protein levels. Despite showing disproportionately high transcript levels, the CNS expressed less Panx2 protein than any other tissues analyzed. Additionally, we showed that Panx2 protein does not localize at the plasma membrane like other gap junction proteins but remains confined within cytoplasmic compartments. Overall, our results demonstrate that the endogenous expression of Panx2 protein is not restricted to the CNS and is more ubiquitous than initially predicted. PMID:25505382

  15. Expression strategies for structural studies of eukaryotic membrane proteins.

    PubMed

    Lyons, Joseph A; Shahsavar, Azadeh; Paulsen, Peter Aasted; Pedersen, Bjørn Panyella; Nissen, Poul

    2016-06-01

    Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy. PMID:27362979

  16. mir-29 regulates Mcl-1 protein expression and apoptosis.

    PubMed

    Mott, J L; Kobayashi, S; Bronk, S F; Gores, G J

    2007-09-13

    Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies, because Mcl-1 is dysregulated in cells with the malignant phenotype. By in silico analysis, we identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated the expression of an Mcl-1 3' untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression, and thereby, apoptosis. PMID:17404574

  17. mir-29 Regulates Mcl-1 Protein Expression and Apoptosis

    PubMed Central

    Mott, Justin L.; Kobayashi, Shogo; Bronk, Steven F.; Gores, Gregory J.

    2008-01-01

    Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies because Mcl-1 is dysregulated in cells with the malignant phenotype. In silico analysis identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and we found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated expression of an Mcl-1 3’ untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to TRAIL cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression and, thereby, apoptosis. PMID:17404574

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

    PubMed Central

    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. PMID:27029048

  19. 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. PMID:27029048

  20. Telomerase expression in noncancerous bronchial epithelia is a possible marker of early development of lung cancer.

    PubMed

    Miyazu, Yuka Matsuoka; Miyazawa, Teruomi; Hiyama, Keiko; Kurimoto, Noriaki; Iwamoto, Yasuo; Matsuura, Hiroo; Kanoh, Koji; Kohno, Nobuoki; Nishiyama, Masahiko; Hiyama, Eiso

    2005-11-01

    Centrally located lung cancers in smokers frequently associated with subsequent primary tumors. We evaluated the telomerase expression chronologically in noncancerous epithelia as a risk factor of susceptibility to lung cancer development. Telomerase protein expression was examined in situ by immunohistochemistry in 26 noncancerous bronchial epithelia adjacent to centrally located early-stage lung cancers in sequential 23 patients treated by photodynamic therapy or surgery among 206 patients who underwent autofluorescence bronchoscopy from 1997 to 2003. Among the 15 lesions in 12 patients treated by photodynamic therapy alone, 11 lesions achieved complete remission after photodynamic therapy, and none of their noncancerous bronchial epithelia was telomerase positive. On the contrary, in the remaining four lesions, either recurrence or secondary lung cancer developed adjacent to the successfully treated primary cancer within 26 months, and the telomerase protein expression in noncancerous epithelia was detected before the secondary cancer development (P < 0.001). The overall relationship of human telomerase reverse transcriptase positivity in noncancerous epithelia and subsequent lung cancer development, including patients treated by radiation or surgery, showed higher significance (P < 0.0001). Histologically "normal" bronchial epithelia in smokers may unphysiologically express telomerase as a field, and such epithelia are likely susceptible to develop lung cancer. We propose that ectopic expression of telomerase in bronchial epithelia may precede transformation in human lung cancer development and that detection of telomerase protein in noncancerous bronchial epithelia will become a useful marker detecting high-risk patients for lung cancer development. PMID:16266979

  1. 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

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

    PubMed

    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

  3. 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.

  4. 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.

  5. Gene expression profiling in spleens of deoxynivalenol-exposed mice: immediate early genes as primary targets.

    PubMed

    Kinser, Shawn; Jia, Qunshan; Li, Maioxing; Laughter, Ashley; Cornwell, Paul; Corton, J Christopher; Pestka, James

    2004-09-24

    Exposure to the trichothecene mycotoxin deoxynivalenol (DON) alters immune functions in vitro and in vivo. To gain further insight into DON's immunotoxic effects, microarrays were used to determine how acute exposure to this mycotoxin modulates gene expression profiles in murine spleen. B6C3F1 mice were treated orally with 25mg/kg body weight DON, and 2h later spleens were collected for macroarray analysis. Following normalization using a local linear regression model, expression of 116 out of 1176 genes was significantly altered compared to average expression levels in all treatment groups. When genes were arranged into an ontology tree to facilitate comparison of expression profiles between treatment groups, DON was found primarily to modulate genes associated with immunity, inflammation, and chemotaxis. Real-time polymerase chain reaction was used to confirm modulation for selected genes. DON was found to induce the cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and IL-11. In analogous fashion, DON upregulated expression of the chemokines macrophage inhibitory protein-2 (MIP-2), cytokine-induced chemoattractant protein-1 (CINC-1), monocyte chemoattractant protein (MCP)-1, MCP-3, and cytokine-responsive gene-2 (CRG-2). c-Fos, Fra-, c-Jun, and JunB, components of the activator protein-1 (AP-1) transcription factor complex, were induced by DON as well as another transcription factor, NR4A1. Four hydrolases were found to be upregulated by DON, including mitogen-activated protein kinase phosphatase 1 (MKP1), catalytic subunit beta isoform (CnAbeta), protein tyrosine phosphatase receptor type J (Ptprj), and protein tyrosine phosphatase nonreceptor type 8 (Ptpn8), whereas three other hydrolases, microsomal epoxide hydrolase (Eph) 1, histidine triad nucleotide binding protein (Hint), and proteosome subunit beta type 8 (Psmb8) were significantly decreased by the toxin. Finally, cysteine-rich protein 61 (CRP61) and heat-shock protein 40 (Hsp40), genes associated with

  6. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Expression dynamics of NADPH oxidases during early zebrafish development.

    PubMed

    Weaver, Cory J; Leung, Yuk Fai; Suter, Daniel M

    2016-07-01

    Nicotinamide dinucleotide phosphate oxidases (NOX) control various cellular signaling cascades. In the nervous system, there is recent evidence that NOX-derived reactive oxygen species (ROS) regulate neurite outgrowth, regeneration, and stem cell proliferation; however, a comprehensive NOX gene expression analysis is missing for all major model systems. Zebrafish embryos provide an excellent model system to study neurodevelopment and regeneration because they develop quickly and are well suited for in vivo imaging and molecular approaches. Although the sequences of five NOX genes (nox1, nox2/cybb, nox4, nox5, and duox) have been identified in the zebrafish genome, nothing is known about their expression pattern. Here, we used quantitative polymerase chain reaction combined with in situ hybridization to develop a catalog of nox1, nox2/cybb, nox5, and duox expression in zebrafish during early nervous system development from 12 to 48 hours post fertilization. We found that expression levels of nox1, nox5, and duox are dynamic during the first 2 days of development, whereas nox2/cybb levels remain remarkably stable. By sectioning in situ hybridized embryos, we found a pattern of broad and overlapping NOX isoform expression at 1 and 1.5 days post fertilization. After 2 days of development, a few brain regions displayed increased NOX expression levels. Collectively, these results represent the first comprehensive analysis of NOX gene expression in the zebrafish and will provide a basis for future studies aimed at determining the functions of NOX enzymes in neurodevelopment and regeneration. J. Comp. Neurol. 524:2130-2141, 2016. © 2015 Wiley Periodicals, Inc. PMID:26662995

  9. 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.

  10. Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

    PubMed Central

    Weidner, Maria; Taupp, Marcus; Hallam, Steven J.

    2010-01-01

    Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system. As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1]. As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3]. Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4]. The vector used here (pPICZαA) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the α-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector. PMID:20186119

  11. 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

  12. Immunological evasion of immediate-early varicella zoster virus proteins.

    PubMed

    Meysman, Pieter; Fedorov, Dmitry; Van Tendeloo, Viggo; Ogunjimi, Benson; Laukens, Kris

    2016-07-01

    The varicella zoster virus (VZV) causes the childhood disease commonly known as chickenpox and can later in life reactivate as herpes zoster. The adaptive immune system is known to play an important role in suppressing VZV reactivation. A central aspect of this system is the presentation of VZV-derived peptides by the major histocompatibility complex (MHC) proteins. Here, we investigate if key VZV proteins have evolved their amino acid sequence to avoid presentation by MHC based on predictive models of MHC-peptide affinity. This study shows that the immediate-early proteins of all characterized VZV strains are profoundly depleted for high-affinity MHC-I-restricted epitopes. The same depletion can be found in its closest animal analog, the simian varicella virus. Further orthology analysis towards other herpes viruses suggests that the protein amino acid frequency is one of the primary drivers of targeted epitope depletion. PMID:27020058

  13. Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

    PubMed Central

    Flick, Kirsten; Ahuja, Sanjay; Chene, Arnaud; Bejarano, Maria Teresa; Chen, Qijun

    2004-01-01

    Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons

  14. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  15. Insulin influenced expression of myelin proteins in diabetic peripheral neuropathy.

    PubMed

    Rachana, Kuruvanthe S; Manu, Mallahalli S; Advirao, Gopal M

    2016-08-26

    Diabetic peripheral neuropathy (DPN) is one of the downstream complications of diabetes. This complication is caused by the deficiency of insulin action and subsequent hyperglycemia, but the details of their pathogenesis remain unclear. Hence, it is of critical importance to understand how such hormonal variation affects the expression of myelin proteins such as myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in the peripheral nerve. An earlier report from our lab has demonstrated the expression of insulin receptors (IR) in Schwann cells (SCs) of sciatic nerve. To assess the neurotrophic role of insulin in diabetic neuropathy, we studied the expression of these myelin proteins under control, DPN and insulin treated DPN subjects at developmental stages. Further, the expression of these myelin proteins was correlated with the expression of insulin receptor. Expression of myelin proteins was significantly reduced in the diabetic model compared to normal, and upregulated in insulin treated diabetic rats. Similarly, an in vitro study was also carried out in SCs grown at high glucose and insulin treated conditions. The expression pattern of myelin proteins in SCs was comparable to that of in vivo samples. In addition, quantitative study of myelin genes by real time PCR has also showed the significant expression pattern change in the insulin treated and non-treated DPN subjects. Taken together, these results corroborate the critical importance of insulin as a neurotrophic factor in demyelinized neurons in diabetic neuropathy. PMID:27373589

  16. Early Differential Expression of Oncostatin M in Obstructive Nephropathy

    PubMed Central

    Truong, Luan D.; Tawil, Ahmad; Wang, Wansheng; Dawson, Sara; Lan, Hui Y.; Zhang, Ping; Garcia, Gabriela E.; Smith, C. Wayne

    2010-01-01

    Interstitial fibrosis plays a major role in progression of renal diseases. Oncostatin M (OSM) is a cytokine that regulates cell survival, differentiation, and proliferation. Renal tissue from patients with chronic obstructive nephropathy was examined for OSM expression. The elevated levels in diseased human kidneys suggested possible correlation between OSM level and kidney tissue fibrosis. Indeed, unilateral ureteral obstruction (UUO), a model of renal fibrosis, increased OSM and OSM receptor (OSM-R) expression in a time-dependent manner within hours following UUO. In vitro, OSM overexpression in tubular epithelial cells (TECs) resulted in epithelial-myofibroblast transdifferentiation. cDNA microarray technology identified up-regulated expression of immune modulators in obstructed compared with sham-operated kidneys. In vitro, OSM treatment up-regulated CC chemokine ligand CCL7, and CXC chemokine ligand (CXCL)-14 mRNA in kidney fibroblasts. In vivo, treatment of UUO mice with neutralizing anti-OSM antibody decreased renal chemokines expression. In conclusion, OSM is up-regulated in kidney tissue early after urinary obstruction. Therefore, OSM might play an important role in initiation of renal fibrogenesis, possibly by inducing myofibroblast transdifferentiation of TECs as well as leukocyte infiltration. This process may, in turn, contribute in part to progression of obstructive nephropathy and makes OSM a promising therapeutic target in renal fibrosis. PMID:20626292

  17. 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

  18. 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. PMID:19156736

  19. Protein expression analyses at the single cell level.

    PubMed

    Ohno, Masae; Karagiannis, Peter; Taniguchi, Yuichi

    2014-01-01

    The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. PMID:25197931

  20. Varicella-zoster virus open reading frame 4 encodes an immediate-early protein with posttranscriptional regulatory properties.

    PubMed Central

    Defechereux, P; Debrus, S; Baudoux, L; Rentier, B; Piette, J

    1997-01-01

    Varicella-zoster virus (VZV) encodes four putative immediate-early proteins based on sequence homology with herpes simplex virus type 1: the products of ORF4, -61, -62, and -63. Until now, only two VZV proteins have been described as being truly expressed with immediate-early kinetics (IE62 and IE63). The ORF4-encoded protein can stimulate gene expression either alone or in synergy with the major regulatory protein IE62. Making use of a sequential combination of transcription and protein synthesis inhibitors (actinomycin D and cycloheximide, respectively), we demonstrated the immediate-early nature of the ORF4 gene product, which can thus be named IE4. The fact that IE4 is expressed with kinetics similar to that of IE62 further underlines the possible cooperation between these two VZV proteins in gene expression. Analysis of the IE4-mediated autologous or heterologous viral gene expression at the mRNA levels clearly indicated that IE4 may have several mechanisms of action. Activation of the two VZV genes tested could occur partly by a posttranscriptional mechanism, since increases in chloramphenicol acetyltransferase (CAT) mRNA levels do not account for the stimulation of CAT activity. On the other hand, stimulation of the human immunodeficiency virus type 1 long terminal repeat- or the cytomegalovirus promoter-associated CAT activity is correlated with an increase in the corresponding CAT mRNA. PMID:9261438

  1. 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. PMID:27581123

  2. 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

  3. 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. PMID:24780627

  4. A Gene Expression Atlas of Early Craniofacial Development

    PubMed Central

    Brunskill, Eric W.; Potter, Andrew S.; Distasio, Andrew; Dexheimer, Phillip; Plassard, Andrew; Aronow, Bruce J.; Potter, S. Steven

    2014-01-01

    We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical micro-regions, 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. PMID:24780627

  5. Translocation of a store of maternal cytoplasmic c-myc protein into nuclei during early development.

    PubMed Central

    Gusse, M; Ghysdael, J; Evan, G; Soussi, T; Méchali, M

    1989-01-01

    The c-myc proto-oncogene is expressed as a maternal protein during oogenesis in Xenopus laevis, namely, in nondividing cells. A delayed translation of c-myc mRNA accumulated in early oocytes results in the accumulation of the protein during late oogenesis. The oocyte c-myc protein is unusually stable and is located in the cytoplasm, contrasting with its features in somatic cells. A mature oocyte contains a maternal c-myc protein stockpile of 4 x 10(5) to 6 x 10(5) times the level in a somatic growing cell. This level of c-myc protein is preserved only during the cleavage stage of the embryo. Fertilization triggers its rapid migration into the nuclei of the cleaving embryo and a change in the phosphorylation state of the protein. The c-myc protein content per nucleus decreases exponentially during the cleavage stage until a stoichiometric titration by the embryonic nuclei is reached during a 0.5-h period at the midblastula stage. Most of the maternal c-myc store is degraded by the gastrula stage. These observations implicate the participation of c-myc in the events linked to early embryonic development and the midblastula transition. Images PMID:2685563

  6. 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

  7. Expression of trisomic proteins in Down syndrome model systems.

    PubMed

    Spellman, Claire; Ahmed, Md Mahiuddin; Dubach, Daphne; Gardiner, Katheleen J

    2013-01-10

    Down syndrome (DS) is the most common genetic aberration leading to intellectual disability. DS results from an extra copy of the long arm of human chromosome 21 (HSA21) and the increased expression of trisomic genes due to gene dosage. While expression in DS and DS models has been studied extensively at the RNA level, much less is known about expression of trisomic genes at the protein level. We have used quantitative Western blotting with antibodies to 20 proteins encoded by HSA21 to assess trisomic protein expression in lymphoblastoid cell lines (LCLs) from patients with DS and in brains from two mouse models of DS. These antibodies have recently become available and the 20 proteins largely have not been investigated previously for their potential contributions to the phenotypic features of DS. Twelve proteins had detectable expression in LCLs and three, CCT8, MX1 and PWP2, showed elevated levels in LCLs derived from patients with DS compared with controls. Antibodies against 15 proteins detected bands of appropriate sizes in lysates from mouse brain cortex. Genes for 12 of these proteins are trisomic in the Tc1 mouse model of DS, but only SIM2 and ZNF295 showed elevated expression in Tc1 cortex when compared with controls. Genes for eight of the 15 proteins are trisomic in the Ts65Dn mouse model of DS, but only ZNF294 was over expressed in cortex. Comparison of trisomic gene expression at the protein level with previous reports at the mRNA level showed many inconsistencies. These may be caused by natural inter-individual variability, differences in the age of mice analyzed, or post-transcriptional regulation of gene dosage effects. These antibodies provide resources for further investigation of the molecular basis of intellectual disability in DS. PMID:23103828

  8. 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. PMID:20457256

  9. 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. PMID:27063333

  10. DNA damage promotes Herpes Simplex Virus-1 protein expression in a neuroblastoma cell line

    PubMed Central

    Volcy, Ketna; Fraser, Nigel W.

    2013-01-01

    Although the induction of the cellular DNA damage response by Herpes simplex virus-1 (HSV-1) infection of epithelial cells in tissue culture promotes productive infection, there has been no experimental observation of the effect of the cellular DNA damage response on HSV-1 infection in vivo or in neuronal derived cell lines in tissue culture. Thus, it has been speculated that the lack of cellular DNA damage induction during infection of neurons may promote latency in these cells. This work examines the profile of HSV-1 promoter induction and protein expression, in the absence or presence of infection; using cellular DNA damage inducing topoisomerase inhibitors (Camptothecin and Etoposide) on a neuroblastoma cell line (C1300) in which HSV-1 infection fails to induce the DNA damage response. In the absence of infection, a plasmid expressing the immediate early ICP0 promoter was the most induced by the DNA damage drug treatments compared to the early (RR) and late (VP16) gene promoters. Similarly, drug treatment of C1300 cells infected with HSV-1 virus showed enhanced protein expression for ICP0, but not ICP4 and VP16 proteins. However, when the cells were infected with a HSV-1 virus defective in the immediate early gene trans-activator VP16 (in814) and treated with the DNA damaging drugs, there was enhanced expression of immediate early and late HSV-1 proteins. Although, viral infection of the neuroblastoma cell alone did not induce DNA damage, cellular DNA damage induced by drug treatments facilitated viral promoter induction and viral protein expression. This implicates a mechanism by which HSV-1 viral genes in a quiescent or latent state may become induced by cellular DNA damage in neuronal cells to facilitate productive infection. PMID:23354549

  11. 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.

  12. 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. PMID:11742876

  13. Protein Expression in a Drosophila Model of Parkinson’s Disease

    PubMed Central

    Xun, Zhiyin; Sowell, Renã A.; Kaufman, Thomas C.; Clemmer, David E.

    2008-01-01

    Liquid chromatographies coupled to mass spectrometry and database analysis techniques are used to carry out a large-scale proteome characterization for a Drosophila model of Parkinson’s disease. Semi-quantitative analysis is performed on A30P α-synuclein expressing transgenic Drosophila and a control lacking the gene at pre-symptomatic, early and advanced disease stages. Changes in gene expression at the level of the proteome are compared with changes reported from published transcriptome measurements. A summary of the comparison indicates that ~44% of transcripts that show changes can also be observed as proteins. However, the patterns of change in protein expression vary substantially compared with the patterns of change observed for corresponding transcripts. In addition, the expression changes of many genes are observed for only transcripts or proteins. Proteome measurements provide evidence for dysregulation of a group of proteins associated with the actin cytoskeleton and mitochondrion at pre-symptomatic and early disease stages that may presage the development of later symptoms. Overall, the proteome measurements provide a view of gene expression that is highly complementary to the insights obtained from the transcriptome. PMID:17203978

  14. A multifunctional protein EWS is essential for early brown fat lineage determination

    PubMed Central

    Park, Jun Hong; Kang, Hong Jun; Kang, Soo Im; Lee, Ji Eun; Hur, Jamie; Ge, Kai; Mueller, Elisabetta; Li, Hongjie; Lee, Byeong-Chel; Lee, Sean Bong

    2013-01-01

    Summary A recent surge in obesity has given impetus to better understand the mechanisms of adipogenesis, particularly brown adipose tissue (BAT) due to its potential utilization for anti-obesity therapy. Postnatal brown adipocytes arise from early muscle-progenitors but how brown fat lineage is determined is not completely understood. Here, we show that a multifunctional protein EWS (Ewing Sarcoma) is essential for determining brown fat lineage during development. BATs from Ews-null embryos and newborns are developmentally arrested. Ews mutant brown preadipocytes fail to differentiate due to loss of Bmp7 expression, a critical early brown adipogenic factor. We demonstrate that EWS, along with its binding partner YBX1 (Y-box binding protein 1), activates Bmp7 transcription. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Remarkably, Ews-null BATs and brown preadipocytes ectopically express myogenic genes. These results demonstrate that EWS is essential for early brown fat lineage determination. PMID:23987512

  15. A multifunctional protein, EWS, is essential for early brown fat lineage determination.

    PubMed

    Park, Jun Hong; Kang, Hong Jun; Kang, Soo Im; Lee, Ji Eun; Hur, Jamie; Ge, Kai; Mueller, Elisabetta; Li, Hongjie; Lee, Byeong-Chel; Lee, Sean Bong

    2013-08-26

    The recent surge in obesity has provided an impetus to better understand the mechanisms of adipogenesis, particularly in brown adipose tissue (BAT) because of its potential utilization for antiobesity therapy. Postnatal brown adipocytes arise from early muscle progenitors, but how brown fat lineage is determined is not completely understood. Here, we show that a multifunctional protein, Ewing Sarcoma (EWS), is essential for determining brown fat lineage during development. BATs from Ews null embryos and newborns are developmentally arrested. Ews mutant brown preadipocytes fail to differentiate due to loss of Bmp7 expression, a critical early brown adipogenic factor. We demonstrate that EWS, along with its binding partner Y-box binding protein 1 (YBX1), activates Bmp7 transcription. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Remarkably, Ews null BATs and brown preadipocytes ectopically express myogenic genes. These results demonstrate that EWS is essential for early brown fat lineage determination. PMID:23987512

  16. Serum MX2 Protein as Candidate Biomarker for Early Pregnancy Diagnosis in Buffalo.

    PubMed

    Buragohain, L; Kumar, R; Nanda, T; Phulia, S K; Mohanty, A K; Kumar, S; Balhara, S; Ghuman, Sps; Singh, I; Balhara, A K

    2016-08-01

    Interferon-tau (IFN-τ)-induced molecular markers such as ubiquitin-like modifier (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and MX2) have generated immense attention towards developing diagnostic tools for early diagnosis of pregnancy in bovine. These molecules are expressed at transcriptional level in peripheral nucleated cells. However, their presence in the serum is still a question mark. This study reports sequential changes in expression of MX2 transcript in whole blood and serum MX2 protein level on days 0, 7, 14, 21, 28 and 35 in pregnant (n = 9) buffalo heifers, and on days 0, 7 and 14 in non-inseminated (n = 8) and inseminated non-pregnant (n = 10) control animals. In non-inseminated and inseminated non-pregnant heifers, the differential expression of MX2 transcript and MX2 protein level remained similar between day 7 and 14 post-oestrus. However, in pregnant heifers, on 14th and 28th day post-insemination MX2 transcript was 16.38 ± 1.57 and 28.16 ± 1.91 times upregulated as compared to day 0. Similarly, serum MX2 protein concentration followed analogous trend as MX2 transcript and increased gradually with the progression of pregnancy. Correlation analysis between expression of MX2 transcript and its serum protein level showed a significant positive correlation in pregnant animals, while it was random in other two groups. Therefore, MX2 surge at transcriptional and serum protein level after day 14-28 of pregnancy in buffalo holds potential for its use in early pregnancy detection. PMID:27393074

  17. 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

  18. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

    PubMed Central

    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. PMID:26327321

  19. GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

    PubMed Central

    Tatham, Amy L.; Crabtree, Mark J.; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J.; Channon, Keith M.

    2009-01-01

    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 (r2 = 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 (r2 = 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. PMID:19286659

  20. 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. PMID:19286659

  1. Small-scale expression of proteins in E. coli.

    PubMed

    Zerbs, Sarah; Giuliani, Sarah; Collart, Frank

    2014-01-01

    Proteins participate in virtually every cellular activity, and a knowledge of protein function is essential for an understanding of biological systems. However, protein diversity necessitates the application of an array of in vivo and in vitro approaches for characterization of the functional and biochemical properties of proteins. Methods that enable production of proteins for in vitro studies are critical for determination of the molecular, kinetic, and thermodynamic properties of these molecules. Ideally, proteins could be purified from the original source; however, the native host is often unsuitable for a number of reasons. Consequently, systems for heterologous protein production are commonly used to produce large amounts of protein. Heterologous expression hosts are chosen using a number of criteria, including genetic tractability, advantageous production or processing characteristics (secretion or posttranslational modifications), or economy of time and growth requirements. The subcloning process also provides an opportunity to introduce purification tags, epitope tags, fusions, truncations, and mutations into the coding sequence that may be useful in downstream purification or characterization applications. Bacterial systems for heterologous protein expression have advantages in ease of use, cost, short generation times, and scalability. These expression systems have been widely used by high-throughput protein production projects and often represent an initial experiment for any expression target. Escherichia coli has been studied for many years as a model bacterial organism and is one of the most popular hosts for heterologous protein expression (Terpe, 2006). Its protein production capabilities have been intensively studied, and the ease of genetic manipulation in this organism has led to the development of strains engineered exclusively for use in protein expression. These resources are widely available from commercial sources and public repositories

  2. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  3. Noninvasive Detection of Trophoblast Protein Signatures Linked to Early Pregnancy Loss using TRIC

    PubMed Central

    Fritz, Rani; Kohan-Ghadr, Hamid-Reza; Bolnick, Jay M.; Bolnick, Alan D.; Kilburn, Brian A.; Diamond, Michael P.; Drewlo, Sascha; Armant, D. Randall

    2015-01-01

    Objective To examine the expression pattern of biomarker proteins in extravillous trophoblast (EVT) cells obtained noninvasively by transcervical retrieval and isolation from the cervix (TRIC) in patients with early pregnancy loss, compared to control patients with uncomplicated term delivery. Design Case-control study. Setting Academic medical center. Patients Endocervical specimens were obtained from ongoing pregnancies at gestational ages of 5 to 10 weeks to generate an archive of EVT cells isolated by TRIC. Interventions Medical records were examined to select specimens from patients with either early pregnancy loss (EPL; N=10) or an uncomplicated term delivery (N=10), matched for gestational age at the time of endocervical sampling. Main Outcome Measures Known serum biomarkers for adverse pregnancy outcome that are expressed by EVT cells were evaluated by semi-quantitative immunocytochemistry, using antibodies against endoglin (ENG), FMS-like tyrosine kinase-1 (FLT-1), alpha-fetoprotein (AFP), pregnancy-associated plasma protein-A (PAPPA), galectin-13 (LGALS13), galectin-14 (LGALS14), and placental growth factor (PGF). Results EVT purity was over 95% in all specimens, based on chorionic gonadotropin expression; however, the number of EVT cells obtained was significantly lower in women with EPL than the control group. There was significant elevation of AFP, ENG, and FLT-1, and significant reduction of PAPP-A, LGALS14, and PGF in the EPL group, compared to controls. Conclusions In this pilot study, EVT cells isolated by TRIC early in gestation exhibit altered protein expression patterns prior to EPL, compared to uncomplicated term pregnancies. PMID:26051097

  4. 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.

  5. Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins

    PubMed Central

    Braun, Ralf J.

    2015-01-01

    Critically impaired protein degradation is discussed to contribute to neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's, and motor neuron diseases. Misfolded, aggregated, or surplus proteins are efficiently degraded via distinct protein degradation pathways, including the ubiquitin-proteasome system, autophagy, and vesicular trafficking. These pathways are regulated by covalent modification of target proteins with the small protein ubiquitin and are evolutionary highly conserved from humans to yeast. The yeast Saccharomyces cerevisiae is an established model for deciphering mechanisms of protein degradation, and for the elucidation of pathways underlying programmed cell death. The expression of human neurotoxic proteins triggers cell death in yeast, with neurotoxic protein-specific differences. Therefore, yeast cell death models are suitable for analyzing the role of protein degradation pathways in modulating cell death upon expression of disease-causing proteins. This review summarizes which protein degradation pathways are affected in these yeast models, and how they are involved in the execution of cell death. I will discuss to which extent this mimics the situation in other neurotoxic models, and how this may contribute to a better understanding of human disorders. PMID:25814926

  6. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  7. 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

  8. 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

  9. Expression of Putative Immune Response Genes during Early Ontogeny in the Coral Acropora millepora

    PubMed Central

    Puill-Stephan, Eneour; Seneca, François O.; Miller, David J.; van Oppen, Madeleine J. H.; Willis, Bette L.

    2012-01-01

    Background Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals. Methodology/Principal Findings Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned. Conclusions/Significance Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of

  10. Early changes in lung gene expression due to high tidal volume.

    PubMed

    Copland, Ian B; Kavanagh, Brian P; Engelberts, Doreen; McKerlie, Colin; Belik, Jaques; Post, Martin

    2003-11-01

    The purpose of this study was to use gene expression profiling to understand how adult rat lung responds to high tidal volume (HV) ventilation in vivo. HV ventilation for 30 minutes did not cause discernable lung injury (in terms of altered mechanics or histology) but caused obvious injury when continued for 90 minutes. However, at 30-minute ventilation, HV caused significant upregulation of 10 genes and suppression of 12 genes. Among the upregulated genes were transcription factors, stress proteins, and inflammatory mediators; the downregulated genes were exemplified by metabolic regulatory genes. On the basis of cluster analysis, we studied Egr-1, c-Jun, heat shock protein 70, and interleukin (IL)-1beta in further detail. Temporal studies demonstrated that Egr-1 and c-Jun were increased early and before heat shock protein 70 and IL-1beta. Spatial studies using in situ hybridization and laser capture microscopy revealed that all four genes were upregulated primarily in the bronchiolar airway epithelium. Furthermore, at 90 minutes of HV ventilation, a significant increase in intracellular IL-1beta protein was observed. Although there are limitations to gene array methodology, the current data suggest a global hypothesis that (1). the effects of HV are cumulative; (2). specific patterns of gene activation and suppression precede lung injury; and (3). alteration of gene expression after mechanical stretch is pathogenic. PMID:12816737

  11. Whole-Embryo Modeling of Early Segmentation in Drosophila Identifies Robust and Fragile Expression Domains

    PubMed Central

    Bieler, Jonathan; Pozzorini, Christian; Naef, Felix

    2011-01-01

    Segmentation of the Drosophila melanogaster embryo results from the dynamic establishment of spatial mRNA and protein patterns. Here, we exploit recent temporal mRNA and protein expression measurements on the full surface of the blastoderm to calibrate a dynamical model of the gap gene network on the entire embryo cortex. We model the early mRNA and protein dynamics of the gap genes hunchback, Kruppel, giant, and knirps, taking as regulatory inputs the maternal Bicoid and Caudal gradients, plus the zygotic Tailless and Huckebein proteins. The model captures the expression patterns faithfully, and its predictions are assessed from gap gene mutants. The inferred network shows an architecture based on reciprocal repression between gap genes that can stably pattern the embryo on a realistic geometry but requires complex regulations such as those involving the Hunchback monomer and dimers. Sensitivity analysis identifies the posterior domain of giant as among the most fragile features of an otherwise robust network, and hints at redundant regulations by Bicoid and Hunchback, possibly reflecting recent evolutionary changes in the gap-gene network in insects. PMID:21767480

  12. Fibrotic gene expression coexists with alveolar proteinosis in early indium lung.

    PubMed

    Noguchi, Shuhei; Eitoku, Masamitsu; Kiyosawa, Hidenori; Suganuma, Narufumi

    2016-08-01

    Occupational inhalation of indium compounds can cause the so-called "indium lung disease". Most affected individuals show pulmonary alveolar proteinosis (PAP) and fibrotic interstitial lung disease. In animal experiments, inhalation of indium tin oxide or indium oxide has been shown to cause lung damage. However, the mechanisms by which indium compounds lead to indium lung disease remain unknown. In this study, we constructed a mouse model of indium lung disease and analyzed gene expression in response to indium exposure. Indium oxide (In2O3, 10 mg/kg, primary particle size <100 nm) was administered intratracheally to C57BL/6 mice (male, 8 weeks of age) twice a week for 8 weeks. Four weeks after the final instillation, histopathological analysis exhibited periodic acid-Schiff positive material in the alveoli, characteristic of PAP. Comprehensive gene expression analysis by RNA-Seq, however, revealed expression of fibrosis-related genes, such as surfactant associated protein D, surfactant associated protein A1, mucin 1, and collagen type I and III, was significantly increased, indicating that fibrotic gene expression progresses in early phase of indium lung. These data supported the latest hypothesis that PAP occurs as an acute phase response and is replaced by fibrosis after long-term latency. PMID:27308969

  13. Mapping the expression of soluble olfactory proteins in the honeybee.

    PubMed

    Dani, Francesca Romana; Iovinella, Immacolata; Felicioli, Antonio; Niccolini, Alberto; Calvello, Maria Antonietta; Carucci, Maria Giovanna; Qiao, Huili; Pieraccini, Giuseppe; Turillazzi, Stefano; Moneti, Gloriano; Pelosi, Paolo

    2010-04-01

    Chemical communication in insects is mediated by soluble binding proteins, belonging to two large families, Odorant-binding Proteins (OBPs) and Chemosensory Proteins (CSPs). Recently, evidence has been provided that OBPs are involved in recognition of chemical stimuli. We therefore decided to investigate the expression of OBPs and CSPs in the honeybee at the protein level, using a proteomic approach. Our results are in agreement with previous reports of expression at the RNA level and show that 12 of the 21 OBPs predicted in the genome of the honeybee Apis mellifera and 2 of the 6 CSPs are present in the foragers' antennae, while the larvae express only three OBPs and a single CSP. MALDI mass spectrometry on crude antennal extracts and MALDI profiling on sections of antennae demonstrated that these techniques can be applied to investigate individual differences in the expression of abundant proteins, such as OBPs and CSPs, as well as to detect the presence of proteins in different regions of the antenna. Finally, as part of a project aimed at the characterization of all OBPs and CSPs of the honeybee, we expressed 5 OBPs and 4 CSPs in a bacterial system and measured their affinity to a number of ligands. Clear differences in their binding spectra have been observed between OBPs, as well as CSPs. PMID:20155982

  14. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes. PMID:26237676

  15. Identification of Bacillus subtilis genes expressed early during sporulation.

    PubMed

    Mathiopoulos, C; Sonenshein, A L

    1989-08-01

    Labelled cDNA transcribed in vitro from early-sporulation RNA was enriched for sporulation-specific sequences by subtractive hybridization to an excess of vegetative RNA and used to probe libraries of Bacillus subtilis chromosomal DNA. From the initial collection of clones that coded for RNAs transcribed preferentially during sporulation, several were subcloned and studied in more detail. It was found that two clones contained sequences (dciA and dciB) that had an undetectable level of transcription during vegetative growth but had transcripts that started to appear no later than eight minutes after induction of sporulation. A third DNA segment (dciC) was expressed at a low level in vegetative cells and increased within four minutes after induction of sporulation. The effects of spoO mutations, i.e. mutations that prevent cells from reaching stage I of the sporulation process, were tested. Induction of the dciA and dciB transcripts was significantly reduced in strains carrying mutations in the spoOA and spoOH genes but not in a spoOB mutant strain. In addition, a product of the abrB locus, a locus in which mutations are known to partially overcome the pleiotropic effect of spoOA and spoOB mutations, seemed to be required for dciA and dciB expression. PMID:2481799

  16. 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.

  17. 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. PMID:23387626

  18. Expression of Eukaryotic Membrane Proteins in Pichia pastoris.

    PubMed

    Hartmann, Lucie; Kugler, Valérie; Wagner, Renaud

    2016-01-01

    A key point when it comes to heterologous expression of eukaryotic membrane proteins (EMPs) is the choice of the best-suited expression platform. The yeast Pichia pastoris has proven to be a very versatile system showing promising results in a growing number of cases. Indeed, its particular methylotrophic characteristics combined to the very simple handling of a eukaryotic microorganism that possesses the majority of mammalian-like machineries make it a very competitive expression system for various complex proteins, in amounts compatible with functional and structural studies. This chapter describes a set of robust methodologies routinely used for the successful expression of a variety of EMPs, going from yeast transformation with the recombinant plasmid to the analysis of the quality and quantity of the proteins produced. PMID:27485335

  19. Transcriptional regulation of vascular bone morphogenetic protein by endothelin receptors in early autoimmune diabetes mellitus.

    PubMed

    Nett, Philipp C; Ortmann, Jana; Celeiro, Jennifer; Haas, Elvira; Hofmann-Lehmann, Regina; Tornillo, Luigi; Terraciano, Luigi M; Barton, Matthias

    2006-04-01

    Endothelin (ET) and bone morphogenic proteins (BMP) have been implicated in the development of micro- and macrovascular complications of type 2 diabetes mellitus due to atherosclerosis. This study investigated vascular BMP-expression during early development of experimental autoimmune diabetes mellitus and whether ET(A) receptors are involved in its regulation, using the selective ET(A) receptor antagonist BSF461314. Specificity of BSF461314 was confirmed through ET-mediated p44/42 mitogen-activated protein kinase (ERK1/2) phosphorylation experiments. For animal studies, non-obese diabetic (NOD) and control mice at 16 weeks of age were treated with BSF461314 for 6 weeks. Plasma glucose levels were measured before and after treatment and vascular gene expression of BMP-2, BMP-7, and BMP-type II receptor was determined in the aorta by quantitative real-time polymerase chain reaction analysis. At the beginning of the study in all animals, plasma glucose levels were within the normal range. After 6 weeks gene expression of vascular BMP-2, BMP-7 and BMP-type II receptor was almost doubled in NOD mice compared with non-diabetic controls (p < 0.05). Concomitant treatment with BSF461314 significantly reduced expression of all BMPs and lowered plasma glucose levels in NOD mice close to controls (all p < 0.05 versus untreated). In conclusion, vascular BMP-2, BMP-7, and BMP-type II receptor expression is upregulated in early stages of autoimmune diabetes mellitus. The data further indicate that ET(A) receptors inhibit diabetes-associated activation of vascular BMPs and regulate plasma glucose levels suggesting that ET(A) receptors might provide a new therapeutic target to interfere with the early development of atherosclerosis in patients with type 1 diabetes mellitus. PMID:16300798

  20. 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

  1. The Early Phagosomal Stage of Francisella tularensis Determines Optimal Phagosomal Escape and Francisella Pathogenicity Island Protein Expression▿

    PubMed Central

    Chong, Audrey; Wehrly, Tara D.; Nair, Vinod; Fischer, Elizabeth R.; Barker, Jeffrey R.; Klose, Karl E.; Celli, Jean

    2008-01-01

    Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins. PMID:18852245

  2. Expression of microtubule-associated protein 2 by reactive astrocytes.

    PubMed Central

    Geisert, E E; Johnson, H G; Binder, L I

    1990-01-01

    After an injury to the central nervous system, a dramatic change in the astrocytes bordering the wound occurs. The most characteristic feature of this process, termed reactive gliosis, is the upregulation of the intermediate filament protein, glial fibrillary acidic protein. In the present study, we show that reactive astrocytes express high levels of microtubule-associated protein 2 (MAP-2), a protein normally found in the somatodendritic compartment of neurons. When sections of injured brain are double-stained with antibodies directed against MAP-2 and glial fibrillary protein, all of the reactive astrocytes are found to contain MAP-2. The high levels of this protein appear to represent a permanent change in reactive astrocytes. In parallel quantitative studies, an elevated level of MAP-2 in the injured brain is confirmed by an immunoblot analysis of injured and normal white matter. This report demonstrates the direct involvement of a microtubule protein in the process of reactive gliosis. Images PMID:1692628

  3. Differential expression of VegT and Antipodean protein isoforms in Xenopus.

    PubMed

    Stennard, F; Zorn, A M; Ryan, K; Garrett, N; Gurdon, J B

    1999-08-01

    The VegT/Antipodean (Apod) gene is important for germ layer formation in Xenopus. To investigate the role of this gene at the protein level, as opposed to the RNA level, we have generated affinity purified polyclonal antibodies to Apod, and for comparison, to the other early T-box proteins Xbrachyury and Eomesodermin. An anti-VegT/Apod antibody reveals that there are two protein isoforms in Xenopus, one that we refer to as VegT and a smaller molecular weight isoform that we refer to as Apod. These isoforms have different N-terminal domains resulting from developmentally regulated alternative splicing of a primary transcript arising from a single VegT/Apod gene. VegT is maternally expressed. Its translation is blocked during oogenesis but the protein is present from the egg until gastrulation in the presumptive endoderm. There is no evidence for zygotic expression of this isoform. Conversely, the Apod protein isoform is expressed only after the onset of zygotic transcription in the presumptive mesoderm and is inducible by activin. We conclude that the developmental role of VegT/Apod is mediated by two different proteins, with entirely different patterns of expression and response to growth factors. PMID:10446268

  4. Expressing and purifying membrane transport proteins in high yield.

    PubMed

    Hale, Calvin C; Hill, Chananada K; Price, Elmer M; Bossuyt, Julie

    2002-01-01

    Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic. PMID:11741710

  5. Differential expression of vascular endothelial growth factor-A isoforms in the mouse uterus during early pregnancy.

    PubMed

    Walter, Lisa M; Rogers, Peter A W; Girling, Jane E

    2010-12-01

    While vascular endothelial growth factor (VEGF)-A mediates endometrial vascular remodelling during early pregnancy in mice, individual VEGF-A isoforms have not been investigated, despite their different biological properties. Using mice as a model, the expression of VEGF-A isoforms and receptors in the mouse uterus during early pregnancy was quantified. It was postulated that selected isoform expression would increase concurrent with increased endometrial endothelial cell proliferation at this time. Uteri were collected on days 1-5 of pregnancy and mRNA expression was quantified by quantitative reverse-transcription polymerase chain reaction, VEGF-A protein by Western blot and VEGF receptor (VEGFR)-2 by immunohistochemistry. The lowest expression of isomers Vegf(120) and Vegf(164) was observed on day 2 of pregnancy, increasing thereafter. Vegfr-2 mRNA expression was significantly higher on days 3-5 of pregnancy relative to days 1-2 (P<0.05). No significant changes were noted in Vegf(188), Nrp1 or Nrp2 mRNA. VEGF(188) protein expression was consistently higher than other isoforms. These data demonstrate differential regulation of VEGF-A isoforms in mouse uterus during early pregnancy. PMID:21050818

  6. Heat-shock protein expression on the membrane of T cells undergoing apoptosis.

    PubMed Central

    Poccia, F; Piselli, P; Vendetti, S; Bach, S; Amendola, A; Placido, R; Colizzi, V

    1996-01-01

    Heat-shock proteins (hsp) represent a highly conserved family of proteins, normally localized in the cytoplasm and nucleus, whose expression is induced in situations involving cell stress. This paper reports the unusual translocation of hsp to the cell membrane of T cells undergoing apoptosis. We observed that glucocorticosteroid-induced thymocyte death is associated to the surface expression of hsp 60 and hsp 70 in a discrete fraction of apoptotic cells. hsp surface expression is closely related to a thymic subset of immature CD3low/- T cells. The expression of surface hsp 60 appears early after treatment with dexamethasone (3 hr) whereas the membrane expression of hsp 70 follows different kinetics and peaks later. Morphological analysis of the hsp+ apoptotic cells suggest that this subset represents late-stage apoptotic cells at their minimal volume before fragmentation into apoptotic bodies. Membrane expression of hsp is also associated with apoptosis in peripheral blood mononuclear cells from AIDS patients cultured in vitro. Altogether, we show that a discrete fraction of cells undergoing apoptosis expresses membrane hsp 60 and hsp 70, supporting the hypothesis that apoptosis causes a radical alteration in the expression of cell surface molecules. Surface hsp expressed during apoptosis may constitute a novel immune-context able to generate packages of self- and exogenous antigens, originating from degradation of altered cells. Images Figure 1 Figure 3 PMID:8707351

  7. Expression of genes encoding extracellular matrix proteins: A macroarray study

    PubMed Central

    FUTYMA, KONRAD; MIOTŁA, PAWEŁ; RÓŻYŃSKA, KRYSTYNA; ZDUNEK, MAŁGORZATA; SEMCZUK, ANDRZEJ; RECHBERGER, TOMASZ; WOJCIEROWSKI, JACEK

    2014-01-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs. PMID:25231141

  8. Genome engineering for improved recombinant protein expression in Escherichia coli.

    PubMed

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-01-01

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review. PMID:25523647

  9. 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

  10. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

    PubMed

    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

  11. Ameloblastin expression and putative autoregulation in mesenchymal cells suggest a role in early bone formation and repair

    PubMed Central

    Tamburstuen, Margareth V.; Reseland, Janne E.; Spahr, Axel; Brookes, Steven J.; Kvalheim, Gunnar; Slaby, Ivan; Snead, Malcolm L.; Lyngstadaas, S. Petter

    2015-01-01

    Ameloblastin is mainly known as a dental enamel protein, synthesized and secreted into developing enamel matrix by the enamel-forming ameloblasts. The function of ameloblastin in tooth development remains unclear, but it has been suggested to be involved in processes varying from regulating crystal growth to activity as a growth factor or partaking in cell signaling. Recent studies suggest that some enamel matrix proteins also might have important functions outside enamel formation. In this context ameloblastin has recently been reported to induce dentin and bone repair, as well as being present in the early bone and cartilage extracellular matrices during embryogenesis. However, what cells express ameloblastin in these tissues still remain unclear. Thus, the expression of ameloblastin was examined in cultured primary mesenchymal cells and in vivo during healing of bone defects in a “proof of concept” animal study. The real time RT-PCR analysis revealed human ameloblastin (AMBN) mRNA expression in human mesenchymal stem cells and primary osteoblasts and chondrocytes. Expression of AMBN mRNA was also confirmed in human CD34 positive cells and osteoclasts. Western and dot blot analysis of cell lysates and medium confirmed the expression and secretion of ameloblastin from mesenchymal stem cells, primary human osteoblasts and chondrocytes. Expression of ameloblastin was also detected in newly formed bone in experimental bone defects in adult rats. Together these findings suggest a role of this protein in early bone formation and repair. PMID:20854943

  12. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system. PMID:23996319

  13. 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.

  14. Early Alterations in Cytokine Expression in Adult Compared to Developing Lung in Mice after Radiation Exposure

    PubMed Central

    Johnston, Carl J.; Hernady, Eric; Reed, Christina; Thurston, Sally W.; Finkelstein, Jacob N.; Williams, Jacqueline P.

    2010-01-01

    To assess early changes in the lung after low-dose radiation exposure that may serve as targets for mitigation of lung injury in the aftermath of a terrorist event, we analyzed cytokine expression after irradiation. Adult mice were studied after whole-lung or total-body irradiation. Mouse pups of different ages were also investigated after total-body irradiation. mRNA abundance was analyzed in tissue and plasma, and pathological changes were assessed. In lung tissue, dose-related changes were seen in IL1B, IL1R2 and CXCR2 mRNA expression at 1 and 6 h after irradiation, concurrent with increases in plasma protein levels of KC/CXCL1 and IL6. However, in the pups, changes in IL1 abundance were not detected until 28 days of age, coincident with the end of postnatal lung growth, although apoptosis was detected at all ages. In conclusion, although cytokines were expressed after low doses of radiation, their role in the progression of tissue response is yet to be determined. They may be candidates for use in marker-based biodosimetry. However, the lack of cytokine induction in early life suggests that different end points (and mitigating treatments) may be required for children. PMID:20334525

  15. Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer

    PubMed Central

    Becker, Marc A.; Ibrahim, Yasir H.; Oh, Annabell S.; Fagan, Dedra H.; Byron, Sara A.; Sarver, Aaron L.; Lee, Adrian V.; Shaw, Leslie M.; Fan, Cheng; Perou, Charles M.; Yee, Douglas

    2016-01-01

    Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer. PMID:26991655

  16. 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. PMID:26815306

  17. 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

  18. Identification of prognostic biomarkers for glioblastomas using protein expression profiling

    PubMed Central

    JUNG, YONG; JOO, KYEUNG MIN; SEONG, DONG HO; CHOI, YOON-LA; KONG, DOO-SIK; KIM, YONGHYUN; KIM, MI HYUN; JIN, JUYOUN; SUH, YEON-LIM; SEOL, HO JUN; SHIN, CHUL SOO; LEE, JUNG-IL; KIM, JONG-HYUN; SONG, SANG YONG; NAM, DO-HYUN

    2012-01-01

    A set of proteins reflecting the prognosis of patients have clinical significance since they could be utilized as predictive biomarkers and/or potential therapeutic targets. With the aim of finding novel diagnostic and prognostic markers for glioblastoma (GBM), a tissue microarray (TMA) library consisting of 62 GBMs and 28 GBM-associated normal spots was constructed. Immunohistochemistry against 78 GBM-associated proteins was performed. Expression levels of each protein for each patient were analyzed using an image analysis program and converted to H-score [summation of the intensity grade of staining (0–3) multiplied by the percentage of positive cells corresponding to each grade]. Based on H-score and hierarchical clustering methods, we divided the GBMs into two groups (n=19 and 37) that had significantly different survival lengths (p<0.05). In the two groups, expression of nine proteins (survivin, cyclin E, DCC, TGF-β, CDC25B, histone H1, p-EGFR, p-VEGFR2/3, p16) was significantly changed (q<0.05). Prognosis-predicting potential of these proteins were validated with another independent library of 82 GBM TMAs and a public GBM DNA microarray dataset. In addition, we determined 32 aberrant or mislocalized subcellular protein expression patterns in GBMs compared with relatively normal brain tissues, which could be useful for diagnostic biomarkers of GBM. We therefore suggest that these proteins can be used as predictive biomarkers and/or potential therapeutic targets for GBM. PMID:22179774

  19. 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.

  20. Dynamic methylation and expression of Oct4 in early neural stem cells.

    PubMed

    Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

    2010-09-01

    Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form 'induced pluripotent stem cells' (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages. PMID:20646110

  1. Early-response cytokine expression in adult middle ear effusions.

    PubMed

    Ondrey, F G; Juhn, S K; Adams, G L

    1998-10-01

    Various cytokines are presently known to be associated with the regulation of inflammatory responses. In pediatric otitis media, cytokines that correlate with various degrees of inflammation are present in middle ear effusions as inflammatory mediators. The present study was undertaken to examine the potential role of the early-response cytokines, interleukin-1beta and tumor necrosis factor-alpha, in adult otitis media. Fifty-nine adults with otitis media underwent tympanocentesis, and the effusion specimens were analyzed for the presence of both cytokines by enzyme-linked immunosorbent assay methods. Eighty-eight percent of the effusions were serous in nature. Sixty-seven percent of the patients had a known history of head and neck malignancy and radiation to the temporal bone. Twelve percent of the effusions were positive for interleukin-1beta expression, compared with 85% of effusions in children with otitis media. Eight percent of the effusions contained tumor necrosis factor-alpha, compared with 85% of those collected in pediatric otitis media. All of the specimens that contained tumor necrosis factor-alpha also contained interleukin-1beta. In the present study, there was no correlation with head and neck malignancy/radiation or the clinical degree of inflammation with the presence of either cytokine. We conclude that adult otitis media is associated with lower expression of an acute inflammatory response, as judged by the levels of interleukin-1beta and tumor necrosis factor-alpha in the effusions. Additionally, adult otitis probably represents a less severe and more chronic inflammatory state in comparison with pediatric otitis media. Further analysis of inflammatory mediators in adult otitis media is necessary to evaluate the contribution of cytokines in relation to various etiologic factors. PMID:9781987

  2. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish

    PubMed Central

    Horstick, Eric J.; Jordan, Diana C.; Bergeron, Sadie A.; Tabor, Kathryn M.; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A.

    2015-01-01

    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. PMID:25628360

  3. Effects of Chemically Modified Messenger RNA on Protein Expression.

    PubMed

    Li, Bin; Luo, Xiao; Dong, Yizhou

    2016-03-16

    Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Here, we describe the synthesis of two sets of chemically modified mRNAs [encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively], evaluation of protein expression, and correlation analysis of expression level under various conditions. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with N1-methylpseudouridine (me(1)ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Consequently, me(1)ψ, 5moU, and ψ are promising nucleotides for chemical modification of mRNAs. PMID:26906521

  4. 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. PMID:24674065

  5. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  6. Control of adenovirus early gene expression: Posttranscriptional control mediated by both viral and cellular gene products

    SciTech Connect

    Katze, M.G.; Persson, H.; Philipson, L.

    1981-09-01

    An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

  7. Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment

    PubMed Central

    Hu, Xiaoming; Zhou, Yuanfei; Yang, Yang; Peng, Jie; Song, Tongxing; Xu, Tao; Wei, Hongkui; Jiang, Siwen; Peng, Jian

    2016-01-01

    Adipose tissue is a key determinant of whole-body metabolism and energy homeostasis. Unravelling the transcriptional regulatory process during adipogenesis is therefore highly relevant from a biomedical perspective. In these studies, zinc finger protein B-cell lymphoma 6 (Bcl6) was demonstrated to have a role in early adipogenesis of mesenchymal stem cells. Bcl6 is enriched in preadipose versus non-preadipose fibroblasts and shows upregulated expression in the early stage of adipogenesis. Gain- and loss-of-function studies revealed that Bcl6 acts as a key regulator of adipose commitment and differentiation both in vitro and ex vivo. RNAi-mediated knockdown of Bcl6 in C3H10T1/2 cells greatly inhibited adipogenic potential, whereas Bcl6 overexpression enhanced adipogenic differentiation. This transcription factor also directly or indirectly targets and controls the expression of some early and late adipogenic regulators (i.e. Zfp423, Zfp467, KLF15, C/EBPδ, C/EBPα and PPARγ). We further identified that Bcl6 transactivated the signal transducers and activators of transcription 1 (STAT1), which was determined as a required factor for adipogenesis. Moreover, overexpression of STAT1 rescued the impairment of adipogenic commitment and differentiation induced by Bcl6 knockdown in C3H10T1/2 cells, thereby confirming that STAT1 is a downstream direct target of Bcl6. This study identifies Bcl6 as a positive transcriptional regulator of early adipose commitment. PMID:27251748

  8. Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment.

    PubMed

    Hu, Xiaoming; Zhou, Yuanfei; Yang, Yang; Peng, Jie; Song, Tongxing; Xu, Tao; Wei, Hongkui; Jiang, Siwen; Peng, Jian

    2016-06-01

    Adipose tissue is a key determinant of whole-body metabolism and energy homeostasis. Unravelling the transcriptional regulatory process during adipogenesis is therefore highly relevant from a biomedical perspective. In these studies, zinc finger protein B-cell lymphoma 6 (Bcl6) was demonstrated to have a role in early adipogenesis of mesenchymal stem cells. Bcl6 is enriched in preadipose versus non-preadipose fibroblasts and shows upregulated expression in the early stage of adipogenesis. Gain- and loss-of-function studies revealed that Bcl6 acts as a key regulator of adipose commitment and differentiation both in vitro and ex vivo RNAi-mediated knockdown of Bcl6 in C3H10T1/2 cells greatly inhibited adipogenic potential, whereas Bcl6 overexpression enhanced adipogenic differentiation. This transcription factor also directly or indirectly targets and controls the expression of some early and late adipogenic regulators (i.e. Zfp423, Zfp467, KLF15, C/EBPδ, C/EBPα and PPARγ). We further identified that Bcl6 transactivated the signal transducers and activators of transcription 1 (STAT1), which was determined as a required factor for adipogenesis. Moreover, overexpression of STAT1 rescued the impairment of adipogenic commitment and differentiation induced by Bcl6 knockdown in C3H10T1/2 cells, thereby confirming that STAT1 is a downstream direct target of Bcl6. This study identifies Bcl6 as a positive transcriptional regulator of early adipose commitment. PMID:27251748

  9. 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

    2015-01-01

    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. PMID

  10. Bovine parotid secretory protein: structure, expression and relatedness to other BPI (bactericidal/permeability-increasing protein)-like proteins.

    PubMed

    Wheeler, T T; Hood, K; Oden, K; McCracken, J; Morris, C A

    2003-08-01

    Members of the family of BPI (bactericidal/permeability-increasing protein)-like proteins are as yet incompletely characterized, particularly in cattle, where full-length sequence information is available for only three of the 13 family members known from other species. Structural bioinformatic analyses incorporating bovine homologues of several members of the BPI-like protein family, including two forms of bovine parotid secretory protein (PSP), showed that this family is also present in cattle. Expression analyses of several members of the BPI-like protein family in cattle, including PSP (Bsp30), von Ebner's minor salivary gland protein (VEMSGP) and lung-specific X protein (LUNX), showed a restricted pattern of expression, consistent with earlier hypotheses that these proteins function in the innate immune response to bacteria. The possible role of bovine PSP in susceptibility to pasture bloat in cattle is discussed. PMID:12887305

  11. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  12. Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3.

    PubMed Central

    Messerle, M; Bühler, B; Keil, G M; Koszinowski, U H

    1992-01-01

    We have previously defined ie3 as a coding region located downstream of the ie1 gene which gives rise to a 2.75-kb immediate-early (IE) transcript. Here we describe the structural organization of the ie3 gene, the amino acid sequence of the gene product, and some of the functional properties of the protein. The 2.75-kb ie3 mRNA is generated by splicing and is composed of four exons. The first three exons, of 300, 111, and 191 nucleotides (nt), are shared with the ie1 mRNA and are spliced to exon 5, which is located downstream of the fourth exon used by the ie1 mRNA. Exon 5 starts 28 nt downstream of the 3' end of the ie1 mRNA and has a length of 1,701 nt. The IE3 protein contains 611 amino acids, the first 99 of which are shared with the ie1 product pp89. The IE3 protein expressed at IE times has a relative mobility of 88 kDa in gels, and a mobility shift to 90 kDa during the early phase is indicative of posttranslational modification. Sequence comparison reveals significant homology of the exon 5-encoded amino acid sequence with the respective sequence of UL 122, a component of the IE1-IE2 complex of human cytomegalovirus (HCMV). This homology is also apparent at the functional level. The IE3 protein is a strong transcriptional activator of the murine cytomegalovirus (MCMV) e1 promoter and shows an autoregulatory function by repression of the MCMV ie1/ie3 promoter. The high degree of conservation between the MCMV ie3 and HCMV IE2 genes and their products with regard to gene structure, amino acid sequence, and protein functions suggests that these genes play a comparable role in the transcriptional control of the two cytomegaloviruses. Images PMID:1309246

  13. Expression and in vitro functional analyses of recombinant Gam1 protein.

    PubMed

    Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

    2015-01-01

    Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. PMID:25450237

  14. Immunohistological study of tight junction protein expression in mal de Meleda.

    PubMed

    Kacem, Monia; Agili, Faouzia; Tounsi, Haifa; Zribi, Hela; Zaraa, Ines; Mokni, Mourad; Boubaker, Samir

    2016-01-01

    Mal de Meleda (MdM, MIM: 248300) is a rare autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis with onset in early infancy. The gene responsible for MdM, ARS, encodes for Secreted Lys6/Plaur domain-containing protein 1 which is essential for epidermal homeostasis. Tight junctions have been proposed to have two mutually exclusive functions: a fence function which prevents the mixing of membrane proteins between the apical and basolateral membranes; and a gate function which controls the paracellular passage of ions and solutes between cells. In this study we report immunohistochemical investigations of tight junction proteins claudin-1 and occludin in MdM Tunisian families. Nine skin biopsies from patients with MdM were analyzed. The control group was formed by skin biopsies belonging to healthy individuals. Immunohistochemical study was performed on fixed sections from biopsies of four microns with the following polyclonal antibodies: anti-claudin-1 and anti-occludin. In control skin, claudin-1 exhibited membrane expression throughout the epidermis with increasing and upward intensity, whereas occludin was detected in the cell membrane of keratinocytes of the stratum granulosum. In MdM skin, claudin-1 was expressed throughout the thickness of the spinous layers with membrane staining, and occludin had cytoplasmic staining in the granular layer. The immunohistochemical expression of TJ proteins in MdM patients harbors premature expression of occludin and decreased expression of claudin-1, highlighting further evidence for disorders in epidermal homeostasis. PMID:26986447

  15. p53 AND MDM2 PROTEIN EXPRESSION IN ACTINIC CHEILITIS

    PubMed Central

    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. PMID:19082401

  16. Beyond protein expression, MOPED goes multi-omics.

    PubMed

    Montague, Elizabeth; Janko, Imre; Stanberry, Larissa; Lee, Elaine; Choiniere, John; Anderson, Nathaniel; Stewart, Elizabeth; Broomall, William; Higdon, Roger; Kolker, Natali; Kolker, Eugene

    2015-01-01

    MOPED (Multi-Omics Profiling Expression Database; http://moped.proteinspire.org) has transitioned from solely a protein expression database to a multi-omics resource for human and model organisms. Through a web-based interface, MOPED presents consistently processed data for gene, protein and pathway expression. To improve data quality, consistency and use, MOPED includes metadata detailing experimental design and analysis methods. The multi-omics data are integrated through direct links between genes and proteins and further connected to pathways and experiments. MOPED now contains over 5 million records, information for approximately 75,000 genes and 50,000 proteins from four organisms (human, mouse, worm, yeast). These records correspond to 670 unique combinations of experiment, condition, localization and tissue. MOPED includes the following new features: pathway expression, Pathway Details pages, experimental metadata checklists, experiment summary statistics and more advanced searching tools. Advanced searching enables querying for genes, proteins, experiments, pathways and keywords of interest. The system is enhanced with visualizations for comparing across different data types. In the future MOPED will expand the number of organisms, increase integration with pathways and provide connections to disease. PMID:25404128

  17. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    PubMed

    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

  18. 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

  19. 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

  20. Beyond protein expression, MOPED goes multi-omics

    PubMed Central

    Montague, Elizabeth; Janko, Imre; Stanberry, Larissa; Lee, Elaine; Choiniere, John; Anderson, Nathaniel; Stewart, Elizabeth; Broomall, William; Higdon, Roger; Kolker, Natali; Kolker, Eugene

    2015-01-01

    MOPED (Multi-Omics Profiling Expression Database; http://moped.proteinspire.org) has transitioned from solely a protein expression database to a multi-omics resource for human and model organisms. Through a web-based interface, MOPED presents consistently processed data for gene, protein and pathway expression. To improve data quality, consistency and use, MOPED includes metadata detailing experimental design and analysis methods. The multi-omics data are integrated through direct links between genes and proteins and further connected to pathways and experiments. MOPED now contains over 5 million records, information for approximately 75 000 genes and 50 000 proteins from four organisms (human, mouse, worm, yeast). These records correspond to 670 unique combinations of experiment, condition, localization and tissue. MOPED includes the following new features: pathway expression, Pathway Details pages, experimental metadata checklists, experiment summary statistics and more advanced searching tools. Advanced searching enables querying for genes, proteins, experiments, pathways and keywords of interest. The system is enhanced with visualizations for comparing across different data types. In the future MOPED will expand the number of organisms, increase integration with pathways and provide connections to disease. PMID:25404128

  1. Argonaute Family Protein Expression in Normal Tissue and Cancer Entities.

    PubMed

    Völler, Daniel; Linck, Lisa; 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

  2. The Expression and Significance of Neuronal Iconic Proteins in Podocytes

    PubMed Central

    Sun, Yu; Zhang, Hongxia; Hu, Ruimin; Sun, Jianyong; Mao, Xing; Zhao, Zhonghua; Chen, Qi; Zhang, Zhigang

    2014-01-01

    Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes. PMID:24699703

  3. Expression of bone matrix proteins in urolithiasis model rats.

    PubMed

    Yasui, T; Fujita, K; Sasaki, S; Sato, M; Sugimoto, M; Hirota, S; Kitamura, Y; Nomura, S; Kohri, K

    1999-08-01

    Urinary calcium stones are a pathological substance, and they show similarities to physiological mineralization and other pathological mineralizations. The expression of messenger (m) RNAs of osteopontin (OPN), matrix Gla protein (MGP), osteonectin (ON) and osteocalcin (OC) in bones and teeth has been described. We previously identified OPN as an important stone matrix protein. In addition, the spontaneous calcification of arteries and cartilage in mice lacking MGP was recently reported, a finding which indicates that MGP has a function as an inhibitor of mineralization. Here, we examined the mRNA expressions of OPN, MGP, ON, and OC in the kidneys of stone-forming model rats administered an oxalate precursor, ethylene glycol (EG) for up to 28 days. The Northern blotting showed that the mRNA expressions of OPN and MGP were markedly increased with the administration of EG, but their expression patterns differed. The OPN mRNA expression reached the maximal level at day 7 after the initiation of the EG treatment and showed no significant difference after 14 and 28 days, whereas the MGP mRNA expression rose gradually to day 28. The in situ hybridization demonstrated that the cell type expressing OPN mRNA was different from that expressing MGP. We suggest that OPN acts on calcification and MGP acts on suppression. PMID:10460895

  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. Simulations of nucleation and early growth stages of protein crystals.

    PubMed Central

    Kierzek, A M; Wolf, W M; Zielenkiewicz, P

    1997-01-01

    Analysis of known protein crystal structures reveals that interaction energies between monomer pairs alone are not sufficient to overcome entropy loss related to fixing monomers in the crystal lattice. Interactions with several neighbors in the crystal are required for stabilization of monomers in the lattice. A microscopic model of nucleation and early growth stages of protein crystals, based on the above observations, is presented. Anisotropy of protein molecules is taken into account by assigning free energies of association (proportional to the buried surface area) to individual monomer-monomer contacts in the lattice. Lattice simulations of the tetragonal lysozyme crystal based on the model correctly reproduce structural features of the movement of dislocation on the (110) crystal face. The dislocation shifts with the speed equal to the one determined experimentally if the geometric probability of correct orientation is set to 10(-5), in agreement with previously published estimates. At this value of orientational probability, the first nuclei, the critical size of which for lysozyme is four monomers, appear in 1 ml of supersaturated solution on a time scale of microseconds. Formation of the ordered phase proceeds through the growth of nuclei (rather then their association) and requires nucleations on the surface at certain stages. Images FIGURE 2 PMID:9251778

  6. Serum-dependent expression of promyelocytic leukemia protein suppresses propagation of influenza virus

    SciTech Connect

    Iki, Shigeo; Yokota, Shin-ichi; Okabayashi, Tamaki; Yokosawa, Noriko; Nagata, Kyosuke; Fujii, Nobuhiro . E-mail: fujii@sapmed.ac.jp

    2005-12-05

    The rate of propagation of influenza virus in human adenocarcinoma Caco-2 cells was found to negatively correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Virus replicated more rapidly at lower FBS concentrations (0 or 2%) than at higher concentrations (10 or 20%) during an early stage of infection. Basal and interferon (IFN)-induced levels of typical IFN-inducible anti-viral proteins, such as 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and MxA, were unaffected by variation in FBS concentrations. But promyelocytic leukemia protein (PML) was expressed in a serum-dependent manner. In particular, the 65 to 70 kDa isoform of PML was markedly upregulated following the addition of serum. In contrast, other isoforms were induced by IFN treatment, and weakly induced by FBS concentrations. Immunofluorescence microscopy indicated that PML was mainly formed nuclear bodies in Caco-2 cells at various FBS concentrations, and the levels of the PML-nuclear bodies were upregulated by FBS. Overexpression of PML isoform consisting of 560 or 633 amino acid residues by transfection of expression plasmid results in significantly delayed viral replication rate in Caco-2 cells. On the other hand, downregulation of PML expression by RNAi enhanced viral replication. These results indicate that PML isoforms which are expressed in a serum-dependent manner suppress the propagation of influenza virus at an early stage of infection.

  7. hTERT protein expression is independent of clinicopathological parameters and c-Myc protein expression in human breast cancer

    PubMed Central

    Elkak, AE; Meligonis, G; Salhab, M; Mitchell, B; Blake, JRS; Newbold, RF; Mokbel, K

    2005-01-01

    Background Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase) subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is some experimental and in vitro evidence that c-Myc may increase hTERT expression. We previously reported no correlation between c-Myc mRNA expression and hTERT mRNA or telomerase activity in human breast cancer. This study aims to examine the correlation between hTERT expression as determined by immunohistochemistry and c-Myc expression, lymph node status, and tumour size and grade in human breast cancer. Materials and methods The immunohistochemical expression of hTERT and c-Myc was investigated in 38 malignant breast tumours. The expression of hTERT was then correlated with the lymph node status, c-Myc expression and other clinicopathological parameters of the tumours. Results hTERT expression was positive in 27 (71%) of the 38 tumours. 15 (79%) of 19 node positive tumours were hTERT positive compared with 11 (63%) of 19 node negative tumours. The expression was higher in node positive tumours but this failed to reach statistical significance (p = 0.388). There was no significant association with tumour size, tumour grade or c-Myc expression. However, hTERT expression correlated positively with patients' age (correlation coefficient = 0.415, p = 0.0097). Conclusion hTERT protein expression is independent of lymph node status, tumour size and grade and c-Myc protein expression in human breast cancer PMID:16202165

  8. Prion protein function and the disturbance of early embryonic development in zebrafish

    PubMed Central

    Nourizadeh-Lillabadi, Rasoul; Press, Charles McL; Alestrøm, Peter

    2011-01-01

    Transmissible Spongiform Encephal-opathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of “anchorless” splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist's view of PrP functions might be clearer at a greater phylogenetic distance. PMID:21628994

  9. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  10. Expression of Yes Associated Protein, YAP, Modulates Survivin Expression in Primary Liver Malignancies

    PubMed Central

    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.

    2012-01-01

    Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) 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 following genetic manipulation of Yes associated protein (YAP), a transcription co-activator. Here we comprehensively documented YAP protein expression in the human liver and primary liver cancers. We showed that nuclear YAP expression is significantly increased in human ICC and HCC. We found that increased YAP protein levels in HCC are due to multiple mechanisms including gene amplification, transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein (IAPs) family, has been reported as an independent prognostic factor for poor survival in both HCC and ICC. We found nuclear YAP expression correlates significantly with nuclear Survivin expression for both ICC and HCC. Furthermore, using mice engineered to conditionally overexpress YAP in the liver, we found Survivin mRNA expression depends upon YAP protein levels. Our findings suggested that YAP contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. PMID:22436626

  11. Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification

    PubMed Central

    O'Shaughnessy-Kirwan, Aoife; Signolet, Jason; Costello, Ita; Gharbi, Sarah; Hendrich, Brian

    2015-01-01

    Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment. PMID:26116663

  12. Early MyD88-dependent induction of interleukin-17A expression during Salmonella colitis.

    PubMed

    Keestra, A Marijke; Godinez, Ivan; Xavier, Mariana N; Winter, Maria G; Winter, Sebastian E; Tsolis, Renée M; Bäumler, Andreas J

    2011-08-01

    The development of T helper 17 (T(H)17) cells is a well-established adaptive mechanism for the production of interleukin-17A (IL-17A), a cytokine involved in neutrophil recruitment. However, pathways contributing to mucosal expression of IL-17A during the initial phase of a bacterial infection have received less attention. Here we used the mouse colitis model of Salmonella enterica serotype Typhimurium infection to investigate the contribution of myeloid differentiation primary response protein 88 (MyD88) to inflammation and mucosal IL-17A expression. Expression of IL-23 in the cecal mucosa during S. Typhimurium colitis was dependent on the presence of MyD88. Furthermore, initial expression of IL-17A at 24 h after S. Typhimurium infection was dependent on MyD88 and the receptor for IL-1β. IL-23 and IL-1β synergized in inducing expression of IL-17A in splenic T cells in vitro. In the intestinal mucosa, IL-17A was produced by three distinct T cell populations, including δγ T cells, T(H)17 cells, and CD4(-)CD8(-) T cells. The absence of IL-1β signaling or IL-17 signaling reduced CXC chemokine expression but did not alter the overall severity of pathological lesions in the cecal mucosa. In contrast, cecal pathology and neutrophil recruitment were markedly reduced in Myd88-deficient mice during the initial phases of S. Typhimurium infection. Collectively, these data demonstrate that MyD88-dependent mechanisms, including an initial expression of IL-17A, are important for orchestrating early inflammatory responses during S. Typhimurium colitis. PMID:21576324

  13. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

  14. Oog1, an oocyte-specific protein, interacts with Ras and Ras-signaling proteins during early embryogenesis

    SciTech Connect

    Tsukamoto, Satoshi; Ihara, Ryo; Aizawa, Akira; Kishida, Shosei; Kikuchi, Akira; Imai, Hiroshi; Minami, Naojiro . E-mail: naojiro@kais.kyoto-u.ac.jp

    2006-05-19

    We previously identified an oocyte-specific gene, Oogenesin 1 (Oog1), that encodes 326 amino acids containing a leucine zipper structure and a leucine-rich repeat. In the present study, to identify the interacting proteins of Oog1, we performed a yeast two-hybrid screening using a GV-oocyte cDNA library and found that Ral guanine nucleotide dissociation stimulator (RalGDS) is the binding partner of Oog1. Coimmunoprecipitation assay confirmed the interaction between Oog1 and RalGDS proteins. Colocalization experiments provide the evidence that the nuclear localization of RalGDS depends on the expression of Oog1. Interestingly, RalGDS protein localized in the nucleus rather than the cytoplasm between late 1-cell and early 2-cell stages, the time when Oog1 localizes in the nucleus. We also examined the interaction between Oog1 and Ras by GST pull-down assay and revealed that Oog1 interacts with Ras in a GTP-dependent manner. These findings suggest a role of Oog1 as a Ras-binding protein.

  15. Gene Expression of Rat Alveolar Type II Cells during Hyperoxia Exposure and Early Recovery

    PubMed Central

    Chen, Zhongming; Chintagari, Narendranath Reddy; Guo, Yujie; Bhaskaran, Manoj; Chen, Jiwang; Gao, Li; Jin, Nili; Weng, Tingting; Liu, Lin

    2007-01-01

    Alveolar epithelial cell (AEC) injury and repair during hyperoxia exposure and recovery have been investigated for decades, but the molecular mechanisms of these processes are not clear. To identify potentially important genes involved in lung injury and repair, we studied the gene expression profiles of isolated AEC II from control, 48-hour hyperoxia-exposed (>95% O2) and 1-7 day recovering rats using a DNA microarray containing 10,000 genes. Fifty genes showed significant differential expression between two or more time points (p<0.05, fold change >2). These genes can be classified into 8 unique gene expression patterns. Real-time PCR verified 14 selected genes in three patterns related to hyperoxia exposure and early recovery. The change in the protein level for two of the selected genes, bmp-4 and retnla, paralleled that of the mRNA level. Many of these genes were found to be involved in cell proliferation and differentiation. In an in vitro AEC trans-differentiation culture model using AEC II isolated from control and 48 hrs hyperoxia-exposed rats, the expression of the cell proliferation and differentiation genes identified above were consistent with their predicted roles in the trans-differentiation of AEC. These data indicate that a coordinated mechanism may control AEC differentiation during in vivo hyperoxia exposure and recovery as well as during in vitro AEC culture. PMID:17640573

  16. Novel Implant Coating Agent Promotes Gene Expression of Osteogenic Markers in Rats during Early Osseointegration

    PubMed Central

    Bougas, Kostas; Jimbo, Ryo; Xue, Ying; Mustafa, Kamal; Wennerberg, Ann

    2012-01-01

    The aim of this study was to evaluate the early bone response around laminin-1-coated titanium implants. Forty-five rats distributed in three equally sized groups were provided with one control (turned) and one test (laminin-1-coated) implant and were sacrificed after 3, 7, and 21 days. Real-time reverse-transcriptase polymerase chain reaction was performed for osteoblast markers (alkaline phosphatase, runt-related transcription factor 2, osteocalcin, type I collagen, and bone morphogenic protein 2), osteoclast markers (cathepsin K and tartrate-resistant acid phosphatase), inflammation markers (tumor necrosis factor α, interleukin 1β and interleukin 10), and integrin β1. Bone implant contact (BIC) and bone area (BA) were assessed and compared to the gene expression. After 3 days, the expression of bone markers was higher for the control group. After 7 days, the expression of integrin β1 and osteogenic markers was enhanced for the test group, while cathepsin K and inflammation markers were down-regulated. No significant differences in BIC or BA were detected between test and control at any time point. As a conclusion, implant coating with laminin-1 altered gene expression in the bone-implant interface. However, traditional evaluation methods, as histomorphometry, were not adequately sensitive to detect such changes due to the short follow-up time. PMID:23193408

  17. The protein expression landscape of the Arabidopsis root

    PubMed Central

    Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

    2012-01-01

    Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

  18. Expression of extracellular matrix proteins in adenomatoid odontogenic tumor.

    PubMed

    Modolo, Filipe; Biz, Michelle Tillmann; Martins, Marília Trierveiller; Machado de Sousa, Suzana Orsini; de Araújo, Ney Soares

    2010-03-01

    Altered expression of extracellular matrix (ECM) components has been reported in several pathologies; however, few ECM proteins have been evaluated in adenomatoid odontogenic tumor (AOT). The aim of this study was to analyze the expression and distribution of the ECM proteoglycans: biglycan and decorin; and glycoproteins: osteonectin, osteopontin, bone sialoprotein and osteocalcin in the AOT. Three-micrometer sections from paraffin-embedded specimens were evaluated employing a streptavidin-biotin immunohistochemical method with the antibodies against the proteins previously cited. Only the osteonectin was expressed in the epithelial cells. The eosinophilic amorphous material and the connective tissue showed expression of all components studied. The calcification foci expressed only osteopontin. In conclusion, the low expression of the components studied in neoplastic epithelial cells suggests that the epithelial cells act probably as stimulators of the expression by the stroma, which in turn can act as agonist or antagonist of the tumor growth. These results suggest that the components studied probably have a key role in the biological behavior of the AOT. PMID:20070486

  19. Protein expression analysis of rat testes induced testicular toxicity with several reproductive toxicants.

    PubMed

    Yamamoto, Toshinori; Fukushima, Tamio; Kikkawa, Rie; Yamada, Hiroshi; Horii, Ikuo

    2005-05-01

    The utilization of safety biomarkers to predict the possibility of compound-related toxicity provides several advantages for drug discovery and development, especially at an early stage. The objectives of this study were to investigate the effects of male reproductive toxicants on protein expression profiles in the rat testes and to identify potential biomarker candidates. Four well-known reproductive toxicants, ethylene glycol monomethyl ether (EGME), cyclophosphamide (CP), sulfasalazine (SASP) and 2,5-hexanedione (2,5-HD), were administered to male rats in a single dose, and protein expression profiles were investigated after 24 hr by two-dimensional gel electrophoresis (2DE). Histopathological examination of the testes and serum concentration analysis were also performed. From the results of the comparison of 2D-gels among different doses of a compound and among compounds, 52, 20, 24 and 111 spots were nominated as differentially expressed spots with EGME, CP, SASP and 2,5-HD treatments, respectively. Several spermatogenesis-involved proteins were identified, including glutathione S-transferase (GST), testis-specific heat shock protein 70-2 (HSP70-2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphatidylethanolamine-binding protein (PEBP). Some of them were altered by more than one compound. In summary, remarkable histopathological findings were observed only in the EGME high-dose group, and most of the protein changes were detected before histopathological changes occurred. Therefore, the proteins identified in this study could potentially serve as biomarkers to evaluate male reproductive toxicity at an early stage of drug discovery and development. PMID:15928459

  20. Spatio-Temporal Gene Expression Profiling during In Vivo Early Ovarian Folliculogenesis: Integrated Transcriptomic Study and Molecular Signature of Early Follicular Growth

    PubMed Central

    Bonnet, Agnes; Servin, Bertrand; Mulsant, Philippe; Mandon-Pepin, Beatrice

    2015-01-01

    Background The successful achievement of early ovarian folliculogenesis is important for fertility and reproductive life span. This complex biological process requires the appropriate expression of numerous genes at each developmental stage, in each follicular compartment. Relatively little is known at present about the molecular mechanisms that drive this process, and most gene expression studies have been performed in rodents and without considering the different follicular compartments. Results We used RNA-seq technology to explore the sheep transcriptome during early ovarian follicular development in the two main compartments: oocytes and granulosa cells. We documented the differential expression of 3,015 genes during this phase and described the gene expression dynamic specific to these compartments. We showed that important steps occurred during primary/secondary transition in sheep. We also described the in vivo molecular course of a number of pathways. In oocytes, these pathways documented the chronology of the acquisition of meiotic competence, migration and cellular organization, while in granulosa cells they concerned adhesion, the formation of cytoplasmic projections and steroid synthesis. This study proposes the involvement in this process of several members of the integrin and BMP families. The expression of genes such as Kruppel-like factor 9 (KLF9) and BMP binding endothelial regulator (BMPER) was highlighted for the first time during early follicular development, and their proteins were also predicted to be involved in gene regulation. Finally, we selected a data set of 24 biomarkers that enabled the discrimination of early follicular stages and thus offer a molecular signature of early follicular growth. This set of biomarkers includes known genes such as SPO11 meiotic protein covalently bound to DSB (SPO11), bone morphogenetic protein 15 (BMP15) and WEE1 homolog 2 (S. pombe)(WEE2) which play critical roles in follicular development but other

  1. PROTEIN EXPRESSION AND SECRETION BY TRICHODERMA REESEI UNDER LOW ENDOGENOUS PROTEIN BACKGROUND

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trichoderma reesei (Hypocrea jecorina) is one of the most commonly used fungi for the manufacturing of industrial enzyme products. The fungus is capable of secreting proteins in levels up to 100 grams per liter. A number of homologous and heterologous proteins have been successfully over-expressed...

  2. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  3. A comparative approach to understanding tissue-specific expression of uncoupling protein 1 expression in adipose tissue.

    PubMed

    Shore, Andrew; Emes, Richard D; Wessely, Frank; Kemp, Paul; Cillo, Clemente; D'Armiento, Maria; Hoggard, Nigel; Lomax, Michael A

    2012-01-01

    The thermoregulatory function of brown adipose tissue (BAT) is due to the tissue-specific expression of uncoupling protein 1 (UCP1) which is thought to have evolved in early mammals. We report that a CpG island close to the UCP1 transcription start site is highly conserved in all 29 vertebrates examined apart from the mouse and xenopus. Using methylation sensitive restriction digest and bisulfite mapping we show that the CpG island in both the bovine and human is largely un-methylated and is not related to differences in UCP1 expression between white and BAT. Tissue-specific expression of UCP1 has been proposed to be regulated by a conserved 5' distal enhancer which has been reported to be absent in marsupials. We demonstrate that the enhancer, is also absent in five eutherians as well as marsupials, monotremes, amphibians, and fish, is present in pigs despite UCP1 having become a pseudogene, and that absence of the enhancer element does not relate to BAT-specific UCP1 expression. We identify an additional putative 5' regulatory unit which is conserved in 14 eutherian species but absent in other eutherians and vertebrates, but again unrelated to UCP1 expression. We conclude that despite clear evidence of conservation of regulatory elements in the UCP1 5' untranslated region, this does not appear to be related to species or tissues-specific expression of UCP1. PMID:23293654

  4. Histone methyltransferase SMYD3 regulates the expression of transcriptional factors during bovine oocyte maturation and early embryonic development.

    PubMed

    Bai, Haidong; Li, Yan; Gao, Haixia; Dong, Yanhua; Han, Pengyong; Yu, Haiquan

    2016-08-01

    Mammalian early embryonic development is controlled by a unique program of gene expression, and involves epigenetic reprogramming of histone modifications and DNA methylation. SET and MYND domain-containing protein 3 (SMYD3) is a histone H3 lysine 4 methyltransferase that plays important roles in transcription regulation. The expression of SMYD3 has been studied in some cancer cell lines. However, its expression in oocytes and embryos has not previously been reported. Here, we detected the SMYD3 mRNA and found that it was expressed throughout bovine oocyte in vitro maturation and early embryonic development. Microinjection of SMYD3 siRNA at germinal vesicle stage decreased the transcription level of NANOG, and blocked the development of in vitro fertilization embryos at 4-8 cell stage. Conversely, Microinjection of SMYD3 siRNA at pronuclear stage did not affect early embryonic development. Our findings suggest that SMYD3 regulates the expression of NANOG, and plays an essential role in bovine early embryonic development. PMID:25563599

  5. Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

    PubMed

    van Opstal, Angélique; Bijvelt, José; van Donselaar, Elly; Humbel, Bruno M; Boonstra, Johannes

    2012-04-01

    Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase. PMID:22251027

  6. Methods and constructs for expression of foreign proteins in photosynthetic organisms

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2002-01-01

    A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

  7. Controlling for Gene Expression Changes in Transcription Factor Protein Networks*

    PubMed Central

    Banks, Charles A. S.; Lee, Zachary T.; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D.; Wen, Zhihui; Hattem, Gaye L.; Seidel, Chris W.; Florens, Laurence; Washburn, Michael P.

    2014-01-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein–protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions. PMID:24722732

  8. Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.

    PubMed

    Schild, Christof; Beyeler, Michael; Lang, Niklaus P; Trueb, Beat

    2009-02-01

    Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP. PMID:19148556

  9. Computational codon optimization of synthetic gene for protein expression

    PubMed Central

    2012-01-01

    Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. PMID:23083100

  10. Expression and Characterization of Recombinant Campylobacter jejuni Chemotactic Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression and Characterization of Recombinant Campylobacter jejuni Chemotactic Proteins Hung-Yueh Yeh*, Kelli L. Hiett, John E. Line, Brian B. Oakley and Bruce S. Seal, Poultry Microbiological Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, Uni...

  11. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  12. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  13. Optimization of Translation Profiles Enhances Protein Expression and Solubility

    PubMed Central

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. PMID:25965266

  14. Quantitative proteomics reveals the novel co-expression signatures in early brain development for prognosis of glioblastoma multiforme

    PubMed Central

    Yu, Xuexin; Feng, Lin; Liu, Dianming; Zhang, Lianfeng; Wu, Bo; Jiang, Wei; Han, Zujing; Cheng, Shujun

    2016-01-01

    Although several researches have explored the similarity across development and tumorigenesis in cellular behavior and underlying molecular mechanisms, not many have investigated the developmental characteristics at proteomic level and further extended to cancer clinical outcome. In this study, we used iTRAQ to quantify the protein expression changes during macaque rhesus brain development from fetuses at gestation 70 days to after born 5 years. Then, we performed weighted gene co-expression network analysis (WGCNA) on protein expression data of brain development to identify co-expressed modules that highly expressed on distinct development stages, including early stage, middle stage and late stage. Moreover, we used the univariate cox regression model to evaluate the prognostic potentials of these genes in two independent glioblastoma multiforme (GBM) datasets. The results showed that the modules highly expressed on early stage contained more reproducible prognostic genes, including ILF2, CCT7, CCT4, RPL10A, MSN, PRPS1, TFRC and APEX1. These genes were not only associated with clinical outcome, but also tended to influence chemoresponse. These signatures identified from embryonic brain development might contribute to precise prediction of GBM prognosis and identification of novel drug targets in GBM therapies. Thus, the development could become a viable reference model for researching cancers, including identifying novel prognostic markers and promoting new therapies. PMID:26895104

  15. Quantitative proteomics reveals the novel co-expression signatures in early brain development for prognosis of glioblastoma multiforme.

    PubMed

    Yu, Xuexin; Feng, Lin; Liu, Dianming; Zhang, Lianfeng; Wu, Bo; Jiang, Wei; Han, Zujing; Cheng, Shujun

    2016-03-22

    Although several researches have explored the similarity across development and tumorigenesis in cellular behavior and underlying molecular mechanisms, not many have investigated the developmental characteristics at proteomic level and further extended to cancer clinical outcome. In this study, we used iTRAQ to quantify the protein expression changes during macaque rhesus brain development from fetuses at gestation 70 days to after born 5 years. Then, we performed weighted gene co-expression network analysis (WGCNA) on protein expression data of brain development to identify co-expressed modules that highly expressed on distinct development stages, including early stage, middle stage and late stage. Moreover, we used the univariate cox regression model to evaluate the prognostic potentials of these genes in two independent glioblastoma multiforme (GBM) datasets. The results showed that the modules highly expressed on early stage contained more reproducible prognostic genes, including ILF2, CCT7, CCT4, RPL10A, MSN, PRPS1, TFRC and APEX1. These genes were not only associated with clinical outcome, but also tended to influence chemoresponse. These signatures identified from embryonic brain development might contribute to precise prediction of GBM prognosis and identification of novel drug targets in GBM therapies. Thus, the development could become a viable reference model for researching cancers, including identifying novel prognostic markers and promoting new therapies. PMID:26895104

  16. Elevated expression of flotillin-1 is associated with lymph node metastasis and poor prognosis in early-stage cervical cancer

    PubMed Central

    Li, Zheng; Yang, Yang; Gao, Yang; Wu, Xiaoliu; Yang, Xielan; Zhu, Yingjie; Yang, Hongying; Wu, Lin; Yang, Chengang; Song, Libing

    2016-01-01

    Accumulating evidence has revealed that the expression of the lipid raft protein flotillin-1 is elevated in various human cancers, but the role flotillin-1 plays in the carcinogenesis of cervical cancer remains unclear. The expression profile of flotillin-1 was assayed using real-time PCR, western blotting, and immunohistochemical (IHC) staining in cervical cancer cell lines and cancer tissues with paired adjacent noncancerous cervical tissues. The expression of flotillin-1 protein was detected by IHC staining in a large cohort of 308 paraffin-embedded cervical cancer tissues. Ectopic expression and the short hairpin RNA interference approach were employed to determine the role of flotillin-1 in cervical cancer cell metastasis and the possible mechanism involved. Flotillin-1 expression protein and mRNA were significantly upregulated in cervical cancer cell lines and cancer tissues; elevated expression of flotillin-1 protein in early-stage cervical cancer was significantly associated with pelvic lymph node metastasis (P < 0.001), and was an independent predictive factor of poor overall survival. Moreover, flotillin-1 up- and downregulation remarkably affected cervical cancer cell motility and invasion, respectively, through epithelial-mesenchymal transition (EMT) regulated by the Wnt/β-catenin and nuclear factor-κB (NF-κB) pathways. Our results suggest that flotillin-1 facilitates cervical cancer cell metastasis through Wnt/β-catenin and NF-κB pathway-regulated EMT and that the flotillin-1 expression profile serves not only as novel predictor of pelvic lymph node metastasis, but also as neoteric risk factor for patients with early-stage cervical cancer. PMID:27073721

  17. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  18. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  19. Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body.

    PubMed

    Seita, Yasunari; Tsukiyama, Tomoyuki; Iwatani, Chizuru; Tsuchiya, Hideaki; Matsushita, Jun; Azami, Takuya; Okahara, Junko; Nakamura, Shinichiro; Hayashi, Yoshitaka; Hitoshi, Seiji; Itoh, Yasushi; Imamura, Takeshi; Nishimura, Masaki; Tooyama, Ikuo; Miyoshi, Hiroyuki; Saitou, Mitinori; Ogasawara, Kazumasa; Sasaki, Erika; Ema, Masatsugu

    2016-01-01

    Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson's disease and Alzheimer's disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research. PMID:27109065

  20. Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body

    PubMed Central

    Seita, Yasunari; Tsukiyama, Tomoyuki; Iwatani, Chizuru; Tsuchiya, Hideaki; Matsushita, Jun; Azami, Takuya; Okahara, Junko; Nakamura, Shinichiro; Hayashi, Yoshitaka; Hitoshi, Seiji; Itoh, Yasushi; Imamura, Takeshi; Nishimura, Masaki; Tooyama, Ikuo; Miyoshi, Hiroyuki; Saitou, Mitinori; Ogasawara, Kazumasa; Sasaki, Erika; Ema, Masatsugu

    2016-01-01

    Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson’s disease and Alzheimer’s disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research. PMID:27109065

  1. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    PubMed

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  2. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  3. Early Evolution of Vertebrate Mybs: An Integrative Perspective Combining Synteny, Phylogenetic, and Gene Expression Analyses.

    PubMed

    Campanini, Emeline B; Vandewege, Michael W; Pillai, Nisha E; Tay, Boon-Hui; Jones, Justin L; Venkatesh, Byrappa; Hoffmann, Federico G

    2015-11-01

    The genes in the Myb superfamily encode for three related transcription factors in most vertebrates, A-, B-, and c-Myb, with functionally distinct roles, whereas most invertebrates have a single Myb. B-Myb plays an essential role in cell division and cell cycle progression, c-Myb is involved in hematopoiesis, and A-Myb is involved in spermatogenesis and regulating expression of pachytene PIWI interacting RNAs, a class of small RNAs involved in posttranscriptional gene regulation and the maintenance of reproductive tissues. Comparisons between teleost fish and tetrapods suggest that the emergence and functional divergence of the Myb genes were linked to the two rounds of whole-genome duplication early in vertebrate evolution. We combined phylogenetic, synteny, structural, and gene expression analyses of the Myb paralogs from elephant shark and lampreys with data from 12 bony vertebrates to reconstruct the early evolution of vertebrate Mybs. Phylogenetic and synteny analyses suggest that the elephant shark and Japanese lamprey have copies of the A-, B-, and c-Myb genes, implying their origin could be traced back to the common ancestor of lampreys and gnathostomes. However, structural and gene expression analyses suggest that their functional roles diverged between gnathostomes and cyclostomes. In particular, we did not detect A-Myb expression in testis suggesting that the involvement of A-Myb in the pachytene PIWI interacting RNA pathway is probably a gnathostome-specific innovation. We speculate that the secondary loss of a central domain in lamprey A-Myb underlies the functional differences between the cyclostome and gnathostome A-Myb proteins. PMID:26475318

  4. Expression of cyr61, a growth factor-inducible immediate-early gene.

    PubMed Central

    O'Brien, T P; Yang, G P; Sanders, L; Lau, L F

    1990-01-01

    A set of immediate-early genes that are rapidly activated by serum or purified platelet-derived growth factor in mouse 3T3 fibroblasts has been previously identified. Among these genes, several are related to known or putative transcription factors and growth factors, supporting the notion that some of these genes encode regulatory molecules important to cell growth. We show here that a member of this set of genes, cyr61 (originally identified by its cDNA 3CH61), encodes a 379-amino-acid polypeptide rich in cysteine residues. cyr61 can be induced through protein kinase C-dependent and -independent pathways. Unlike many immediate-early genes that are transiently expressed, the cyr61 mRNA is accumulated from the G0/G1 transition through mid-G1. This expression pattern is due to persistent transcription, while the mRNA is rapidly turned over during the G0/G1 transition and in mid-G1 at the same rate. In logarithmically growing cells, the cyr61 mRNA level is constant throughout the cell cycle. Cyr61 contains an N-terminal secretory signal sequence; however, it is not detected in the culture medium by immunoprecipitation. Cyr61 is synthesized maximally at 1 to 2 h after serum stimulation and has a short half-life within the cell. Images PMID:2355916

  5. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    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 potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  6. Expression and localization of X11 family proteins in neurons.

    PubMed

    Motodate, Rika; Saito, Yuhki; Hata, Saori; Suzuki, Toshiharu

    2016-09-01

    The X11/Mint family of proteins comprises X11/X11α/Mint1, X11L/X11β/Mint2, and X11L2/X11γ/Mint3. Each of these molecules is an adaptor protein that contains a phosphotyrosine interaction/binding (PI/PTB) and two PDZ domains in its carboxy-terminal region. X11/Mint family members associate with a broad spectrum of membrane proteins, including Alzheimer's β-amyloid precursor protein (APP), alcadeins, and low density lipoprotein receptor proteins, as well as various cytoplasmic proteins including Arf, kalirin-7, and Munc18. In particular, X11 and X11L are thought to play various roles in the regulation of neural functions in brain. Nevertheless, the protein levels and respective localization of individual family members remain controversial. We analyzed the protein levels of X11 and X11L in the corresponding single- and double-knockout mice. X11 and X11L did not exhibit obvious changes of their protein levels when the other was absent, especially in cerebrum in which they were widely co-expressed. In cerebellum, X11 and X11L localized in characteristic patterns in various types of neurons, and X11 protein level increased without an obvious ectopic localization in X11L-knockout mice. Interestingly, only X11L protein existed specifically in brain, whereas, contrary to the accepted view, X11 protein was detected at the highest levels in brain but was also strongly detected in pancreas, testis, and paranephros. Together, our results indicate that both X11 and X11L exert largely in brain neurons, but X11 may also function in peripheral tissues. PMID:27268412

  7. Correlation of gene expression and protein production rate - a system wide study

    PubMed Central

    2011-01-01

    Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR). PMID:22185473

  8. LC–MS Based Detection of Differential Protein Expression

    PubMed Central

    Tuli, Leepika; Ressom, Habtom W.

    2010-01-01

    While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics. Significant technical advances at both sample preparation/separation and mass spectrometry levels have revolutionized comprehensive proteome analysis. Moreover, automation and robotics for sample handling process permit multiple sampling with high throughput. For LC-MS based quantitative proteomics, sample preparation turns out to be critical step, as it can significantly influence sensitivity of downstream analysis. Several sample preparation strategies exist, including depletion of high abundant proteins or enrichment steps that facilitate protein quantification but with a compromise of focusing on a smaller subset of a proteome. While several experimental strategies have emerged, certain limitations such as physiochemical properties of a peptide/protein, protein turnover in a sample, analytical platform used for sample analysis and data processing, still imply challenges to quantitative proteomics. Other aspects that make analysis of a proteome a challenging task include dynamic nature of a proteome, need for efficient and fast analysis of protein due to its constant modifications inside a cell, concentration range of proteins that exceed dynamic range of a single analytical method, and absence of appropriate bioinformatics tools for analysis of large volume and high dimensional data. This paper gives an overview of various LC-MS methods currently used in quantitative proteomics and their potential for detecting differential protein expression. Fundamental steps such as sample preparation, LC separation, mass spectrometry, quantitative assessment and protein identification are discussed. For quantitative assessment of protein expression, both label and label free approaches are evaluated for their set of merits and demerits. While most of these methods edge on providing

  9. Flow cytometry of p53 protein expression in some hematological malignancies.

    PubMed

    Koníková, E; Kusenda, J; Babusíková, O

    1999-01-01

    p53 is a tumor suppressor gene encoding a nuclear phosphoprotein that plays an important role in the control of normal cell proliferation. We have tried to establish the value of the p53 protein expression in peripheral blood (PB) and/or bone marrow (BM) cells of patients with some hematological malignancies. A recently developed fixation/permeabilization method was modified for flow cytometric assessment of p53 protein expression using two anti-p53 monoclonal antibodies. p53 quantitation expressed as molecules of equivalent soluble fluorochrome per cell (MESF) providing valuable data contributing to a more precise definition of leukemic cells, was also applied. Our findings showed higher percentage of p53 expression in cells of AML patients at the time of diagnosis opposite to the controls. These data, in association with immunophenotype of cells, accompanied diagnosis of relapse or definition of remission after allogeneic BM transplantation. We observed also elevated levels of p53 protein at initial diagnosis of early B-ALL. According to our results quantitation of p53 protein allows better characterization of selected population of BM cells and should be used for the monitoring of blast persistence during and after therapy and might also be one of the methods to indicate early relapse. Percentage of p53 protein positivity varied in our group of B-CLL patients tested in connection with progression of disease. We documented also one case of Burkitt's lymphoma with high percentage of p53 positivity. Measurement of p53 protein expression by flow cytometry may be of clinical importance by indicating levels of positivity. Our results suggest, that p53 alteration is frequently involved at initial diagnosis of AML, in some T-cell disorders and on the contrary more frequently during early B-ALL relapse, in advanced stages of B-CLL and in Burkitt's lymphoma. p53 protein quantitation is of value to ascertain malignancy and provides additional parameter suitable for the

  10. Properties of a novel gene isolated from a Hodgkin's disease cell line that is expressed early during lymphoid cell activation

    SciTech Connect

    Bennett, J.S.; Tredway, T.L.; Dizikes, G.J.; Nawrocki, J.F. Hines Veterans Administration Hospital, IL )

    1994-03-01

    The authors have isolated a novel 667-bp cDNA clone, designated epag, from a Hodgkin's-disease cell line-derived library that is expressed in association with T cell activation and which is not related to any known gene family. By using reverse transcription/PCR, the authors have demonstrated that epag mRNA is expressed as early as 1 h after stimulation of normal PBMCs with anti-CD3. The levels of mRNA peaked by 4 h, and no expression was detectable by 12 h postactivation or in resting cells incubated in culture without activation. Expression of epag was also detected in PMA- and PHA-stimulated, but not in nonstimulated Jurkat cells, and overall its expression in transformed cell lines of hemopoietic origin is highly restricted. Sequence analysis of multiple independent cDNA clones showed that epag expressed in the Hodgkin's-disease cell line L428 is identical to the gene expressed in normal activated PBMC. Epag expression was detected by reverse transcription/PCR in RNA preparations made from various normal nonlymphoid tissues. Computer analysis of the sequence identified an open reading frame encoding a putative protein of 13.2 kDa initiating at a CUG translational codon. In vitro translation and Western blot analysis with anti-peptide serum supported this analysis. The authors hypothesize that epag functions as an early signal that helps mediate the activation of T cells. 63 refs., 11 figs.

  11. Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

    PubMed

    Murphy, Noelle; Gaynor, Katherine U; Rowan, Simon C; Walsh, Sinead M; Fabre, Aurelie; Boylan, John; Keane, Michael P; McLoughlin, Paul

    2016-03-01

    Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury. PMID:26765958

  12. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle. PMID:26813519

  13. Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

    PubMed

    Nguyen, Tien Dung; Moon, Sunok; Nguyen, Van Ngoc Tuyet; Gho, Yunsil; Chandran, Anil Kumar Nalini; Soh, Moon-Soo; Song, Jong Tae; An, Gynheung; Oh, Sung Aeong; Park, Soon Ki; Jung, Ki-Hong

    2016-09-01

    Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant. PMID:27356912

  14. Dynamic expression of the vertebrate-specific protein Nucks during rodent embryonic development.

    PubMed

    Drosos, Yiannis; Kouloukoussa, Mirsini; Ostvold, Anne Carine; Havaki, Sophia; Katsantoni, Eleni; Marinos, Evangelos; Aleporou-Marinou, Vassiliki

    2014-01-01

    The nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) is a highly phosphorylated nuclear protein that is overexpressed in many types of cancer. The flexibility of NUCKS and its extensive posttranslational modifications indicate that it is multifunctional, and its expression in most cell types suggests a housekeeping function. However, spatiotemporal expression of the Nucks protein during rodent development has not been reported. Thus, we investigated the expression of both the Nucks mRNA and protein during rat and mouse development by immunohistochemistry, in situ hybridization, Western immunoblotting, and reverse-transcription PCR analysis. We also used BLAST analysis against expressed sequence tag databases to determine whether a NUCKS homologue is expressed in invertebrate organisms. We found that Nucks expression increased during the initial stages of embryonic development, and then gradually decreased until birth in all tissues except the nervous tissue and muscle fibers. Interestingly, the expression of Nucks was very strong in migrating neural crest cells at E13.5 and ectoderm-derived tissues. In most tissues analyzed, the levels of Nucks correlated with the levels of Bax and activated caspase-3, which are indicative of apoptosis. Moreover, Nucks was upregulated very early during neuronal apoptosis in vitro. Expression analysis revealed that no transcript with close homology to the Nucks gene was present in invertebrates. The expression of Nucks in both proliferating and quiescent cells and its correlation with Bax levels and apoptosis strongly suggest that Nucks plays complex roles in cell homeostasis. Furthermore, the lack of homology in invertebrate organisms indicates a specific role for Nucks in vertebrate embryogenesis. PMID:24140890

  15. Early post parturient changes in milk acute phase proteins.

    PubMed

    Thomas, Funmilola C; Waterston, Mary; Hastie, Peter; Haining, Hayley; Eckersall, P David

    2016-08-01

    The periparturient period is one of the most critical periods in the productive life of a dairy cow, and is the period when dairy cows are most susceptible to developing new intramammary infections (IMI) leading to mastitis. Acute phase proteins (APP) such as haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) have been detected in milk during mastitis but their presence in colostrum and milk in the immediate postpartum period has had limited investigation. The hypothesis was tested that APP are a constituent of colostrum and milk during this period. Enzyme linked immunosorbent assays (ELISAs) were used to determine each APP's concentration in colostrum and milk collected daily from the first to tenth day following calving in 22 Holstein-Friesian dairy cows. Haptoglobin was assessed in individual quarters and composite milk samples while M-SAA3 and CRP concentration were determined in composite milk samples. Change in Hp in relation to the high abundance proteins during the transition from colostrum to milk were evaluated by 1 and 2 dimension electrophoresis and western blot. In 80% of the cows all APPs were detected in colostrum on the first day following parturition at moderately high levels but gradually decreased to minimal values in the milk by the 6th day after calving. The remaining cows (20%) showed different patterns in the daily milk APP concentrations and when an elevated level is detected could reflect the presence of IMI. Demonstration that APP are present in colostrum and milk following parturition but fall to low levels within 4 days means that elevated APP after this time could be biomarkers of post parturient mastitis allowing early intervention to reduce disease on dairy farms. PMID:27600971

  16. Expression of Exocytosis Proteins in Rat Supraoptic Nucleus Neurones

    PubMed Central

    Tobin, V.; Schwab, Y.; Lelos, N.; Onaka, T.; Pittman, Q. J.; Ludwig, M.

    2012-01-01

    In magnocellular neurones of the supraoptic nucleus (SON), the neuropeptides vasopressin and oxytocin are synthesised and packaged into large dense-cored vesicles (LDCVs). These vesicles undergo regulated exocytosis from nerve terminals in the posterior pituitary gland and from somata/dendrites in the SON. Regulated exocytosis of LDCVs is considered to involve the soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) complex [comprising vesicle associated membrane protein 2 (VAMP-2), syntaxin-1 and soluble N-ethylmaleimide attachment protein-25 (SNAP-25)] and regulatory proteins [such as synaptotagmin-1, munc-18 and Ca2+-dependent activator protein for secretion (CAPS-1)]. Using fluorescent immunocytochemistry and confocal microscopy, in both oxytocin and vasopressin neurones, we observed VAMP-2, SNAP-25 and syntaxin-1-immunoreactivity in axon terminals. The somata and dendrites contained syntaxin-1 and other regulatory exocytosis proteins, including munc-18 and CAPS-1. However, the distribution of VAMP-2 and synaptotagmin-1 in the SON was limited to putative pre-synaptic contacts because they co-localised with synaptophysin (synaptic vesicle marker) and had no co-localisation with either oxytocin or vasopressin. SNAP-25 immunoreactivity in the SON was limited to glial cell processes and was not detected in oxytocin or vasopressin somata/dendrites. The present results indicate differences in the expression and localisation of exocytosis proteins between the axon terminals and somata/dendritic compartment. The absence of VAMP-2 and SNAP-25 immunoreactivity from the somata/dendrites suggests that there might be different SNARE protein isoforms expressed in these compartments. Alternatively, exocytosis of LDCVs from somata/dendrites may use a different mechanism from that described by the SNARE complex theory. PMID:21988098

  17. Long Interspersed Element-1 Protein Expression Is a Hallmark of Many Human Cancers

    PubMed Central

    Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S.; Hruban, Ralph H.; Iacobuzio-Donahue, Christine A.; Maitra, Anirban; Torbenson, Michael S.; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S.; Montgomery, Elizabeth A.; Gabrielson, Edward; Netto, George J.; Lotan, Tamara L.; De Marzo, Angelo M.; Westra, William; Binder, Zev A.; Orr, Brent A.; Gallia, Gary L.; Eberhart, Charles G.; Boeke, Jef D.; Harris, Chris R.; Burns, Kathleen H.

    2014-01-01

    Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1–encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1–encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. PMID:24607009

  18. Early responses of silkworm midgut to microsporidium infection--A Digital Gene Expression analysis.

    PubMed

    Yue, Ya-Jie; Tang, Xu-Dong; Xu, Li; Yan, Wei; Li, Qian-Long; Xiao, Sheng-Yan; Fu, Xu-Liang; Wang, Wei; Li, Nan; Shen, Zhong-Yuan

    2015-01-01

    Host-pathogen interactions are complex processes, which have been studied extensively in recent years. In insects, the midgut is a vital organ of digestion and nutrient absorption, and also serves as the first physiological and immune barrier against invading pathogenic microorganisms. Our focus is on Nosema bombycis, which is a pathogen of silkworm pebrine and causes great economic losses to the silk industry. A complete understanding of the host response to infection by N. bombycis and the interaction between them is necessary to prevent this disease. Silkworm midgut infected with N. bombycis is a good model to investigate the early host responses to microsporidia infection and the interaction between the silkworm and the microsporidium. Using Digital Gene Expression analysis, we investigated the midgut transcriptome profile of P50 silkworm larvae orally inoculated with N. bombycis. At 6, 12, 18, 24, 48, 72, and 96 h post-infection (hpi), 247, 95, 168, 450, 89, 80, and 773 DEGs were identified, respectively. KEGG pathway analysis showed the influence of N. bombycis infection on many biological processes including folate biosynthesis, spliceosome, nicotinate and nicotinamide metabolism, protein export, protein processing in endoplasmic reticulum, lysosome, biosynthesis of amino acids, ribosome, and RNA degradation. In addition, a number of differentially expressed genes involved in the immune response were identified. Overall, the results of this study provide an understanding of the strategy used by silkworm as a defense against the invasion by N. bombycis. Similar interactions between hosts and pathogens infection may exist in other species. PMID:25315610

  19. Tools to cope with difficult-to-express proteins.

    PubMed

    Saccardo, Paolo; Corchero, José Luís; Ferrer-Miralles, Neus

    2016-05-01

    The identification of DNA coding sequences contained in the genome of many organisms coupled to the use of high throughput approaches has fueled the field of recombinant protein production. Apart from basic research interests, the growing relevance of this field is highlighted by the global sales of the top ten biopharmaceuticals on the market, which exceeds the trillion USD in a steady increasing tendency. Therefore, the demand of biological compounds seems to have a long run on the market. One of the most popular expression systems is based on Escherichia coli cells which apart from being cost-effective counts with a large selection of resources. However, a significant percentage of the genes of interest are not efficiently expressed in this system, or the expressed proteins are accumulated within aggregates, degraded or lacking the desired biological activity, being finally discarded. In some instances, expressing the gene in a homologous expression system might alleviate those drawbacks but then the process usually increases in complexity and is not as cost-effective as the prokaryotic systems. An increasing toolbox is available to approach the production and purification of those difficult-to-express proteins, including different expression systems, promoters with different strengths, cultivation media and conditions, solubilization tags and chaperone coexpression, among others. However, in most cases, the process follows a non-integrative trial and error strategy with discrete success. This review is focused on the design of the whole process by using an integrative approach, taken into account the accumulated knowledge of the pivotal factors that affect any of the key processes, in an attempt to rationalize the efforts made in this appealing field. PMID:27079572

  20. Expression of interleukin-17RC protein in normal human tissues

    PubMed Central

    Ge, Dongxia; You, Zongbing

    2008-01-01

    Background Interleukin-17 (IL-17) cytokines and receptors play an important role in many autoimmune and inflammatory diseases. IL-17 receptors IL-17RA and IL-17RC have been found to form a heterodimer for mediating the signals of IL-17A and IL-17F cytokines. While the function and signaling pathway of IL-17RA has been revealed, IL-17RC has not been well characterized. The function and signaling pathway of IL-17RC remain largely unknown. The purpose of the present study was to systematically examine IL-17RC protein expression in 53 human tissues. Results IL-17RC expression in 51 normal human tissues and two benign tumors (i.e., lymphangioma and parathyroid adenoma) on the tissue microarrays was determined by immunohistochemical staining, using two polyclonal antibodies against IL-17RC. IL-17RC protein was expressed in many cell types including the myocardial cells, vascular and lymphatic endothelial cells, glandular cells (of the adrenal, parathyroid, pituitary, thyroid, pancreas, parotid salivary, and subepidermal glands), epithelial cells (of the esophagus, stomach, intestine, anus, renal tubule, breast, cervix, Fallopian tube, epididymis, seminal vesicle, prostate, gallbladder, bronchus, lung, and skin), oocytes in the ovary, Sertoli cells in the testis, motor neurons in the spinal cord, autonomic ganglia and nerves in the intestine, skeletal muscle cells, adipocytes, articular chondrocytes, and synovial cells. High levels of IL-17RC protein expression were observed in most vascular and lymphatic endothelium and squamous epithelium. The epithelium of the breast, cervix, Fallopian tube, kidney, bladder and bronchus also expressed high levels of IL-17RC, so did the glandular cells in the adrenal cortex, parotid salivary and subepidermal glands. In contrast, IL-17RC protein was not detectable in the smooth muscle cells, fibroblasts, antral mucosa of the stomach, mucosa of the colon, endometrium of the uterus, neurons of the brain, hepatocytes, or lymphocytes

  1. Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids

    PubMed Central

    L'Espérance, Sylvain; Bachvarova, Magdalena; Tetu, Bernard; Mes-Masson, Anne-Marie; Bachvarov, Dimcho

    2008-01-01

    Background Chemotherapy (CT) resistance in ovarian cancer (OC) is broad and encompasses diverse unrelated drugs, suggesting more than one mechanism of resistance. To better understand the molecular mechanisms controlling the immediate response of OC cells to CT exposure, we have performed gene expression profiling in spheroid cultures derived from six OC cell lines (OVCAR3, SKOV3, TOV-112, TOV-21, OV-90 and TOV-155), following treatment with 10,0 μM cisplatin, 2,5 μM paclitaxel or 5,0 μM topotecan for 72 hours. Results Exposure of OC spheroids to these CT drugs resulted in differential expression of genes associated with cell growth and proliferation, cellular assembly and organization, cell death, cell cycle control and cell signaling. Genes, functionally involved in DNA repair, DNA replication and cell cycle arrest were mostly overexpressed, while genes implicated in metabolism (especially lipid metabolism), signal transduction, immune and inflammatory response, transport, transcription regulation and protein biosynthesis, were commonly suppressed following all treatments. Cisplatin and topotecan treatments triggered similar alterations in gene and pathway expression patterns, while paclitaxel action was mainly associated with induction of genes and pathways linked to cellular assembly and organization (including numerous tubulin genes), cell death and protein synthesis. The microarray data were further confirmed by pathway and network analyses. Conclusion Most alterations in gene expression were directly related to mechanisms of the cytotoxics actions in OC spheroids. However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment. Similarly, overexpression of different tubulin genes upon exposure to paclitaxel could represent an early compensatory effect to this drug action. Finally, multicellular growth conditions that are

  2. A Protein Microarray Signature of Autoantibody Biomarkers for the Early Detection of Breast Cancer

    PubMed Central

    Anderson, Karen S.; Sibani, Sahar; Wallstrom, Garrick; Qiu, Ji; Mendoza, Eliseo A.; Raphael, Jacob; Hainsworth, Eugenie; Montor, Wagner R.; Wong, Jessica; Park, Jin G.; Lokko, Naa; Logvinenko, Tanya; Ramachandran, Niroshan; Godwin, Andrew K.; Marks, Jeffrey; Engstrom, Paul; LaBaer, Joshua

    2011-01-01

    Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins.. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4,988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a pre-screen was performed to eliminate uninformative antigens. Sera from stage I–III IBC (n=53) and healthy women (n=53) were screened for AAb to all 4,988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p<0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p<0.05). These 761 antigens were screened using an independent set of IBC sera (n=51) and sera from women with benign breast disease (BBD) (n=39). From these, 119 antigens had a partial area under the ROC curve (p<0.05), with sensitivities ranging from 9–40% at >91% specificity. 28 of these antigens were confirmed using an independent serum cohort (n=51 cases/38 controls, p<0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC=0.756). These are potential biomarkers for the early detection of breast cancer. PMID:20977275

  3. Survivin and related proteins in canine mammary tumors: immunohistochemical expression.

    PubMed

    Bongiovanni, L; Romanucci, M; Malatesta, D; D'Andrea, A; Ciccarelli, A; Della Salda, L

    2015-03-01

    Survivin is reexpressed in most human breast cancers, where its expression has been associated with tumor aggressiveness, poor prognosis, and poor response to therapy. Survivin expression was evaluated in 41 malignant canine mammary tumors (CMTs) by immunohistochemistry, in relation to histological grade and stage, and correlated with that of some related molecules (β-catenin, caspase 3, heat shock proteins) to understand their possible role in canine mammary tumorigenesis. An increase in nuclear survivin expression, compared with healthy mammary glands, was observed in CMTs, where nuclear immunolabeling was related to the presence of necrosis. No statistically significant relation was found between the expression of the investigated molecules and the histological grade or stage. The present study may suggest an important involvement of survivin in CMT tumorigenesis. Its overexpression in most of the cases evaluated might suggest that targeting survivin in CMTs may be a valid anticancer therapy. PMID:24686389

  4. Expression and serological reactivity of hemorrhagic enteritis virus hexon protein.

    PubMed

    Lobová, Dana; Celer, Vladimír

    2016-05-01

    The aim of this work was to express the recombinant hexon protein of the hemorrhagic enteritis virus, to establish the diagnostic value of this protein for serological detection of antibodies in turkey serum samples and to assess seroprevalence of the infection in the Czech Republic. The N' terminal part of the hexon protein was expressed in a bacterial expression system and used as an antigen in an ELISA test for the detection of hemorrhagic enteritis virus specific antibodies in turkey sera. Validation of the test was performed by comparison with a commercially available ELISA test. Serological reactivity was assessed on a panel of 126 turkey sera by a newly developed ELISA test. Serum samples were taken from turkey farms with the history of hemorrhagic enteritis virus infection, from farms with animals free of infection, and from turkey farms following vaccination. Both ELISA kits gave identical results (100 %) with the tested sera. ELISA based on the recombinant hexon protein thus proved useful and cheaper for detection of antibodies in turkey flocks infected with the hemorrhagic enteritis virus. PMID:26471497

  5. Human testis expresses a specific poly(A)-binding protein.

    PubMed

    Féral, C; Guellaën, G; Pawlak, A

    2001-05-01

    In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids. PMID:11328870

  6. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders. PMID:26975317

  7. Small-molecule inhibitors of ERK-mediated immediate early gene expression and proliferation of melanoma cells expressing mutated BRaf

    PubMed Central

    Samadani, Ramin; Zhang, Jun; Brophy, Amanda; Oashi, Taiji; Priyakumar, U. Deva; Raman, E. Prabhu; St John, Franz J.; Jung, Kwan-Young; Fletcher, Steven; Pozharski, Edwin; MacKerell, Alexander D.; Shapiro, Paul

    2015-01-01

    Constitutive activation of the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) are central to regulating the proliferation and survival of many cancer cells. The current inhibitors of ERK1/2 target ATP binding or the catalytic site and are therefore limited in their utility for elucidating the complex biological roles of ERK1/2 through its phosphorylation and regulation of over 100 substrate proteins. To overcome this limitation, a combination of computational and experimental methods was used to identify low-molecular-mass inhibitors that are intended to target ERK1/2 substrate-docking domains and selectively interfere with ERK1/2 regulation of substrate proteins. In the present study, we report the identification and characterization of compounds with a thienyl benzenesulfonate scaffold that were designed to inhibit ERK1/2 substrates containing an F-site or DEF (docking site for ERK, FXF) motif. Experimental evidence shows the compounds inhibit the expression of F-site containing immediate early genes (IEGs) of the Fos family, including c-Fos and Fra1, and transcriptional regulation of the activator protein-1 (AP-1) complex. Moreover, this class of compounds selectively induces apoptosis in melanoma cells containing mutated BRaf and constitutively active ERK1/2 signalling, including melanoma cells that are inherently resistant to clinically relevant kinase inhibitors. These findings represent the identification and initial characterization of a novel class of compounds that inhibit ERK1/2 signalling functions and their potential utility for elucidating ERK1/2 and other signalling events that control the growth and survival of cancer cells containing elevated ERK1/2 activity. PMID:25695333

  8. Small-molecule inhibitors of ERK-mediated immediate early gene expression and proliferation of melanoma cells expressing mutated BRaf.

    PubMed

    Samadani, Ramin; Zhang, Jun; Brophy, Amanda; Oashi, Taiji; Priyakumar, U Deva; Raman, E Prabhu; St John, Franz J; Jung, Kwan-Young; Fletcher, Steven; Pozharski, Edwin; MacKerell, Alexander D; Shapiro, Paul

    2015-05-01

    Constitutive activation of the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) are central to regulating the proliferation and survival of many cancer cells. The current inhibitors of ERK1/2 target ATP binding or the catalytic site and are therefore limited in their utility for elucidating the complex biological roles of ERK1/2 through its phosphorylation and regulation of over 100 substrate proteins. To overcome this limitation, a combination of computational and experimental methods was used to identify low-molecular-mass inhibitors that are intended to target ERK1/2 substrate-docking domains and selectively interfere with ERK1/2 regulation of substrate proteins. In the present study, we report the identification and characterization of compounds with a thienyl benzenesulfonate scaffold that were designed to inhibit ERK1/2 substrates containing an F-site or DEF (docking site for ERK, FXF) motif. Experimental evidence shows the compounds inhibit the expression of F-site containing immediate early genes (IEGs) of the Fos family, including c-Fos and Fra1, and transcriptional regulation of the activator protein-1 (AP-1) complex. Moreover, this class of compounds selectively induces apoptosis in melanoma cells containing mutated BRaf and constitutively active ERK1/2 signalling, including melanoma cells that are inherently resistant to clinically relevant kinase inhibitors. These findings represent the identification and initial characterization of a novel class of compounds that inhibit ERK1/2 signalling functions and their potential utility for elucidating ERK1/2 and other signalling events that control the growth and survival of cancer cells containing elevated ERK1/2 activity. PMID:25695333

  9. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins. PMID:25851716

  10. Gene expression profile and synovial microcirculation at early stages of collagen-induced arthritis

    PubMed Central

    Gierer, Philip; Ibrahim, Saleh; Mittlmeier, Thomas; Koczan, Dirk; Moeller, Steffen; Landes, Jürgen; Gradl, Georg; Vollmar, Brigitte

    2005-01-01

    A better understanding of the initial mechanisms that lead to arthritic disease could facilitate development of improved therapeutic strategies. We characterized the synovial microcirculation of knee joints in susceptible mouse strains undergoing intradermal immunization with bovine collagen II in complete Freund's adjuvant to induce arthritis (i.e. collagen-induced arthritis [CIA]). Susceptible DBA1/J and collagen II T-cell receptor transgenic mice were compared with CIA-resistant FVB/NJ mice. Before onset of clinical symptoms of arthritis, in vivo fluorescence microscopy of knee joints revealed marked leucocyte activation and interaction with the endothelial lining of synovial microvessels. This initial inflammatory cell response correlated with the gene expression profile at this disease stage. The majority of the 655 differentially expressed genes belonged to classes of genes that are involved in cell movement and structure, cell cycle and signal transduction, as well as transcription, protein synthesis and metabolism. However, 24 adhesion molecules and chemokine/cytokine genes were identified, some of which are known to contribute to arthritis (e.g. CD44 and neutrophil cytosolic factor 1) and some of which are novel in this respect (e.g. CC chemokine ligand-27 and IL-13 receptor α1). Online in vivo data on synovial tissue microcirculation, together with gene expression profiling, emphasize the potential role played by early inflammatory events in the development of arthritis. PMID:15987489

  11. Reelin-immunoreactive neurons in entorhinal cortex layer II selectively express intracellular amyloid in early Alzheimer's disease.

    PubMed

    Kobro-Flatmoen, Asgeir; Nagelhus, Anne; Witter, Menno P

    2016-09-01

    The onset of Alzheimer's disease (AD) is associated with subtle pathological changes including increased intracellular expression of amyloid-β (Aβ). A structure affected particularly early in the course of AD is the entorhinal cortex, where neuronal death in layer II is observed already at initial stages. Neurons in EC-layer II, particularly those that express the protein Reelin, give rise to projections to the hippocampal dentate gyrus and this projection shows severe loss of synaptic contacts during early-stage AD. Given this anatomical specificity, we sought to determine whether increased intracellular expression of Aβ is selectively associated with Reelin-immunoreactive neurons in layer II of the entorhinal cortex. Here we report that in a transgenic rat model, which mimics the onset and distribution of extracellular amyloid deposits seen in human AD subjects, expression of intracellular Aβ in entorhinal layer II selectively occurs in Reelin-immunoreactive neurons during the early, pre-plaque stage. This Reelin-Aβ association is also present in human subjects with AD-related pathological changes, even in early disease stages. These findings strongly indicate that Reelin-immunoreactive neurons in entorhinal layer II play a crucial role during the initial stages of AD, and may therefore lead to refined hypotheses concerning the origin of this devastating condition. PMID:27195475

  12. Alternative Eukaryotic Expression Systems for the Production of Proteins and Protein Complexes.

    PubMed

    Gómez, Sara; López-Estepa, Miguel; Fernández, Francisco J; Suárez, Teresa; Vega, M Cristina

    2016-01-01

    Besides the most established expression hosts, several eukaryotic microorganisms and filamentous fungi have also been successfully used as platforms for the production of foreign proteins. Filamentous fungi and Dictyostelium discoideum are two prominent examples. Filamentous fungi, typically Aspergillus and Trichoderma, are usually employed for the industrial production of enzymes and secondary metabolites for food processing, pharmaceutical drugs production, and textile and paper applications, with multiple products already accepted for their commercialization. The low cost of culture medium components, high secretion capability directly to the extracellular medium, and the intrinsic ability to produce post-translational modifications similar to the mammalian type, have promoted this group as successful hosts for the expression of proteins, including examples from phylogenetically distant groups: humans proteins such as IL-2, IL-6 or epithelial growth factor; α-galactosidase from plants; or endoglucanase from Cellulomonas fimi, among others. D. discoideum is a social amoeba that can be used as an expression platform for a variety of proteins, which has been extensively illustrated for cytoskeletal proteins. New vectors for heterologous expression in D. discoideum have been recently developed that might increase the usefulness of this system and expand the range of protein classes that can be tackled. Continuous developments are ongoing to improve strains, promoters, production and downstream processes for filamentous fungi, D. discoideum, and other alternative eukaryotic hosts. Either for the overexpression of individual genes, or in the coexpression of multiples genes, this chapter illustrates the enormous possibilities offered by these groups of eukaryotic organisms. PMID:27165325

  13. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  14. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  15. High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

    PubMed

    Chen, W J; Moomaw, J F; Overton, L; Kost, T A; Casey, P J

    1993-05-01

    The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme. PMID:8486655

  16. Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

    SciTech Connect

    Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.; Bilimoria, Shaen L.

    2008-01-20

    Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV{sub XS}; 400 {mu}g/ml), UV-irradiated virus (CIV{sub UV}; 10 {mu}g/ml) and CVPE (CIV protein extract; 10 {mu}g/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 {mu}g/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i. CIV{sub UV} or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV{sub UV} particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV{sub UV}, CIV{sub XS} or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae

  17. Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells

    PubMed Central

    Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

    2016-01-01

    The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS. PMID:26934632

  18. HIV-1 Tat Protein Enhances Expression and Function of Breast Cancer Resistance Protein.

    PubMed

    Zhou, Yancong; Zhang, Kun; Yin, Xiaojie; Nie, Qichang; Ma, Yonggang

    2016-01-01

    ATP binding cassette (ABC) transporters can transfer a variety of antiviral agents from the cytoplasm to body fluid, which results in a reduced intracellular concentration of the drugs. Proteins of HIV-1, e.g., Tat and gp120, altered some types of ABC transporter expression in brain microvascular endothelial cells and astrocytes. However, the effect of Tat on ABC transporters in T lymphocytes is unclear. In this study the status of breast cancer resistance protein (BCRP) in Tat expressing cell lines was examined with real-time PCR and flow cytometry. It was found that HIV-1 Tat protein upregulated BCRP expression and enhanced efflux mediated by BCRP significantly, which could inhibit antiviral drugs from entering infected cells and interfere with the therapeutic effect of HAART. PMID:26367065

  19. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    PubMed Central

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  20. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  1. Improving Protein Expression Prediction Using Extra Features and Ensemble Averaging

    PubMed Central

    Fernandes, Armando; Vinga, Susana

    2016-01-01

    The article focus is the improvement of machine learning models capable of predicting protein expression levels based on their codon encoding. Support vector regression (SVR) and partial least squares (PLS) were used to create the models. SVR yields predictions that surpass those of PLS. It is shown that it is possible to improve the models predictive ability by using two more input features, codon identification number and codon count, besides the already used codon bias and minimum free energy. In addition, applying ensemble averaging to the SVR or PLS models also improves the results even further. The present work motivates the test of different ensembles and features with the aim of improving the prediction models whose correlation coefficients are still far from perfect. These results are relevant for the optimization of codon usage and enhancement of protein expression levels in synthetic biology problems. PMID:26934190

  2. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  3. Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27.

    PubMed Central

    Panagiotidis, C A; Lium, E K; Silverstein, S J

    1997-01-01

    The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4. PMID:8995681

  4. Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

    PubMed Central

    Grosso, L E; Parks, W C; Wu, L J; Mecham, R P

    1991-01-01

    A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor. Images Fig. 2. Fig. 3. PMID:1996952

  5. Expression of extracellular matrix proteins in cervical squamous cell carcinoma--a clinicopathological study.

    PubMed Central

    Goldberg, I; Davidson, B; Lerner-Geva, L; Gotlieb, W H; Ben-Baruch, G; Novikov, I; Kopolovic, J

    1998-01-01

    AIM: To evaluate the intracellular and peritumoral expression of matrix proteins in squamous cell carcinoma of the uterine cervix using immunohistochemistry. METHODS: 71 squamous cell carcinomas and 10 controls were stained for laminin, fibronectin, and collagen IV. Cytoplasmic staining in tumour cells and peritumoral deposition of matrix proteins were evaluated. The association between staining results and patient age, tumour stage, histological grade, and survival was studied. RESULTS: Positive cytoplasmic staining for laminin, fibronectin, and collagen IV was observed in 17 (23.9%), 27 (38%), and 10 (14.1%) cases, respectively. Staining for laminin was most pronounced in the invasive front of tumour islands, while for fibronectin and collagen IV it appeared to be diffuse. Peritumoral staining for laminin and collagen IV was detected in 12 cases (16.9%). Early stage (Ia1-Ia2) tumours were uniformly negative for all three proteins. Cytoplasmic staining for laminin correlated with positive staining for fibronectin and collagen IV, and with the presence of a peritumoral deposition of collagen IV and laminin. There was no correlation with any of the three markers between staining results and patient age, stage, grade, or survival. CONCLUSIONS: Expression of extracellular matrix proteins in some cervical squamous cell carcinomas might reflect the enhanced ability of these tumours to modify the peritumoral stroma. This ability seems to be absent in early stage tumours. The correlation between intracytoplasmic and peritumoral expression of matrix proteins supports the evidence of their synthesis by tumour cells. However, this property did not correlate with disease outcome in this study. Images PMID:10023343

  6. EARLY SENESCENCE1 Encodes a SCAR-LIKE PROTEIN2 That Affects Water Loss in Rice1[OPEN

    PubMed Central

    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

    2015-01-01

    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. PMID:26243619

  7. Live Imaging Fluorescent Proteins in Early Mouse Embryos

    PubMed Central

    Xenopoulos, Panagiotis; Nowotschin, Sonja; Hadjantonakis, Anna-Katerina

    2016-01-01

    Mouse embryonic development comprises highly dynamic and coordinated events that drive key cell lineage specification and morphogenetic events. These processes involve cellular behaviors including proliferation, migration, apoptosis, and differentiation, each of which is regulated both spatially and temporally. Live imaging of developing embryos provides an essential tool to investigate these coordinated processes in three-dimensional space over time. For this purpose, the development and application of genetically encoded fluorescent protein (FP) reporters has accelerated over the past decade allowing for the high-resolution visualization of developmental progression. Ongoing efforts are aimed at generating improved reporters, where spectrally distinct as well as novel FPs whose optical properties can be photomodulated, are exploited for live imaging of mouse embryos. Moreover, subcellular tags in combination with using FPs allow for the visualization of multiple subcellular characteristics, such as cell position and cell morphology, in living embryos. Here, we review recent advances in the application of FPs for live imaging in the early mouse embryo, as well as some of the methods used for ex utero embryo development that facilitate on-stage time-lapse specimen visualization. PMID:22341233

  8. Distinct patterns of gene and protein expression elicited by organophosphorus pesticides in Caenorhabditis elegans

    PubMed Central

    Lewis, John A; Szilagyi, Maria; Gehman, Elizabeth; Dennis, William E; Jackson, David A

    2009-01-01

    Background The wide use of organophosphorus (OP) pesticides makes them an important public health concern. Persistent effects of exposure and the mechanism of neuronal degeneration are continuing issues in OP toxicology. To elucidate early steps in the mechanisms of OP toxicity, we studied alterations in global gene and protein expression in Caenorhabditis elegans exposed to OPs using microarrays and mass spectrometry. We tested two structurally distinct OPs (dichlorvos and fenamiphos) and employed a mechanistically different third neurotoxicant, mefloquine, as an out-group for analysis. Treatment levels used concentrations of chemical sufficient to prevent the development of 10%, 50% or 90% of mid-vulval L4 larvae into early gravid adults (EGA) at 24 h after exposure in a defined, bacteria-free medium. Results After 8 h of exposure, the expression of 87 genes responded specifically to OP treatment. The abundance of 34 proteins also changed in OP-exposed worms. Many of the genes and proteins affected by the OPs are expressed in neuronal and muscle tissues and are involved in lipid metabolism, cell adhesion, apoptosis/cell death, and detoxification. Twenty-two genes were differentially affected by the two OPs; a large proportion of these genes encode cytochrome P450s, UDP-glucuronosyl/UDP-glucosyltransferases, or P-glycoproteins. The abundance of transcripts and the proteins they encode were well correlated. Conclusion Exposure to OPs elicits a pattern of changes in gene expression in exposed worms distinct from that of the unrelated neurotoxicant, mefloquine. The functional roles and the tissue location of the genes and proteins whose expression is modulated in response to exposure is consistent with the known effects of OPs, including damage to muscle due to persistent hypercontraction, neuronal cell death, and phase I and phase II detoxification. Further, the two different OPs evoked distinguishable changes in gene expression; about half the differences are in

  9. Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin.

    PubMed

    George, Caroline L S; White, Misty L; O'Neill, Marsha E; Thorne, Peter S; Schwartz, David A; Snyder, Jeanne M

    2003-12-01

    Chronically inhaled endotoxin, which is ubiquitous in many occupational and domestic environments, can adversely affect the respiratory system resulting in an inflammatory response and decreased lung function. Surfactant-associated protein A (SP-A) is part of the lung innate immune system and may attenuate the inflammatory response in various types of lung injury. Using a murine model to mimic occupational exposures to endotoxin, we hypothesized that SP-A gene expression and protein would be elevated in response to repeat exposure to inhaled grain dust and to purified lipopolysaccharide (LPS). Our results demonstrate that repeat exposure to inhaled endotoxin, either in the form of grain dust or purified LPS, results in increased whole lung SP-A gene expression and type II alveolar epithelial cell hyperplasia, whereas SP-A protein levels in lung lavage fluid are decreased. Furthermore, these alterations in SP-A gene activity and protein metabolism are dependent on an intact endotoxin signaling system. PMID:12922979

  10. Serum protein-expression profiling using the ProteinChip biomarker system.

    PubMed

    Gilbert, Kate; Figueredo, Sharel; Meng, Xiao-Ying; Yip, Christine; Fung, Eric T

    2004-01-01

    Protein-expression profiling of serum is a common approach to the discovery of potential diagnostic and therapeutic markers of disease. Like any other proteome, the serum proteome is characterized by protein expression across a large dynamic range. This single facet requires the employment of fractionation procedures prior to detection of protein. The authors use a combination of conventional column chromatography with array-based chromatography to simplify the serum proteome into subproteomes, thus providing a greater representation of the serum proteome. Robotics is employed to increase the throughput of sample processing. These procedures result in large amounts of data that are analyzed through a series of preprocessing and postprocessing steps. A well-designed serum profiling project can therefore result in the discovery of statistically sound, clinically meaningful protein biomarkers. PMID:15020796

  11. Differential expression of ribosomal proteins in myelodysplastic syndromes.

    PubMed

    Rinker, Elizabeth B; Dueber, Julie C; Qualtieri, Julianne; Tedesco, Jason; Erdogan, Begum; Bosompem, Amma; Kim, Annette S

    2016-02-01

    Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond-Blackfan anaemia, Shwachman-Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non-5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed. PMID:26408650

  12. Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

    PubMed Central

    van Eekelen, Mark; Overvoorde, John; van Rooijen, Carina; den Hertog, Jeroen

    2010-01-01

    Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs. PMID:20838449

  13. Insulin-like growth factor (IGF) and IGF binding protein gene expression in multicystic renal dysplasia.

    PubMed

    Matsell, D G; Bennett, T; Armstrong, R A; Goodyer, P; Goodyer, C; Han, V K

    1997-01-01

    Multicystic dysplastic kidney disease is the most common form of renal dysplasia that leads to ESRD in children. This study describes the histopathological changes of multicystic dysplasia that occur from early fetal life to the postnatal period. At 14 wk gestation, early cystic enlargement of various segments of the nephron have been identified, in addition to a displaced metanephric blastema adjacent to zones of normal nephrogenesis. At later stages, the predominant features include cyst enlargement with marked fibromuscular collars, architectural disorganization, and replacement of the interstitium with a disarray of mesenchymal tissue. This study investigated the expression of the mRNA encoding the insulin-like growth factors (IGF) and IGF binding proteins (IGFBP) and have demonstrated IGF-II, IGFBP-2, and IGFBP-3 to be altered. Apart from their expression in the displaced metanephric blastema, both IGF-II and IGFBP-2 were overexpressed in abnormal tissue elements in all kidneys from fetal to postnatal life. IGF-II gene expression was localized to mesenchymal tissue, specifically in the periductal fibromuscular collars. IGFBP-2 mRNA was found to be expressed exclusively in the cyst epithelia of all cysts at all ages studied, whereas IGFBP-3 mRNA was absent from these epithelia. This study details the failure of normal IGF expression in the development of multicystic renal dysplasia and suggests a role for the IGF system in the progressive histopathological changes of this disorder. PMID:9013452

  14. Proteomic analysis of the differentially expressed proteins by airborne nanoparticles.

    PubMed

    Park, Seul Ki; Jeon, Yu Mi; Son, Bu Soon; Youn, Hyung Sun; Lee, Mi Young

    2011-07-01

    Airborne nanoparticles with thermodynamic diameters less than 56 nm (PM(0.056)) were collected using a Moudi cascade impactor, and the differentially expressed proteins upon exposure to the airborne nanoparticles were identified in human bronchial epithelial cells. More than 600 protein spots were detected on the two-dimensional gel, and the identified 13 of these proteins showed notable changes. Nine were up-regulated and four were down-regulated following treatment with the airborne nanoparticles. Notably, malignant transformation-associated multiple forms of keratins, epigenetic regulation-related MBD1-containing chromatin associated factor 2, epithelial malignancy-related vimentin and exocytosis-related annexin A2 were changed upon exposure to airborne nanoparticle PM(0.056). PMID:21491466

  15. Mechanism of expression of the rat HCNP precursor protein gene.

    PubMed

    Tohdoh, N; Tojo, S; Kimura, M; Ishii, T; Ojika, K

    1997-04-01

    The hippocampal cholinergic neurostimulating peptide (HCNP), isolated from hippocampal tissue of 10- to 12-day-old rats, enhances the in vitro synthesis of acetylcholine in medial septal tissue explants. The HCNP precursor is a 21 kDa protein that binds hydrophobic ligands and Mg-ATP, and is associated with the opioid-binding protein. We employed an HCNP-precursor cDNA as probe to clone the genomic DNA, used for mapping of the exon-intron structure of the gene. We also determined the nucleotide structure of the promoter region of the rat HCNP precursor protein gene. By using S1 mapping and CAT as a reporter, we found multiple promoters that were aligned in the 5' untranslated region. In addition, the presence of several putative enhancer binding sequences were tested by electrophoresis mobility shift assays. Northern blot analysis revealed that the gene is expressed in a variety of rat tissues and various subregions of the brain. These results suggest that HCNP-precursor gene expression is regulated by a general transactivation factor such as SP1, and that the specific presence of the bioactive HCNP in certain tissues results from post-translational events such as proteolytic processing of the precursor protein, which takes place predominantly in the hippocampus of young rats. PMID:9105667

  16. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. PMID:20347821

  17. Gi/o proteins: expression for direct activation enquiry.

    PubMed

    Di Cesare Mannelli, Lorenzo; Pacini, Alessandra; Toscano, Annarita; Fortini, Martina; Berti, Debora; Ghelardini, Carla; Galeotti, Nicoletta; Baglioni, Piero; Bartolini, Alessandro

    2006-05-01

    G protein-mediated pathways are fundamental mechanisms of cell signaling. In this paper, the expression and the characterization of the alphai1, alphai3, alphao1, beta1, and gamma2 subunits of the human G protein are described. This approach was developed to evaluate the G protein activation profile of new compounds. pCR-TOPO T7 vectors, engineered to contain the target sequences, were used to transform Escherichia coli competent cells. Subunits were over-expressed in a preparative scale as fusion proteins with a six-histidine tag, and subsequently purified by metal chelate chromatography. Afterward, the His-tag was removed by enterokinase digestion, and the secondary structures of the recombinant subunits were analyzed by circular dichroism. To assess the functionality of the subunits, the rate of GTP hydrolysis and GTPgammaS binding were evaluated both in the absence and in the presence of two modulators: the peptidic activator Mastoparan and the non-peptidic activator N-dodecyl-lysinamide (ML250). Tests were conducted on isolated alpha-subunit and on heterotrimeric alphabetagamma complex, alone or reconstituted in phospholipidic vesicles. Our results show that recombinant subunits are stable, properly folded and, fully active, which makes them suitable candidates for functional studies. PMID:16364655

  18. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  19. Regulation of RAG-2 protein expression in avian thymocytes.

    PubMed Central

    Ferguson, S E; Accavitti, M A; Wang, D D; Chen, C L; Thompson, C B

    1994-01-01

    The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells. Images PMID:7935443

  20. Expression pattern of Protein Kinase C ϵ during mouse embryogenesis

    PubMed Central

    2013-01-01

    Background Protein kinase C epsilon (PKCϵ) belongs to the novel PKC subfamily, which consists of diacylglycerol dependent- and calcium independent-PKCs. Previous studies have shown that PKCϵ is important in different contexts, such as wound healing or cancer. In this study, we contribute to expand the knowledge on PKCϵ by reporting its expression pattern during murine midgestation using the LacZ reporter gene and immunostaining procedures. Results Sites showing highest PKCϵ expression were heart at ealier stages, and ganglia in older embryos. Other stained domains included somites, bone, stomach, kidney, and blood vessels. Conclusions The seemingly strong expression of PKCϵ in heart and ganglia shown in this study suggests a important role of this isoform in the vascular and nervous systems during mouse development. However, functional redundancy with other PKCs during midgestation within these domains and others reported here possibly exists since PKCϵ deficient mice do not display obvious embryonic developmental defects. PMID:23639204

  1. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

    PubMed

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  2. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

    PubMed Central

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  3. Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera.

    PubMed

    Calvello, M; Brandazza, A; Navarrini, A; Dani, F R; Turillazzi, S; Felicioli, A; Pelosi, P

    2005-04-01

    The expression of chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) in individuals of different castes and ages have been monitored in three species of social hymenopterans, Polistes dominulus (Hymenoptera, Vespidae), Vespa crabro (Hymenoptera, Vespidae) and Apis mellifera (Hymenoptera, Apidae), using PCR with specific primers and polyclonal antibodies. In the paper wasp P. dominulus, OBP is equally expressed in antennae, wings and legs of all castes and ages, while CSP is often specifically present in antennae and in some cases also in legs. In the vespine species V. crabro CSP is antennal specific, while OBP is also expressed in legs and wings. The three CSPs and the five OBPs of A. mellifera show a complex pattern of expression, where both classes of proteins include members specifically expressed in antennae and others present in other parts of the body. These data indicate that at least in some hymenopteran species CSPs are specifically expressed in antennae and could perform roles in chemosensory perception so far assigned only to OBPs. PMID:15763466

  4. Unique expression of cytoskeletal proteins in human soft palate muscles.

    PubMed

    Shah, Farhan; Berggren, Diana; Holmlund, Thorbjörn; Levring Jäghagen, Eva; Stål, Per

    2016-03-01

    The human oropharyngeal muscles have a unique anatomy with diverse and intricate functions. To investigate if this specialization is also reflected in the cytoarchitecture of muscle fibers, intermediate filament proteins and the dystrophin-associated protein complex have been analyzed in two human palate muscles, musculus uvula (UV) and musculus palatopharyngeus (PP), with immunohistochenmical and morphological techniques. Human limb muscles were used as reference. The findings show that the soft palate muscle fibers have a cytoskeletal architecture that differs from the limb muscles. While all limb muscles showed immunoreaction for a panel of antibodies directed against different domains of cytoskeletal proteins desmin and dystrophin, a subpopulation of palate muscle fibers lacked or had a faint immunoreaction for desmin (UV 11.7% and PP 9.8%) and the C-terminal of the dystrophin molecule (UV 4.2% and PP 6.4%). The vast majority of these fibers expressed slow contractile protein myosin heavy chain I. Furthermore, an unusual staining pattern was also observed in these fibers for β-dystroglycan, caveolin-3 and neuronal nitric oxide synthase nNOS, which are all membrane-linking proteins associated with the dystrophin C-terminus. While the immunoreaction for nNOS was generally weak or absent, β-dystroglycan and caveolin-3 showed a stronger immunostaining. The absence or a low expression of cytoskeletal proteins otherwise considered ubiquitous and important for integration and contraction of muscle cells indicate a unique cytoarchitecture designed to meet the intricate demands of the upper airway muscles. It can be concluded that a subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties, and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles. PMID:26597319

  5. Amyloid Precursor Protein Expression Modulates Intestine Immune Phenotype

    PubMed Central

    Puig, Kendra L.; Swigost, Adam J.; Zhou, Xudong; Sens, MaryAnn; Combs, Colin K.

    2014-01-01

    Amyloid precursor protein (APP) is widely expressed across many tissue and cell types. Proteolytic processing of the protein gives rise to a plethora of protein fragments with varied biological activities. Although a large amount of data has been generated describing the metabolism of the protein in neurons, its role in regulating the phenotype of other cells remains unclear. Based upon prior work demonstrating that APP regulates the activation phenotype of monocytic lineage cells, we hypothesized that APP can regulate macrophage activation phenotype in tissues other than brain. Ileums of the small intestines from C57BL6/J wild type and APP−/− mice were compared as a representative tissue normally associated with abundant macrophage infiltration. APP−/− intestines demonstrated diminished CD68 immunoreactivity compared to wild type mice. This correlated with significantly less cycloxygenase-2 (cox-2), CD68, CD40, CD11c, and βIII-tubulin protein levels. Peritoneal macrophage from APP−/− mice demonstrated decreased in vitro migratory ability compared to wild type cells and diminished basal KC cytokine secretion. Whereas, APP−/− intestinal macrophage had an increase in basal KC cytokine secretion compared to wild type cells. Conversely, there was a significant decrease in multiple cytokine levels in APP−/− compared to wild type ileums. Finally, APP−/− mice demonstrated impaired absorption and increased motility compared to wild type mice. These data demonstrate the APP expression regulates immune cell secretions and phenotype and intestinal function. This data set describes a novel function for this protein or its metabolites that may be relevant not only for Alzheimer’s disease but a range of immune-related disorders. PMID:22124967

  6. Expression and Localization of Lung Surfactant Proteins in Human Testis

    PubMed Central

    Wagner, Walter; Matthies, Cord; Ruf, Christian; Hartmann, Arndt; Garreis, Fabian; Paulsen, Friedrich

    2015-01-01

    Background Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions. Objective The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated. Results Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig. Conclusion Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP's was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor. PMID:26599233

  7. Transcriptome Sequencing, De Novo Assembly and Differential Gene Expression Analysis of the Early Development of Acipenser baeri

    PubMed Central

    Song, Wei; Jiang, Keji; Zhang, Fengying; Lin, Yu; Ma, Lingbo

    2015-01-01

    The molecular mechanisms that drive the development of the endangered fossil fish species Acipenser baeri are difficult to study due to the lack of genomic data. Recent advances in sequencing technologies and the reducing cost of sequencing offer exclusive opportunities for exploring important molecular mechanisms underlying specific biological processes. This manuscript describes the large scale sequencing and analyses of mRNA from Acipenser baeri collected at five development time points using the Illumina Hiseq2000 platform. The sequencing reads were de novo assembled and clustered into 278167 unigenes, of which 57346 (20.62%) had 45837 known homologues proteins in Uniprot protein databases while 11509 proteins matched with at least one sequence of assembled unigenes. The remaining 79.38% of unigenes could stand for non-coding unigenes or unigenes specific to A. baeri. A number of 43062 unigenes were annotated into functional categories via Gene Ontology (GO) annotation whereas 29526 unigenes were associated with 329 pathways by mapping to KEGG database. Subsequently, 3479 differentially expressed genes were scanned within developmental stages and clustered into 50 gene expression profiles. Genes preferentially expressed at each stage were also identified. Through GO and KEGG pathway enrichment analysis, relevant physiological variations during the early development of A. baeri could be better cognized. Accordingly, the present study gives insights into the transcriptome profile of the early development of A. baeri, and the information contained in this large scale transcriptome will provide substantial references for A. baeri developmental biology and promote its aquaculture research. PMID:26359664

  8. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    PubMed Central

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  9. Anesthesia-induced hypothermia mediates decreased ARC gene and protein expression through ERK/MAPK inactivation

    PubMed Central

    Whittington, Robert A.; Bretteville, Alexis; Virág, László; Emala, Charles W.; Maurin, Thomas O.; Marcouiller, François; Julien, Carl; Petry, Franck R.; El-Khoury, Noura B.; Morin, Françoise; Charron, Jean; Planel, Emmanuel

    2013-01-01

    Several anesthetics have been reported to suppress the transcription of a number of genes, including Arc, also known as Arg3.1, an immediate early gene that plays a significant role in memory consolidation. The purpose of this study was to explore the mechanism of anesthesia-mediated depression in Arc gene and protein expression. Here, we demonstrate that isoflurane or propofol anesthesia decreases hippocampal Arc protein expression in rats and mice. Surprisingly, this change was secondary to anesthesia-induced hypothermia. Furthermore, we confirm in vivo and in vitro that hypothermia per se is directly responsible for decreased Arc protein levels. This effect was the result of the decline of Arc mRNA basal levels following inhibition of ERK/MAPK by hypothermia. Overall, our results suggest that anesthesia-induced hypothermia leads to ERK inhibition, which in turns decreases Arc levels. These data give new mechanistic insights on the regulation of immediate early genes by anesthesia and hypothermia. PMID:24045785

  10. Constraints imposed by nonfunctional protein-protein interactions on gene expression and proteome size

    NASA Astrophysics Data System (ADS)

    Zhang, Jingshan; Maslov, Sergei; Shakhnovich, Eugene

    2009-03-01

    Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing nonfunctional interactions with promiscuous nonfunctional partners. Here we show how nonfunctional interactions limit the proteome diversity and the average concentration of co-expressed and co-localized proteins. We use yeast compartments to verify our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, while keeping individual protein concentrations sufficiently low to reduce nonfunctional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity, and differentiation.

  11. A systematic approach for testing expression of human full-length proteins in cell-free expression systems

    PubMed Central

    Langlais, Claudia; Guilleaume, Birgit; Wermke, Nadja; Scheuermann, Tina; Ebert, Lars; LaBaer, Joshua; Korn, Bernhard

    2007-01-01

    Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield. PMID:17915018

  12. The novel protein DELAYED PALE-GREENING1 is required for early chloroplast biogenesis in Arabidopsis thaliana.

    PubMed

    Liu, Dong; Li, Weichun; Cheng, Jianfeng

    2016-01-01

    Chloroplast biogenesis is one of the most important subjects in plant biology. In this study, an Arabidopsis early chloroplast biogenesis mutant with a delayed pale-greening phenotype (dpg1) was isolated from a T-DNA insertion mutant collection. Both cotyledons and true leaves of dpg1 mutants were initially albino but gradually became pale green as the plant matured. Transmission electron microscopic observations revealed that the mutant displayed a delayed proplastid-to-chloroplast transition. Sequence and transcription analyses showed that AtDPG1 encodes a putatively chloroplast-localized protein containing three predicted transmembrane helices and that its expression depends on both light and developmental status. GUS staining for AtDPG1::GUS transgenic lines showed that this gene was widely expressed throughout the plant and that higher expression levels were predominantly found in green tissues during the early stages of Arabidopsis seedling development. Furthermore, quantitative real-time RT-PCR analyses revealed that a number of chloroplast- and nuclear-encoded genes involved in chlorophyll biosynthesis, photosynthesis and chloroplast development were substantially down-regulated in the dpg1 mutant. These data indicate that AtDPG1 plays an essential role in early chloroplast biogenesis, and its absence triggers chloroplast-to-nucleus retrograde signalling, which ultimately down-regulates the expression of nuclear genes encoding chloroplast-localized proteins. PMID:27160321

  13. The novel protein DELAYED PALE-GREENING1 is required for early chloroplast biogenesis in Arabidopsis thaliana

    PubMed Central

    Liu, Dong; Li, Weichun; Cheng, Jianfeng

    2016-01-01

    Chloroplast biogenesis is one of the most important subjects in plant biology. In this study, an Arabidopsis early chloroplast biogenesis mutant with a delayed pale-greening phenotype (dpg1) was isolated from a T-DNA insertion mutant collection. Both cotyledons and true leaves of dpg1 mutants were initially albino but gradually became pale green as the plant matured. Transmission electron microscopic observations revealed that the mutant displayed a delayed proplastid-to-chloroplast transition. Sequence and transcription analyses showed that AtDPG1 encodes a putatively chloroplast-localized protein containing three predicted transmembrane helices and that its expression depends on both light and developmental status. GUS staining for AtDPG1::GUS transgenic lines showed that this gene was widely expressed throughout the plant and that higher expression levels were predominantly found in green tissues during the early stages of Arabidopsis seedling development. Furthermore, quantitative real-time RT-PCR analyses revealed that a number of chloroplast- and nuclear-encoded genes involved in chlorophyll biosynthesis, photosynthesis and chloroplast development were substantially down-regulated in the dpg1 mutant. These data indicate that AtDPG1 plays an essential role in early chloroplast biogenesis, and its absence triggers chloroplast-to-nucleus retrograde signalling, which ultimately down-regulates the expression of nuclear genes encoding chloroplast-localized proteins. PMID:27160321

  14. Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins

    PubMed Central

    2013-01-01

    Background A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity. Results In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full) - immunodominant antigens of Mycobacterium tuberculosis - were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone. Conclusions We reached the goal of purifying large quantities of soluble antigens effective in generating immunological response against M. tuberculosis by a robust, controlled, scalable and economically feasible production process. PMID:24252280

  15. Shared Understanding and Idiosyncratic Expression in Early Vocabularies

    ERIC Educational Resources Information Center

    Mayor, Julien; Plunkett, Kim

    2014-01-01

    To what extent do toddlers have shared vocabularies? We examined CDI data collected from 14,607 infants and toddlers in five countries and measured the amount of variability between individual lexicons during development for both comprehension and production. Early lexicons are highly overlapping. However, beyond 100 words, toddlers share more…

  16. Expression of proteoglycan core proteins in human bone marrow stroma.

    PubMed Central

    Schofield, K P; Gallagher, J T; David, G

    1999-01-01

    Heparan sulphate proteoglycans (HSPGs) present on the surface of bone marrow stromal cells and in the extracellular matrix (ECM) have important roles in the control of adhesion and growth of haemopoietic stem and progenitor cells. The two main groups of proteoglycans which contain heparan sulphate chains are members of the syndecan and glypican families. In this study we have identified the main surface membrane and matrix-associated HSPGs present in normal human bone marrow stroma formed in long-term culture. Proteoglycans were extracted from the adherent stromal layers and treated with heparitinase and chondroitinase ABC. The core proteins were detected by Western blotting using antibodies directed against syndecans-1-4, glypican-1 and the ECM HSPG, perlecan. Stromal cell expression at the RNA level was detected by Northern blotting and by reverse transcription PCR. Glypican-1, syndecan-3 and syndecan-4 were the major cell-membrane HSPG species and perlecan was the major ECM proteoglycan. There was no evidence for expression of syndecan-1 protein. Syndecan-3 was expressed mainly as a variant or processed 50-55 kDa core protein and in lower amounts as the characteristic 125 kDa core protein. These results suggest that syndecan-3, syndecan-4 and glypican-1 present on the surface of marrow stromal cells, together with perlecan in the ECM, may be responsible for creating the correct stromal 'niche' for the maintenance and development of haemopoietic stem and progenitor cells. The detection of a variant form of syndecan-3 as a major stromal HSPG suggests a specific role for this syndecan in haemopoiesis. PMID:10527946

  17. Protein expression and characterization of SEP3 from Arabidopsis thaliana.

    PubMed

    Shi, Q; Zhou, J; Wang, P; Lin, X; Xu, Y

    2015-01-01

    SEPALLATA (SEP) MADS-box genes play crucial roles in the regulation of floral growth and development. They are required for the specification of sepals, petals, stamens, and carpels as well as for floral determinacy. SEPs perform their functions through the formation of homo- or hetero-polymers, which are the molecular basis of floral quartets. In vitro assays indicated that SEP3 forms a tetramer after binding to DNA, but it is unclear whether DNA binding induces the tetramer, because SEP3 is often reported to form a dimer. Here, we analyzed the oligomeric status of SEP3 domains in the absence of the DNA-binding MADS-box domain. The truncated SEP3 was constructed as a fusion protein and expressed in prokaryotic cells. The purified protein fragment displayed as a tetramer in the size exclusion chromatographic column, and a glutaraldehyde cross-linking assay demonstrated that the protein contained a dimer unit. Yeast two-hybrid tests further verified that the fragments form homologous polymers in vivo, and that the K domain is involved in tetramer formation. Current results imply that the SEP3 protein regulates the formation of flower meristems using the tetramer as a unit, and that the DNA-binding MADS-box is dispensable for polymer formation. The C-terminal region does not contribute to homo-tetramer formation, but it may be reserved to glue other proteins. PMID:26505403

  18. Preliminary identification of differentially expressed tear proteins in keratoconus

    PubMed Central

    Wasinger, Valerie C.; Pye, David C.; Willcox, Mark D. P.

    2013-01-01

    Purpose To examine the proteins differentially expressed in the tear film of people with keratoconus and normal subjects. Methods Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microflow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. Results Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobulin receptor (ninefold), fibrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. Conclusions The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies reflect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options. PMID:24194634

  19. Protein expression strategies in Tobacco necrosis virus-D.

    PubMed

    Chkuaseli, Tamari; Newburn, Laura R; Bakhshinyan, David; White, K Andrew

    2015-12-01

    Tobacco necrosis virus (TNV-D) has a plus-strand RNA genome that is neither 5' capped nor 3' poly-adenylated. Instead, it utilizes a 3' cap-independent translational enhancer (3'CITE) located in its 3' untranslated region (UTR) for translation of its proteins. We have examined the protein expression strategies used by TNV-D and our results indicate that: (i) a base pairing interaction between conserved ACCA and UGGU motifs in the genomic 5'UTR and 3'CITE, respectively, is not required for efficient plant cell infection, (ii) similar potential 5'UTR-3'CITE interactions in the two viral subgenomic mRNAs are not needed for efficient translation of viral proteins in vitro, (iii) a small amount of capsid protein is translated from the viral genome by a largely 3'CITE-independent mechanism, (iv) the larger of two possible forms of capsid protein is efficiently translated, and (v) p7b is translated from subgenomic mRNA1 by a leaky scanning mechanism. PMID:26402375

  20. Circulating promyelocytes and low levels of CD16 expression on polymorphonuclear leukocytes accompany early-onset periodontitis.

    PubMed Central

    Nemoto, E; Nakamura, M; Shoji, S; Horiuchi, H

    1997-01-01

    Early-onset periodontitis (EOP) is characterized by rapidly progressive alveolar bone loss, chemotactic defects of neutrophils, and significant familial aggregation. We found immature myeloid lineage cells, defined as promyelocytes, in the peripheral blood in patients with EOP. A hematological examination of peripheral blood cells showed normal reference values regarding cell proportions. Flow cytometry revealed significantly lower expression of CD16, a glycosylphosphatidylinositol (GPI)-anchored protein, on peripheral neutrophils in patients compared with those in age- and sex-matched healthy controls, whereas the levels of CD11a and CD11b expression were similar. The chemotactic response of neutrophils was lower toward not only formyl-methionyl-leucyl-phenylalanine but also complement fragment C5a than that of healthy controls. The expression of another GPI-anchored protein, CD14, was equally expressed by controls and patients. Therefore, the low level of CD16 expression was not due to the incomplete synthesis of the GPI anchor. GPI anchors of CD16 on neutrophils from controls and patients were both partially resistant to phosphatidylinositol-specific phospholipase C. The presence of promyelocytes in peripheral blood, low expression of CD16, and low chemotactic response of neutrophils suggest that patients with EOP have an abnormal maturation system in myeloid lineage cells in the bone marrow, which may be associated with the onset and course of EOP. PMID:9284170

  1. HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation

    PubMed Central

    Johansson, Cecilia; Somberg, Monika; Li, Xiaoze; Backström Winquist, Ellenor; Fay, Joanna; Ryan, Fergus; Pim, David; Banks, Lawrence; Schwartz, Stefan

    2012-01-01

    We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2. PMID:22617423

  2. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins

    PubMed Central

    2013-01-01

    Background Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. Results The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. Conclusion The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags

  3. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

    PubMed Central

    Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

    2016-01-01

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

  4. Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells.

    PubMed Central

    Chapman, B S; Thayer, R M; Vincent, K A; Haigwood, N L

    1991-01-01

    A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells. Images PMID:1650459

  5. Early gene expression in Pseudomonas fluorescens exposed to a polymetallic solution.

    PubMed

    Gómez-Sagasti, María T; Becerril, José M; Epelde, Lur; Alkorta, Itziar; Garbisu, Carlos

    2015-02-01

    The molecular response of Pseudomonas fluorescens cells exposed to a mixture of heavy metals remains largely unknown. Here, we studied the temporal changes in the early gene expression of P. fluorescens cells exposed to three doses of a polymetallic solution over two exposure times, through the application of a customized cDNA microarray. At the lowest metal dose (MD/4), we observed a repression of the Hsp70 chaperone system, MATE and MFS transporters, TonB membrane transporter and histidine kinases, together with an overexpression of metal transport (ChaC, CopC), chemotaxis and glutamine synthetase genes. At the intermediate metal dose (MD), several amino acid transporters, a response regulator (CheY), a TonB-dependent receptor and the mutT DNA repair gene were repressed; by contrast, an overexpression of genes associated with the antioxidative stress system and the transport of chelates and sulfur was observed. Finally, at the highest metal dose (4MD), a repression of genes encoding metal ion transporters, drug resistance and alginate biosynthesis was found, together with an overexpression of genes encoding antioxidative proteins, membrane transporters, ribosomal proteins, chaperones and proteases. It was concluded that P. fluorescens cells showed, over exposure time, a highly complex molecular response when exposed to a polymetallic solution, involving mechanisms related with chemotaxis, signal transmission, membrane transport, cellular redox state, and the regulation of transcription and ribosomal activity. PMID:25754557

  6. Calcitonin Induces Expression of the Inducible cAMP Early Repressor in Osteoclasts

    PubMed Central

    Yang, Maobin; Kream, Barbara E.

    2010-01-01

    The cAMP response element modulator gene (Crem) encodes a variety of transcriptional regulators including the inducible cAMP early repressor, ICER. We previously showed that Crem knockout mice, which are deficient in CREM and ICER factors, display slightly increased long bone mass and decreased osteoclast number. These data are consistent with the notion that Crem regulates bone mass in part through an effect on osteoclast formation and/or function. Since ICER is strongly induced by cAMP, we asked whether the calcium-regulating hormone calcitonin, which stimulates cAMP production and inhibits osteoclastic bone resorption, could induce ICER in osteoclasts. The monocytic cell line RAW264.7 was treated with receptor activator of NF-κB ligand (RANKL) to induce osteoclast formation. Calcitonin caused a time- and dose-dependent induction of ICER mRNA and an increase in ICER protein abundance in RANKL-treated RAW264.7 cells. Calcitonin also induced ICER mRNA and protein in osteoclasts derived from primary mouse bone marrow cell cultures. Calcitonin-treated osteoclasts showed immunoreactivity with an anti-CREM antibody. Calcitonin decreased the activity of wild type and Crem knockout osteoclasts in vitro, and this inhibitory effect was greater in Crem knockout osteoclasts. Furthermore, calcitonin decreased calcitonin receptor mRNA expression in wild type osteoclasts but not in Crem knockout osteoclasts. These data suggest that calcitonin induction of ICER in osteoclasts might regulate osteoclast activity. PMID:19016003

  7. An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

    PubMed Central

    Furutani, Masahiro; Hata, Jun-Ichi; Shomura, Yasuhito; Itami, Keisuke; Yoshida, Takao; Izumoto, Yoshitaka; Togi, Akiko; Ideno, Akira; Yasunaga, Takuo; Miki, Kunio; Maruyama, Tadashi

    2005-01-01

    The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin α-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3′ end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21–25 kDa) toxic to host cells or two antibody fragments (25–36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities. PMID:15659368

  8. Gene expression analysis of early and late maturation stage rat enamel organ

    PubMed Central

    LACRUZ, RODRIGO S.; SMITH, CHARLES E.; CHEN, YI-BU; HUBBARD, MICHAEL J.; HACIA, JOSEPH G.; PAINE, MICHAEL L.

    2011-01-01

    Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH and resorption of extracellular enamel proteins. During maturation, ameloblasts change from a tall, thin and highly polarized organization characteristic of the secretory stage, to a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early and late maturation stages from rat incisor and analyzed global gene expression profiles at each stage. Sixty three candidate genes were identified with potential roles in the maturation process. qPCR was used to confirm results from this genome-wide analysis in a subset of genes. Enriched transcripts in late maturation (n= 38) included those associated with lysosomal activity, solute carrier transport and calcium signaling. Cellular responses to oxidative stress, proton transport, cell death and immune system-related transcripts were also up-regulated. Transcripts down-regulated in the late maturation stage (n= 25) included those with functions related to cell adhesion, cell signaling, and T-cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and so provide the bases for future functional investigations into the mechanistic basis of enamel mineralization. PMID:22243241

  9. Comprehensive analysis of TCP transcription factors and their expression during cotton (Gossypium arboreum) fiber early development

    PubMed Central

    Ma, Jun; Liu, Fang; Wang, Qinglian; Wang, Kunbo; Jones, Don C.; Zhang, Baohong

    2016-01-01

    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. PMID:26857372

  10. Expression of recombinant green fluorescent protein in Bacillus methanolicus.

    PubMed

    Nilasari, Dewi; Dover, Nir; Rech, Sabine; Komives, Claire

    2012-01-01

    Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein. PMID:22275315

  11. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

    PubMed

    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01

    The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

  12. Monotreme Lactation Protein Is Highly Expressed in Monotreme Milk and Provides Antimicrobial Protection

    PubMed Central

    Enjapoori, Ashwantha Kumar; Grant, Tom R.; Nicol, Stewart C.; Lefèvre, Christophe M.; Nicholas, Kevin R.; Sharp, Julie A.

    2014-01-01

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female’s abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples. PMID:25245409

  13. Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection.

    PubMed

    Enjapoori, Ashwantha Kumar; Grant, Tom R; Nicol, Stewart C; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

    2014-10-01

    Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female's abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples. PMID:25245409

  14. Early and persistent up-regulated expression of renal cortical osteopontin in experimental hydronephrosis.

    PubMed Central

    Diamond, J. R.; Kees-Folts, D.; Ricardo, S. D.; Pruznak, A.; Eufemio, M.

    1995-01-01

    The mechanical disturbance after unilateral ureteral obstruction (UUO) is a nonimmune stimulus that is capable of eliciting a florid macrophage infiltration of the kidney and subsequent post-inflammatory renal scarring. Osteopontin has potential chemoattractant activity and, for this reason, we delineated the kinetics of its expression in the renal cortex of rats with UUO. Whole body X-irradiation and reversal of UUO were utilized as interventional maneuvers to give additional pathobiological insight into this protein's role in the response of the kidneys to ureteral obstruction. Increased osteopontin mRNA levels in obstructed kidneys versus contralateral unobstructed specimens were evident as early as 4 hours after UUO and steadily increased at 12, 24, 48, and 96 hours after UUO. Both X-irradiation and reversal of UUO failed to significantly modulate renal cortical osteopontin mRNA expression at all of the above time points. Paralleling the increments in renal cortical osteopontin mRNA levels were significant elevations in the cortical renal interstitial macrophage number, which was significantly diminished by previous X-irradiation but not reversal of UUO. Focal labeling of osteopontin was noted in both tubular and Bowman's capsular epithelium in obstructed kidneys as early as 4 hours after UUO, whereas, in the contralateral unobstructed specimens, there was only faint staining in Bowman's capsule. By 96 hours after UUO, obstructed kidneys exhibited intense, diffuse staining for osteopontin in both tubules and Bowman's capsule. Osteopontin's immunolocalization was not modulated by X-irradiation or reversal of UUO. These data support the contention that osteopontin is involved in the accumulation of macrophages within the peritubular and periglomerular interstitium in the obstructed renal cortex. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7778684

  15. Immunohistochemical evaluation of mx protein expression in canine encephalitides.

    PubMed

    Porter, B F; Ambrus, A; Storts, R W

    2006-11-01

    Mx proteins are a group of interferon-induced GTPases whose expression has been demonstrated in a number of human viral infections and in some idiopathic inflammatory diseases. In this study, the expression of Mx protein was evaluated in known viral, nonviral, and idiopathic encephalitides in the dog via immunohistochemistry using an antibody against human MxA. All 12 cases of confirmed viral encephalitis, including 7 cases of canine distemper, 4 cases of canine herpesvirus, and 1 case of rabies, were Mx positive. In canine distemper cases, staining was particularly strong and a variety of cell types were positive, including astrocytes, macrophages/microglia, and neurons. Immunoreactivity for Mx protein was evident in a few cases of nonviral infectious encephalitis, including neosporosis (1/1), Chagas disease (2/3), aspergillosis (1/2), and encephalitozoonosis (1/1). Consistent staining was observed in most cases of idiopathic encephalitis, including granulomatous meningoencephalomyelitis (7/7), necrotizing meningoencephalitis of pug dogs (6/7), and necrotizing encephalitis of the Yorkshire Terrier (3/3) and Maltese (1/1) breeds. Mx staining was negative in 5 normal dog brains; 3 cases of cryptococcosis; and single cases of blastomycosis, protothecosis, and bacterial meningitis. PMID:17099155

  16. Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

    PubMed Central

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  17. Transient expression of proteins by hydrodynamic gene delivery in mice.

    PubMed

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  18. Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

    PubMed

    Boiocchi, C; Monti, M C; Osera, C; Mallucci, G; Pistono, C; Ferraro, O E; Nosari, G; Romani, A; Cuccia, M; Govoni, S; Pascale, A; Montomoli, C; Bergamaschi, R

    2016-09-15

    Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity. PMID:27609295

  19. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  20. Protein tyrosine phosphatases expression during development of mouse superior colliculus.

    PubMed

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-12-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis. PMID:19727691

  1. Transformation of Escherichia coli and protein expression using lipoplex mimicry.

    PubMed

    Yun, Chul-Ho; Bae, Chun-Sik; Ahn, Taeho

    2016-11-01

    We investigated a "one-step" method for transformation of and protein expression in Escherichia coli (E. coli) using a complex of n-stearylamine, a cationic lipid, and plasmid DNA, which mimics lipoplex-based approaches. When E. coli cells were treated with the cationic lipid-plasmid complex, the transformation efficiencies were in the range of approximately 2-3 × 10(6) colony-forming units. Further increase in the efficiency was obtained by co-treatment with calcium chloride (or rubidium chloride) and the complexes. Moreover, after DNA transfer, E. coli cells successfully expressed plasmid-encoded proteins such as cytochrome P450s and glutathione-S-transferase without overnight incubation of the cells to form colonies, an indispensable step in other bacterial transformation methods. In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step. PMID:27416742

  2. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    PubMed

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy. PMID:26037116

  3. Proteomic analysis of serum proteins in triple transgenic Alzheimer's disease mice: implications for identifying biomarkers for use to screen potential candidate therapeutic drugs for early Alzheimer's disease.

    PubMed

    Sui, Xiaojing; Ren, Xiaohu; Huang, Peiwu; Li, Shuiming; Ma, Quan; Ying, Ming; Ni, Jiazuan; Liu, Jianjun; Yang, Xifei

    2014-01-01

    Alzheimer's disease (AD) is the most common fatal neurodegenerative disease affecting the elderly worldwide. There is an urgent need to identify novel biomarkers of early AD. This study aims to search for potential early protein biomarkers in serum from a triple transgenic (PS1M146V/APPSwe/TauP301L) mouse model. Proteomic analysis via two-dimensional fluorescence difference gel electrophoresis was performed on serum samples from wild-type (WT) and triple transgenic mice that were treated with or without coenzyme Q10 (CoQ10) (800 mg/kg body weight/day), a powerful endogenous antioxidant displaying therapeutic benefits against AD pathology and cognitive impairment in multiple AD mouse models, for a period of three months beginning at two months of age. A total of 15 differentially expressed serum proteins were identified between the WT and AD transgenic mice. The administration of CoQ10 was found to alter the changes in the differentially expressed serum proteins by upregulating 10 proteins and down-regulating 10 proteins. Among the proteins modulated by CoQ10, clusterin and α-2-macroglobulin were validated via ELISA assay. These findings revealed significant changes in serum proteins in the AD mouse model at an early pathological stage and demonstrated that administration of CoQ10 could modulate these changes in serum proteins. Our study suggested that these differentially expressed serum proteins could serve as potential protein biomarkers of early AD and that screening for potential candidate AD therapeutic drugs and monitoring of therapeutic effects could be performed via measurement of the changes in these differentially expressed serum proteins. PMID:24496070

  4. A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

    PubMed

    Wennier, Sonia T; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  5. A Novel Naturally Occurring Tandem Promoter in Modified Vaccinia Virus Ankara Drives Very Early Gene Expression and Potent Immune Responses

    PubMed Central

    Wennier, Sonia T.; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  6. Label-Free Proteomic Analysis of Protein Changes in the Striatum during Chronic Ethanol Use and Early Withdrawal

    PubMed Central

    Ayers-Ringler, Jennifer R.; Oliveros, Alfredo; Qiu, Yanyan; Lindberg, Daniel M.; Hinton, David J.; Moore, Raymond M.; Dasari, Surendra; Choi, Doo-Sup

    2016-01-01

    The molecular mechanisms underlying the neuronal signaling changes in alcohol addiction and withdrawal are complex and multifaceted. The cortico-striatal circuit is highly implicated in these processes, and the striatum plays a significant role not only in the early stages of addiction, but in the developed-addictive state as well, including withdrawal symptoms. Transcriptional analysis is a useful method for determining changes in gene expression, however, the results do not always accurately correlate with protein levels. In this study, we employ label-free proteomic analysis to determine changes in protein expression within the striatum during chronic ethanol use and early withdrawal. The striatum, composed primarily of medium spiny GABAergic neurons, glutamatergic and dopaminergic nerve terminals and astrocytes, is relatively homogeneous for proteomic analysis. We were able to analyze more than 5000 proteins from both the dorsal (caudate and putamen) and ventral (nucleus accumbens) striatum and identified significant changes following chronic intermittent ethanol exposure and acute (8 h) withdrawal compared to ethanol naïve and ethanol exposure groups respectively. Our results showed significant changes in proteins involved in glutamate and opioid peptide signaling, and also uncovered novel pathways including mitochondrial function and lipid/cholesterol metabolism, as revealed by changes in electron transport chain proteins and RXR activation pathways. These results will be useful in the development of novel treatments for alcohol withdrawal and thereby aid in recovery from alcohol use disorder. PMID:27014007

  7. Differential expression of murine adult hemoglobins in early ontogeny

    SciTech Connect

    Wawrzyniak, C.J.; Lewis, S.E.; Popp, R.A.

    1985-01-01

    A hemoglobin mutation is described that permits study of the expression of the two adult ..beta..-globin genes throughout fetal and postnatal development. Mice with a mutation at the Hbb/sup s/, ..beta..-globin locus, were used to study the relative levels of ..beta..-s2major and ..beta..-sminor globins specified by the mutant Hbb/sup s2/ haplotype during development. At 11.5 days of gestation ..beta..-sminor comprised over 80% and ..beta..-s2major under 20% of the adult beta-globin. The relative level of ..beta..-sminor decreased through fetal development; at birth ..beta..-sminor represented 33.7% of the ..beta..-globin. The adult values of 71.0% ..beta..-s2major and 29.0% ..beta..-sminor globin are expressed in mice six days after birth. Because the two ..beta..-globin genes are expressed in mice of the Hbb/sup 2s/ haplotype, both the ..beta..-smajor and ..beta..-sminor genes must be expressed in mice of the Hbb/sup s/ haplotype. Expression of the ..beta..-sminor gene is elevated to 35.6% in Hbb/sup s2/ mice that have been bled repeatedly. Thus, the 5' ..beta..-s2major and 3' ..beta..-sminor genes of the Hbb/sup s2/ haplotype and, presumably the 5' ..beta..-smajor and 3' ..beta..-sminor genes of the Hbb/sup s/ haplotype, are regulated independently and are homologous to the 5' ..beta..-dmajor and 3' ..beta..-dminor genes of the Hbb/sup d/ haplotype. Mice of the Hbb/sup s2/ haplotype are better than mice of the Hbb/sup d/ haplotytpe for studying the mechanisms of hemoglobin switching because the Hbb/sup s2/ each of the three embryonic and two adult hemoglobins can be separated by electrophoresis. 17 refs., 3 figs.

  8. Elevated expressions of myeloid-related proteins-8 and -14 are danger biomarkers for lupus nephritis.

    PubMed

    Tantivitayakul, P; Benjachat, T; Somparn, P; Leelahavanichkul, A; Kittikovit, V; Hirankarn, N; Pisitkun, T; Avihingsanon, Y

    2016-01-01

    Myeloid-related proteins, MRP-8 and -14, which have been identified as molecules that mediate the danger signaling in innate immune response, are also known as the DAMPs (damage associated molecular pattern molecules). The proteins were found in infiltrating macrophages and neutrophils at inflammatory sites. Their expression was correlated with severe forms of glomerulonephritis. Therefore, this study examined whether or not MRP-8 and -14 can be used as biomarkers for identifying severely active lupus nephritis (LN). Total blood leukocyte samples and renal biopsy tissues from a prospective cohort of LN patients were used to determine mRNA and protein expression levels of MRP-8 and -14. The mRNA levels of MRP-8 and -14 in total blood leukocytes were significantly higher in active LN patients than quiescent LN patients and healthy controls. Moreover, the mRNA levels of MRP-8 and -14 in the total blood leukocytes and kidney tissues were significantly correlated with therapeutic response and the mRNA expression levels in the kidney were associated with an early loss of the kidney function. MRP-8 and -14 can be used as non-invasive prognostic biomarkers in patients with LN. PMID:26223295

  9. Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development.

    PubMed

    Marcorelles, Pascale; Gillet, Danièle; Friocourt, Gaëlle; Ledé, Françoise; Samaison, Laura; Huguen, Geneviève; Ferec, Claude

    2012-03-01

    Sterility due to bilateral destruction in utero or in early infancy resulting in congenital absence of the vas deferens is the rule in male patients with cystic fibrosis. To understand the developmental pattern of this anomaly, the microscopic morphology of the male excretory system was analyzed during development and the expression of the cystic fibrosis transmembrane conductance regulator protein was explored by immunohistochemistry. We observed that cystic fibrosis fetuses had no excretory ducts agenesis or obstruction until 22 weeks of gestation. However, a focal inflammatory pattern and mucinous plugs in the oldest cystic fibrosis case suggested a disruptive mechanism. Immunolabeling of cytoplasmic epithelial cystic fibrosis transmembrane conductance regulator protein was demonstrated in all cystic fibrosis and control cases with a similar pattern of expression of the protein between age-matched controls and cystic fibrosis cases. At midgestation, an apical intensification appeared in both cystic fibrosis and control cases and was stable during the remainder of fetal life. No gradient of intensity could be detected between the different segments of the excretory tract. These findings are different from those reported in adults. The absence of any morphologic anomaly until 22 weeks of gestation, the focal destruction of the epithelial structures during the second trimester, and the chronological pattern of expression of cystic fibrosis transmembrane conductance regulator are of interest for a better understanding of the pathophysiology of this disease. PMID:21840567

  10. Protein needs early in life and long-term health.

    PubMed

    Michaelsen, Kim F; Greer, Frank R

    2014-03-01

    The objective of this review was to summarize selected health aspects of protein intake during the first 2 y of life. During this period there is a marked increase in protein intake from an intake of ∼ 5% of energy from protein (PE%) in an exclusively breastfed infant to ∼ 15 PE% when complementary foods have been introduced. At this age, mean protein intake is ∼ 3 times as high as the physiologic requirement, but some children receive 4-5 times their physiologic requirement. Protein from cow milk constitutes a main part of protein intake in toddlers and seems to have a specific effect on insulin-like growth factor I concentrations and growth. Meat has a high protein content, but the small amounts of meat needed to ensure good iron status have less impact on total protein intake. The difference in protein intake between breastfed and formula-fed infants is likely to play a role in the difference between breastfed and formula-fed infants. There is emerging evidence that high protein intake during the first 2 y of life is a risk factor for later development of overweight and obesity. It therefore seems prudent to avoid a high protein intake during the first 2 y of life. This could be accomplished by decreasing the upper allowable limit of the protein content of infant formulas for the first year of life and limiting the intake of cow milk in the second year of life. PMID:24452233

  11. Characterization of WWP1 protein expression in skeletal muscle of muscular dystrophy chickens.

    PubMed

    Imamura, Michihiro; Nakamura, Akinori; Mannen, Hideyuki; Takeda, Shin'ichi

    2016-02-01

    A missense mutation in the gene encoding WWP1 was identified as the most promising candidate responsible for chicken muscular dystrophy (MD) by genetic linkage analysis. WWP1 is a HECT-type E3 ubiquitin protein ligase composed of 922 amino acids, which contains 4 tandem WW domains that interact with the proline-rich peptide motifs of target proteins. The missense mutation changes arginine 441 that is located in the centre of the WW domains into glutamine (R441Q), which potentially affects the function of the WWP1 protein. Here, we show that WWP1 is detected as ∼130-kDa protein that localizes to various structures, such as the plasma membrane (sarcolemma), sarcoplasmic reticulum, mitochondria and nucleus, in normal chicken skeletal muscle. However, in MD chickens, the mutant WWP1 protein was markedly degraded and was absent in the sarcolemma. These changes were also observed in the muscles of chickens in early pre-pathological states. Moreover, in vitro expression analysis showed significant degradation of mutant, but not wild-type WWP1, specifically in myogenic cells. Altogether, our data revealed that the R441Q missense mutation in the WWP1 protein causes degradation and loss of the sarcolemmal localization of WWP1, which may play a role in the pathogenesis of chicken MD. PMID:26314333

  12. Piwi proteins and piRNAs in mammalian oocytes and early embryos: From sample to sequence

    PubMed Central

    Rosenkranz, David; Han, Chung-Ting; Roovers, Elke F.; Zischler, Hans; Ketting, René F.

    2015-01-01

    The role of the Piwi/piRNA pathway during mammalian oogenesis has remained enigmatic thus far, especially since experiments with Piwi knockout mice did not reveal any phenotypic defects in female individuals. This is in striking contrast with results obtained from other species including flies and zebrafish. In mouse oocytes, however, only low levels of piRNAs are found and they are not required for their function. We recently demonstrated dynamic expression of PIWIL1, PIWIL2, and PIWIL3 during mammalian oogenesis and early embryogenesis. In addition, small RNA analysis of human, crab-eating macaque and cattle revealed that piRNAs are also expressed in the female germline and closely resemble piRNAs from testis. Here, we thoroughly describe the experimental and computational methods that we applied for the generation, processing and analyses of next generation sequencing (NGS) data associated with our study on Piwi proteins and piRNAs in mammalian oocytes and embryos (Roovers et al., 2015). The complete sequence data is available at NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession GSE64942. PMID:26484274

  13. Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

    PubMed Central

    Qi, Qian-Rong; Xie, Qing-Zhen; Liu, Xue-Li; Zhou, Yun

    2014-01-01

    Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion. PMID:25133541

  14. Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)

    SciTech Connect

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Li, Jun

    2013-10-11

    Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

  15. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    PubMed Central

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  16. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters.

    PubMed

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present "DyCluster", a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  17. Reduced expression of aquaporins in human intestinal mucosa in early stage inflammatory bowel disease

    PubMed Central

    Ricanek, Petr; Lunde, Lisa K; Frye, Stephan A; Støen, Mari; Nygård, Ståle; Morth, Jens P; Rydning, Andreas; Vatn, Morten H; Amiry-Moghaddam, Mahmood; Tønjum, Tone

    2015-01-01

    Objectives The aim of this study was to investigate the relationship between aquaporin (AQP) water channel expression and the pathological features of early untreated inflammatory bowel disease (IBD) in humans. Methods Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days, were examined at the local hospital. Colonoscopy with biopsies was performed and blood samples were taken. Patients who did not meet the diagnostic criteria for IBD and who displayed no evidence of infection or other pathology in the gut were included as symptomatic non-IBD controls. AQP1, 3, 4, 5, 7, 8, and 9 messenger RNA (mRNA) levels were quantified in biopsies from the distal ileum and colon by quantitative real-time polymerase chain reaction. Protein expression of selected AQPs was assessed by confocal microscopy. Through multiple alignments of the deduced amino acid sequences, the putative three-dimensional structures of AQP1, 3, 7, and 8 were modeled. Results AQP1, 3, 7, and 8 mRNAs were detected in all parts of the intestinal mucosa. Notably, AQP1 and AQP3 mRNA levels were reduced in the ileum of patients with Crohn’s disease, and AQP7 and AQP8 mRNA levels were reduced in the ileum and the colon of patients with ulcerative colitis. Immunofluorescence confocal microscopy showed localization of AQP3, 7, and 8 at the mucosal epithelium, whereas the expression of AQP1 was mainly confined to the endothelial cells and erythrocytes. The reduction in the level of AQP3, 7, and 8 mRNA was confirmed by immunofluorescence, which also indicated a reduction of apical immunolabeling for AQP8 in the colonic surface epithelium and crypts of the IBD samples. This could indicate loss of epithelial polarity in IBD, leading to disrupted barrier function. Conclusion AQPs 1 and 8 and the aquaglyceroporins AQPs 3 and 7 are the AQPs predominantly expressed in the lower intestinal tract of humans. Their expression is

  18. Design of riboregulators for control of cyanobacterial (Synechocystis) protein expression.

    PubMed

    Abe, Koichi; Sakai, Yuta; Nakashima, Saki; Araki, Masataka; Yoshida, Wataru; Sode, Koji; Ikebukuro, Kazunori

    2014-02-01

    Cyanobacteria are attractive host bacteria for biofuel production because they can covert CO2 to biofuel lipids using only sunlight, water, and inorganic ions. For genetically engineering an ideal cyanobacterium, a synthetic biological approach is promising but few genetic components have been characterized in cyanobacteria. Here for controlling cyanobacterial protein expression, we constructed riboregulators, that one of the post-transcriptional regulators composed of RNAs. Riboregulators harboring a ribosome-binding site suitable for Synechocystis sp. were designed by trial and error using Escherichia coli as host bacteria. The designed riboregulators were effective in Synechocystis sp. as well as E. coli with slight interference on growth only observed in E. coli. They will therefore be useful tools for controlling target gene expression. PMID:24068508

  19. Dynamic Alterations of miR-34c Expression in the Hypothalamus of Male Rats after Early Adolescent Traumatic Stress

    PubMed Central

    Li, Chuting; Liu, Yuan; Liu, Dexiang; Jiang, Hong; Pan, Fang

    2016-01-01

    Several types of microRNA (miRNA) overexpression in the brain are associated with stress. One of the targets of miR-34c is the stress-related corticotrophin releasing factor receptor 1 mRNA (CRFR1 mRNA). Here we will probe into the short-term effect and long-term effect of early adolescent traumatic stress on the expression of miR-34c and CRFR1 mRNA. Traumatic stress was established by electric foot shock for six consecutive days using 28-day rats. The anxiety-like behaviors, memory damage, CRFR1 protein, CRFR1 mRNA, and miR-34c expression were detected in our study. The results of our study proved that exposure to acute traumatic stress in early adolescent can cause permanent changes in neural network, resulting in dysregulation of CRFR1 expression and CRFR1 mRNA and miR-34c expression in hypothalamus, anxiety-like behavior, and memory impairment, suggesting that the miR-34c expression in hypothalamus may be an important factor involved in susceptibility to PTSD. PMID:26925271

  20. Satb1 Ablation Alters Temporal Expression of Immediate Early Genes and Reduces Dendritic Spine Density during Postnatal Brain Development

    PubMed Central

    Balamotis, Michael A.; Tamberg, Nele; Woo, Young Jae; Li, Jingchuan; Davy, Brian

    2012-01-01

    Complex behaviors, such as learning and memory, are associated with rapid changes in gene expression of neurons and subsequent formation of new synaptic connections. However, how external signals are processed to drive specific changes in gene expression is largely unknown. We found that the genome organizer protein Satb1 is highly expressed in mature neurons, primarily in the cerebral cortex, dentate hilus, and amygdala. In Satb1-null mice, cortical layer morphology was normal. However, in postnatal Satb1-null cortical pyramidal neurons, we found a substantial decrease in the density of dendritic spines, which play critical roles in synaptic transmission and plasticity. Further, we found that in the cerebral cortex, Satb1 binds to genomic loci of multiple immediate early genes (IEGs) (Fos, Fosb, Egr1, Egr2, Arc, and Bdnf) and other key neuronal genes, many of which have been implicated in synaptic plasticity. Loss of Satb1 resulted in greatly alters timing and expression levels of these IEGs during early postnatal cerebral cortical development and also upon stimulation in cortical organotypic cultures. These data indicate that Satb1 is required for proper temporal dynamics of IEG expression. Based on these findings, we propose that Satb1 plays a critical role in cortical neurons to facilitate neuronal plasticity. PMID:22064485

  1. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    SciTech Connect

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory . E-mail: vchinchar@microbio.umsmed.edu

    2007-02-20

    Frog virus 3 (FV3) is a large DNA virus that encodes {approx} 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-II{alpha}). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-II{alpha} triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

  2. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    NASA Astrophysics Data System (ADS)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  3. Activity, Expression and Function of a Second Drosophila Protein Kinase a Catalytic Subunit Gene

    PubMed Central

    Melendez, A.; Li, W.; Kalderon, D.

    1995-01-01

    The DC2 gene was isolated previously on the basis of sequence similarity to DCO, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development. PMID:8601490

  4. Maternal High Fat Diet Affects Offspring’s Vitamin K-Dependent Proteins Expression Levels

    PubMed Central

    Lanham, Stuart; Cagampang, Felino R.; Oreffo, Richard O. C.

    2015-01-01

    Studies suggest bone growth & development and susceptibility to vascular disease in later life are influenced by maternal nutrition, during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including Osteocalcin, Matrix-gla protein, Periostin, and Gas6, in bone and vascular development. This study extends the analysis of VKDPs previously conducted in 6 week old offspring, into offspring of 30 weeks of age, to assess the longer term effects of a maternal and postnatal high fat (HF) diet on VKDP expression. Overall a HF maternal diet and offspring diet exacerbated the bone changes observed. Sex specific and tissue specific differences were observed in VKDP expression for both aorta and femoral tissues. In addition, significant correlations were observed between femoral OCN, Periostin Gas6, and Vkor expression levels and measures of femoral bone structure. Furthermore, MGP, OCN, Ggcx and Vkor expression levels correlated to mass and fat volume, in both sexes. In summary the current study has highlighted the importance of the long-term effects of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure. PMID:26381752

  5. Insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells: regulation by filamentous actin and requirement for de novo protein synthesis.

    PubMed

    Kim, J J; Jaffe, R C; Fazleabas, A T

    1999-02-01

    Stromal fibroblasts in the primate endometrium undergo dramatic morphological and biochemical changes in response to pregnancy. This transformation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells from the baboon endometrium of nonpregnant animals were cultured and subsequently treated with cytochalasin D to disrupt actin filaments. In response to cytochalasin D treatment, cells contracted and became rounded as early as 10 min after the initiation of treatment. When cytochalasin D was removed, cells reverted back to their original fibroblastic shape within 1 h. After cells were treated with cytochalasin D for 5 h, addition of (Bu)2cAMP and/or hormones (estradiol, medroxyprogesterone acetate, and relaxin) resulted in the expression of IGFBP-1 messenger RNA and protein within 24 h. Cells with an intact cytoskeleton did not express detectable levels of IGFBP-1 in response to hormones and/or (Bu)2cAMP. Furthermore, the addition of cycloheximide inhibited expression of IGFBP-1 in cytochalasin D-treated cells. Stromal cells were also isolated from early pregnant and simulated pregnant animals. Within 48 h, cells from both the pregnant and simulated pregnant animals produced IGFBP-1 in response to hormones and/or (Bu)2cAMP. In these studies, IGFBP-1 expression was also inhibited by cycloheximide. These studies suggest that induction of IGFBP-1 requires an intermediary protein and that alterations in the cytoskeleton may be involved. PMID:9927334

  6. Evaluation of photodynamic therapy in adhesion protein expression

    PubMed Central

    PACHECO-SOARES, CRISTINA; MAFTOU-COSTA, MAIRA; DA CUNHA MENEZES COSTA, CAROLINA GENÚNCIO; DE SIQUEIRA SILVA, ANDREZA CRISTINA; MORAES, KAREN C.M.

    2014-01-01

    Photodynamic therapy (PDT) is a treatment modality that has clinical applications in both non-neoplastic and neoplastic diseases. PDT involves a light-sensitive compound (photosensitizer), light and molecular oxygen. This procedure may lead to several different cellular responses, including cell death. Alterations in the attachment of cancer cells to the substratum and to each other are important consequences of photodynamic treatment. PDT may lead to changes in the expression of cellular adhesion structure and cytoskeleton integrity, which are key factors in decreasing tumor metastatic potential. HEp-2 cells were photosensitized with aluminum phthalocyanine tetrasulfonate and zinc phthalocyanine, and the proteins β1-integrin and focal adhesion kinase (FAK) were assayed using fluorescence microscopy. The verification of expression changes in the genes for FAK and β1 integrin were performed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that HEp-2 cells do not express β-integrin or FAK 12 h following PDT. It was concluded that the PDT reduces the adhesive ability of HEp-2 cells, inhibiting their metastatic potential. The present study aimed to analyze the changes in the expression and organization of cellular adhesion elements and the subsequent metastatic potential of HEp-2 cells following PDT treatment. PMID:25013490

  7. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    PubMed Central

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  8. High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis

    PubMed Central

    Doldo, Elena; Costanza, Gaetana; Ferlosio, Amedeo; Pompeo, Eugenio; Agostinelli, Sara; Bellezza, Guido; Mazzaglia, Donatella; Giunta, Alessandro; Sidoni, Angelo; Orlandi, Augusto

    2015-01-01

    Purpose Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain. Experimental design CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells. Results CRBP-1High expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1High status and dedifferentiation. CRBP-1-transfected (CRBP-1+) A549 grew more than CRBP-1− A549 cells. At >1μM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1+ than in CRBP-1− A549 cells. CRBP-1+ A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARβ and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1+ A549 cells. Conclusion CRBP-1High expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1High expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients. PMID:26807202

  9. Differential Expression of Placental Villous Angiopoietin-1 and -2 During Early, Mid and Late Baboon Pregnancy

    PubMed Central

    Babischkin, J. S.; Suresch, D. L.; Pepe, G. J.; Albrecht, E. D.

    2009-01-01

    Although vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and Ang-2 have important roles in angiogenesis, very little is known about the regulation of these factors in the villous placenta during human pregnancy. In the present study, to investigate whether placental expression of Ang-1, Ang-2 and VEGF was altered in a cell-specific manner with advancing baboon gestation, the mRNA levels of these growth factors were determined by RT-PCR in cells isolated by Percoll gradient centrifugation from and protein localization assessed by immunocytochemistry in the villous placenta at early (day 60), mid (day 100) and late (day 170, term is 184 days) baboon gestation. Mean (± SE) Ang-1 mRNA levels, relative to 18S rRNA, in villous syncytiotrophoblast (3.92 ± 0.68) and cytotrophoblast (1.31 ± 0.31) cell fractions were highest on day 60 of gestation, then decreased by approximately 2.5-fold (P<0.05) to 1.39 ± 0.29 and 0.49 ±0.07, respectively, on day 170. Moreover, Ang-1 mRNA levels in the villous stromal cells and Ang-2 mRNA levels in all placental villous cell fractions were similar on days 60, 100, and 170 of gestation. In contrast to Ang-1 and Ang-2, placental villous cytotrophoblast VEGF mRNA levels were increased 2.94 fold (P<0.05) between mid (0.67 ± 0.15) and late (1.97 ± 0.49) gestation. A corresponding decrease in Ang-1, absence of change in Ang-2, and increase in VEGF protein immunocytochemical expression were exhibited in placental trophoblast with advancing baboon pregnancy. Ang-1/-2 and the angiopoietin Tie-2 receptor were expressed in vascular endothelial cells of the villous placenta, indicating that these blood vessel cells are a major site of ligand-receptor interaction for angiogenesis during primate pregnancy. We conclude that there is a cell-specific differential change in placental villous trophoblast expression of VEGF, Ang-1, and Ang-2 which we propose is important in regulating angiogenesis in the villous placenta during

  10. Extracellular matrix protein expression is brain region dependent.

    PubMed

    Dauth, Stephanie; Grevesse, Thomas; Pantazopoulos, Harry; Campbell, Patrick H; Maoz, Ben M; Berretta, Sabina; Parker, Kevin Kit

    2016-05-01

    In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region-specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin-R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin-R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin-R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309-1336, 2016. © 2016 Wiley Periodicals, Inc. PMID:26780384

  11. Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

    PubMed Central

    2013-01-01

    Introduction Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. Methods Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. Results Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice

  12. Characterization of starch synthase I and II expressed in early developing seeds of kidney bean (Phaseolus vulgaris L.).

    PubMed

    Senoura, Takeshi; Isono, Naoto; Yoshikawa, Motoyo; Asao, Ayako; Hamada, Shigeki; Watanabe, Kenji; Ito, Hiroyuki; Matsui, Hirokazu

    2004-09-01

    Plant starch synthase (SS) contributes to the elongation of glucan chains during starch biosynthesis and hence plays an essential role in determining the fine structure of amylopectin. To elucidate the role of SS activity in the formation of amylopectin in kidney bean (Phaseolus vulgaris L.), a study was undertaken to isolate cDNA clones for SS and to characterize the enzymatic properties of the coded recombinant enzymes. Two SS cDNAs, designated pvss1 and pvss21, which were isolated from early developing seeds, encoded SSI and SSII (designated PvSSI and PvSSII-1) that displayed significant identity (more than 65%) with other SSI and SSII members, respectively. RNA gel blot analysis indicated that both transcripts accumulate in leaves and developing seeds at the early stage. Immunoblot analysis with antisera raised against both recombinant proteins (rPvSSI and rPvSSII-1) showed that the accumulation of both proteins parallels the gene expression profiles, although both were detectable only in starch-granule fractions. Recombinant enzymes expressed by Escherichia coli cells showed distinct chain-length specificities for the extension of glucan chains. Our results suggest that these SS isozymes for synthesis of transitory starch are also responsible for synthesis of storage starch in early developing seeds of kidney bean. PMID:15388972

  13. Expression of Quaking RNA-Binding Protein in the Adult and Developing Mouse Retina

    PubMed Central

    Aono, Kentaro; Kawashima, Togo; Inoue, Kiyoshi; Ku, Li; Feng, Yue; Koike, Chieko

    2016-01-01

    Quaking (QKI), which belongs to the STAR family of KH domain-containing RNA-binding proteins, functions in pre-mRNA splicing, microRNA regulation, and formation of circular RNA. QKI plays critical roles in myelinogenesis in the central and peripheral nervous systems and has been implicated neuron-glia fate decision in the brain; however, neither the expression nor function of QKI in the neural retina is known. Here we report the expression of QKI RNA-binding protein in the developing and mature mouse retina. QKI was strongly expressed by Müller glial cells in both the developing and adult retina. Intriguingly, during development, QKI was expressed in early differentiating neurons, such as the horizontal and amacrine cells, and subsequently in later differentiating bipolar cells, but not in photoreceptors. Neuronal expression was uniformly weak in the adult. Among QKI isoforms (5, 6, and 7), QKI-5 was the predominantly expressed isoform in the adult retina. To study the function of QKI in the mouse retina, we examined quakingviable(qkv) mice, which have a dysmyelination phenotype that results from deficiency of QKI expression and reduced numbers of mature oligodendrocytes. In homozygous qkv mutant mice (qkv/qkv), the optic nerve expression levels of QKI-6 and 7, but not QKI-5 were reduced. In the retina of the mutant homozygote, QKI-5 levels were unchanged, and QKI-6 and 7 levels, already low, were also unaffected. We conclude that QKI is expressed in developing and adult Müller glia. QKI is additionally expressed in progenitors and in differentiating neurons during retinal development, but expression weakened or diminished during maturation. Among QKI isoforms, we found that QKI-5 predominated in the adult mouse retina. Since Müller glial cells are thought to share properties with retinal progenitor cells, our data suggest that QKI may contribute to maintaining retinal progenitors prior to differentiation into neurons. On the other hand, the expression of QKI in

  14. Maternal Smoking History Enhances the Expression of Placental Growth Factor in Invasive Trophoblasts at Early Gestation Despite Cessation of Smoking

    PubMed Central

    Kawashima, Akihiro; Koide, Keiko; Hasegawa, Junichi; Arakaki, Tatsuya; Takenaka, Shin; Maruyama, Daisuke; Matsuoka, Ryu; Sekizawa, Akihiko

    2015-01-01

    Maternal smoking during early pregnancy is associated with a reduced risk for preeclampsia even after smoking cessation during pregnancy. Although the pathophysiology of preeclampsia has not been establishe