WorldWideScience

Sample records for bioactive recombinant human

  1. Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

    Science.gov (United States)

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

    2015-11-01

    With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

  2. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

    Directory of Open Access Journals (Sweden)

    Bin Yang

    Full Text Available BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. CONCLUSIONS/SIGNIFICANCE: Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

  3. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

    Science.gov (United States)

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

    2011-03-16

    There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

  4. Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1

    DEFF Research Database (Denmark)

    Ellis, April L; Pan, Wensheng; Yang, Guang

    2010-01-01

    perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. RESULTS: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess...

  5. Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

    OpenAIRE

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

    2011-01-01

    BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study,...

  6. Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system.

    Science.gov (United States)

    Zhao, Hua; Tang, Jiayong; Cao, Lei; Jia, Gang; Long, Dingbiao; Liu, Guangmang; Chen, Xiaoling; Cai, Jingyi; Shang, Haiying

    2015-09-01

    Parasin I (PI) is a 19 amino acid peptide with potent antimicrobial activities against a broad spectrum of microorganisms and is a good candidate for development as a novel antimicrobial agent. The objective of this study was to express and characterize a codon optimized parasin I peptide fused with human lysozyme (hLY). A 513 bp cDNA fragment encoding the mature hLY protein and parasin I peptide was designed and synthesized according to the codon bias of Pichia pastoris. A 4×Gly flexible amino acid linker with an enterokinase cleavage (DDDDK) was designed to link the PI to the C-terminal of hLY. The codon optimized recombinant hLY-PI was cloned into the pPICZαA vector and expressed in P. pastoris. The over-expressed extracellular rehLY-PI was purified using Ni sepharose affinity column and exhibited a molecular mass of approximately 18 kDa. After digested with enterokinase the rehLY-PI protein release its corresponding rehLY and rePI, with molecular mass of 16 kDa and 2 kDa, respectively, on Tricine-SDS-PAGE. The released rehLY exhibited similar lytical activity against Micrococcus lysodeikticus to its commercial hLY. The digested rehLY-PI product exhibited antimicrobial activities against Bacillus subtilis, Staphylococcus aureus and Escherichia coli, and synergism has been found between the released rePI and rehLY. In conclusion, we successfully optimized a rehLY-PI fusion protein encoding gene and over-expressed the rehLY-PI in P. pastoris. The recombination protein digested with enterokinase released functional hLY and antimicrobial parasin I, which demonstrates a potential for future use as an animal feed additive to partly replace antibiotic. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

    DEFF Research Database (Denmark)

    Jensen, Lena Vinther; Lademann, Ulrik Axel; Andersen, Elisabeth Veyhe

    2014-01-01

    as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N......-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1......), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1...

  8. Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells.

    Science.gov (United States)

    Zhang, Bo; Li, Angdi; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2016-06-10

    Proteinaceous bioactive substances and pharmaceuticals are most conveniently administered orally. However, the facing problems are the side effects of proteolytic degradation and denaturation in the gastrointestinal tract. In recent years, lactic acid bacteria (LAB) have been verified to be a promising delivery vector for susceptible functional proteins and drugs. KiSS-1 peptide, a cancer suppressor, plays a critical role in inhibiting cancer metastasis and its activity has been confirmed by direct administration. However, whether this peptide can be functionally expressed in LAB and exert activity on cancer cells, thus providing a potential alternative administration manner in the future, has not been demonstrated. A recombinant Lactococcus lactis strain NZ9000-401-kiss1 harboring a plasmid containing the gene of the tumor metastasis-inhibiting peptide KiSS1 was constructed. After optimization of the nisin induction conditions, the recombinant strain efficiently secreted KiSS1 with a maximum detectable amount of 27.9 μg/ml in Dulbecco's Modified Eagle medium. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and would healing assays, respectively, indicated that the secreted KiSS1 peptide remarkably inhibited HT-29 cell proliferation and migration. Furthermore, the expressed KiSS1 was shown to induce HT-29 cell morphological changes, apoptosis and reduce the expression of matrix metalloproteinase 9 (MMP-9) at both mRNA and protein levels. A recombinant L. lactis NZ9000-401-kiss1 successfully expressing the human kiss1 was constructed. The secreted KiSS1 peptide inhibited human HT-29 cells' proliferation and migration probably by invoking, or mediating the cell-apoptosis pathway and by down regulating MMP-9 expression, respectively. Our results suggest that L. lactis is an ideal cell factory for secretory expression of tumor metastasis-inhibiting peptide KiSS1, and the KiSS1-producing L. lactis strain may serve as a new tool for cancer therapy in

  9. High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

    Science.gov (United States)

    Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun

    2014-01-01

    Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

  10. Expression and characterization of recombinant human serum ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-14

    Nov 14, 2011 ... Key words: C-peptide, human serum albumin, recombinant fusion protein, Pichia pastoris, bioactivity, biological half-time. ... lines were purchased from Cell bank of Chinese academy of sciences (Shanghai, China). .... agarose electrophoresis and DNA sequencing (data was not shown). Expression and ...

  11. Bioactive factors in human milk.

    Science.gov (United States)

    Hamosh, M

    2001-02-01

    This article reviews the bioactive components of human milk. Special emphasis is given to immune and nonimmune protective function of major and minor nutrients in human milk. Immune modulating components, such as cytokines, nucleotides, hormones, and growth factors, are discussed. Milk enzymes with digestive function in the newborn are reviewed.

  12. Production of Bioactive Recombinant Bovine Chymosin in Tobacco Plants

    Directory of Open Access Journals (Sweden)

    Zheng-Yi Wei

    2016-04-01

    Full Text Available Chymosin (also known as rennin plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA. The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen.

  13. In vitro bioactivation of 3-(N-phenylamino)propane-1,2-diol by human and rat liver microsomes and recombinant P450 enzymes. Implications for toxic oil syndrome

    NARCIS (Netherlands)

    Martinez-Cabot, A.; Morato, A.; Commandeur, J.N.M.; Vermeulen, N.P.E.; Messeguer, A.

    2007-01-01

    Toxic oil syndrome (TOS) was a massive food-borne intoxication that occurred in Spain in 1981. Epidemiological studies imputed 3-(N-phenylamino) propane-1,2-diol (PAP) derivatives as the toxic agents. The in vitro bioactivation of PAP by rat and human liver microsomes was studied. In both cases,

  14. Human Milk Composition: Nutrients and Bioactive Factors

    Science.gov (United States)

    Ballard, Olivia; Morrow, Ardythe L.

    2013-01-01

    Synopsis The composition of human milk is the biologic norm for infant nutrition. Human milk also contains many hundreds to thousands of distinct bioactive molecules that protect against infection and inflammation and contribute to immune maturation, organ development, and healthy microbial colonization. Some of these molecules, e.g., lactoferrin, are being investigated as novel therapeutic agents. A dynamic, bioactive fluid, human milk changes in composition from colostrum to late lactation, and varies within feeds, diurnally, and between mothers. Feeding infants with expressed human milk is increasing. Pasteurized donor milk is now commonly provided to high risk infants and most mothers in the U.S. express and freeze their milk at some point in lactation for future infant feedings. Many milk proteins are degraded by heat treatment and freeze-thaw cycles may not have the same bioactivity after undergoing these treatments. This article provides an overview of the composition of human milk, sources of its variation, and its clinical relevance. PMID:23178060

  15. Dietary bioactive peptides: Human studies.

    Science.gov (United States)

    Bouglé, Dominique; Bouhallab, Saïd

    2017-01-22

    Current opinion strongly links nutrition and health. Among nutrients, proteins, and peptides which are encrypted in their sequences and released during digestion could play a key role in improving health. These peptides have been claimed to be active on a wide spectrum of biological functions or diseases, including blood pressure and metabolic risk factors (coagulation, obesity, lipoprotein metabolism, and peroxidation), gut and neurological functions, immunity, cancer, dental health, and mineral metabolism. A majority of studies involved dairy peptides, but the properties of vegetal, animal, and sea products were also assessed. However, these allegations are mainly based on in vitro and experimental studies which are seldom confirmed in humans. This review focused on molecules which were tested in humans, and on the mechanisms explaining discrepancies between experimental and human studies.

  16. Recombinant human endostatin reduces hypertrophic scar ...

    African Journals Online (AJOL)

    Background: Recombinant human endostatin (Endostar) has been widely used to suppress angiogenesis in carcinoma patients. ... Cite as: Wang P, Jiang L-Z, Xue B. Recombinant human endostatin reduces hypertrophic scar formation in rabbit ear model through ... wounds on the tail of each ear were discarded because.

  17. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  18. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  19. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  20. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    Science.gov (United States)

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Protein Crystal Recombinant Human Insulin

    Science.gov (United States)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  2. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen ...

  3. Recombinant human activated protein C (Xigris)

    NARCIS (Netherlands)

    Levi, M. [=Marcel M.; de Jonge, E.; van der Poll, T.

    2002-01-01

    An impaired function of the protein C pathway plays a central role in the pathogenesis of sepsis. Administration of human recombinant activated protein C (Xigris) may restore the dysfunctional anticoagulant mechanism and prevent amplification and propagation of thrombin generation and formation of

  4. Therapeutic implications of recombinant human erythropoietin in ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-12-29

    Dec 29, 2006 ... The introduction of recombinant human erythropoietin (RHUEPO) has revolutionised the treatment strategies for patients suffering with anaemia of chronic renal disease and chronic heart failure. Clinical studies and several observational evidences have demonstrated that RHUEPO is also useful in various.

  5. Simplified Method to Produce Human Bioactive Leukemia Inhibitory Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Houman Kahroba

    2016-07-01

    Full Text Available Background Human leukemia inhibitory factor (hLIF is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor. Objectives In this study we want to purify recombinant human leukemia inhibitory factor in single simple method. Materials and Methods This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl β-D-1-thiogalactopyranoside (IPTG used for induction. Single step immobilized metal affinity chromatography (IMAC used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P ˂ 0.05 that is significant for our work. Results Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines. Conclusions We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application.

  6. Molecular Dynamics and Bioactivity of a Novel Mutated Human ...

    African Journals Online (AJOL)

    Purpose: To design and evaluate a novel human parathyroid hormone (hPTH) analog. Methods: Mutation stability prediction was processed on hPTH, docked the mutant hPTH with its receptor, and then proceeded with molecular dynamics using Discovery Studio 3.5 software package for the complex. The bioactivity of the ...

  7. Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.

    Science.gov (United States)

    Bals, Carola; Schambach, Axel; Meyer, Johann; Scheper, Thomas; Rinas, Ursula

    2011-03-10

    Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Silk-Silk Interactions between Silkworm Fibroin and Recombinant Spider Silk Fusion Proteins Enable the Construction of Bioactive Materials.

    Science.gov (United States)

    Nilebäck, Linnea; Chouhan, Dimple; Jansson, Ronnie; Widhe, Mona; Mandal, Biman B; Hedhammar, My

    2017-09-20

    Natural silk is easily accessible from silkworms and can be processed into different formats suitable as biomaterials and cell culture matrixes. Recombinant DNA technology enables chemical-free functionalization of partial silk proteins through fusion with peptide motifs and protein domains, but this constitutes a less cost-effective production process. Herein, we show that natural silk fibroin (SF) can be used as a bulk material that can be top-coated with a thin layer of the recombinant spider silk protein 4RepCT in fusion with various bioactive motifs and domains. The coating process is based on a silk assembly to achieve stable interactions between the silk types under mild buffer conditions. The assembly process was studied in real time by quartz crystal microbalance with dissipation. Coatings, electrospun mats, and microporous scaffolds were constructed from Antheraea assama and Bombyx mori SFs. The morphology of the fibroin materials before and after coating with recombinant silk proteins was analyzed by scanning electron microscopy and atomic force microscopy. SF materials coated with various bioactive 4RepCT fusion proteins resulted in directed antibody capture, enzymatic activity, and improved cell attachment and spreading, respectively, compared to pristine SF materials. The herein-described procedure allows a fast and easy route for the construction of bioactive materials.

  9. Bioactivation of the heterocyclic aromatic amine 2-amino-3-methyl-9H-pyrido [2,3-b]indole (MeA alpha C) in recombinant test systems expressing human xenobiotic-metabolizing enzymes

    DEFF Research Database (Denmark)

    Glatt, H.; Pabel, U.; Meinl, W.

    2004-01-01

    2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and some metabolites were investigated for mutagenicity in mammalian cell lines and bacterial strains engineered for the expression of human enzymes. MeAalphaC induced gene mutations (studied at the hprt locus) in Chinese hamster V79-derived cel...

  10. Sweetness characterization of recombinant human lysozyme.

    Science.gov (United States)

    Matano, Mami; Nakajima, Kana; Kashiwagi, Yutaka; Udaka, Shigezo; Maehashi, Kenji

    2015-10-01

    Lysozyme, a bacteriolytic enzyme, is widely distributed in nature and is a component of the innate immune system. It is established that chicken egg lysozyme elicits sweetness. However, the sweetness of human milk lysozyme, which is vital for combating microbial infections of the gastrointestinal tract of breast-fed infants, has not been characterized. This study aimed to assess the elicitation of sweetness using recombinant mammalian lysozymes expressed in Pichia pastoris. Recombinant human lysozyme (h-LZ) and other mammalian lysozymes of mouse, dog, cat and bovine milk elicited similar sweetness as determined using a sensory test, whereas bovine stomach lysozyme (bs-LZ) did not. Assays of cell cultures showed that h-LZ activated the human sweet taste receptor hT1R2/hT1R3, whereas bs-LZ did not. Point mutations confirmed that the sweetness of h-LZ was independent of enzyme activity and substrate-binding sites, although acidic amino acid residues of bs-LZ played a significant role in diminishing sweetness. Therefore, we conclude that elicitation of sweetness is a ubiquitous function among all lysozymes including mammalian lysozymes. These findings may provide novel insights into the biological implications of T1R2/T1R3-activation by mammalian lysozyme in the oral cavity and gastrointestinal tract. However, the function of lysozyme within species lacking the functional sweet taste receptor gene, such as cat, is currently unknown. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Phytoestrogens as Bioactive Compounds with Beneficial Effects for Human Health

    OpenAIRE

    Silva, Ana Cristina Ferrão; Guiné, Raquel

    2017-01-01

    Phytoestrogens, also called estrogens, are bioactive compounds original from plants. They are similar in structure and functionality to the estrogenic hormones in animals. It has been documented that these compounds have several effects on the human body, namely in terms of carbohydrate, protein, lipid and mineral metabolism. Some of the most known effects of these substances are related to their roles in the women’s reproductive system. The dietary phytoestrogens are present in vegetable...

  12. Soluble variants of human recombinant glutaminyl cyclase.

    Directory of Open Access Journals (Sweden)

    Cristiana Castaldo

    Full Text Available Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of (1H-(15N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer's disease.

  13. Cranberries and Their Bioactive Constituents in Human Health12

    Science.gov (United States)

    Blumberg, Jeffrey B.; Camesano, Terri A.; Cassidy, Aedin; Kris-Etherton, Penny; Howell, Amy; Manach, Claudine; Ostertag, Luisa M.; Sies, Helmut; Skulas-Ray, Ann; Vita, Joseph A.

    2013-01-01

    Recent observational and clinical studies have raised interest in the potential health effects of cranberry consumption, an association that appears to be due to the phytochemical content of this fruit. The profile of cranberry bioactives is distinct from that of other berry fruit, being rich in A-type proanthocyanidins (PACs) in contrast to the B-type PACs present in most other fruit. Basic research has suggested a number of potential mechanisms of action of cranberry bioactives, although further molecular studies are necessary. Human studies on the health effects of cranberry products have focused principally on urinary tract and cardiovascular health, with some attention also directed to oral health and gastrointestinal epithelia. Evidence suggesting that cranberries may decrease the recurrence of urinary tract infections is important because a nutritional approach to this condition could lower the use of antibiotic treatment and the consequent development of resistance to these drugs. There is encouraging, but limited, evidence of a cardioprotective effect of cranberries mediated via actions on antioxidant capacity and lipoprotein profiles. The mixed outcomes from clinical studies with cranberry products could result from interventions testing a variety of products, often uncharacterized in their composition of bioactives, using different doses and regimens, as well as the absence of a biomarker for compliance to the protocol. Daily consumption of a variety of fruit is necessary to achieve a healthy dietary pattern, meet recommendations for micronutrient intake, and promote the intake of a diversity of phytochemicals. Berry fruit, including cranberries, represent a rich source of phenolic bioactives that may contribute to human health. PMID:24228191

  14. Micronucleus formation in cultured human keratinocytes: Involvement of intercellular bioactivation.

    Science.gov (United States)

    van Pelt, F N; Haring, R M; Weterings, P J

    1991-01-01

    Micronucleus formation in cultured human keratinocytes was studied after exposure to benzo[a]pyrene, cyclophosphamide and 12-O-tetradecanoylphorbol-13-acetate without the addition of an exogenous metabolizing system. The first two agents need bioactivation by specific isoenzymes of cytochrome P-450 to form genotoxic intermediates. Benzo[a]pyrene induced the micronucleus formation in both uninduced and Aroclor 1254-pretreated cultures. Clastogenic effects of cyclophosphamide were observed only in Aroclor 1254-pretreated cells. The tumour promotor 12-O-tetradecanoylphorbol-13-acetate did not affect the frequency of micronuclei in human keratinocytes. The data indicate that cultured human keratinocytes can be used to study the tissue-specific response to genotoxic agents as well as interindividual variation in biotransformation capacity.

  15. Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation.

    Science.gov (United States)

    Xie, H-J; Yasar, U; Lundgren, S; Griskevicius, L; Terelius, Y; Hassan, M; Rane, A

    2003-01-01

    The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes. A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles. CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting. 4-Hydroxy-CPA and hydroxy-bupropion were quantified by using HPLC and LC-MS, respectively. 7-Ethoxy-4-trifluoromethyl coumarin O-deethylase activity was measured fluorometrically. The frequencies of CYP2B6*5 and CYP2B6*6 mutant alleles were 9.0 and 16.4%, respectively. CYP2B6 protein expression was detected in 80% of the samples, with a large variation (0.003-2.234, arbitrary units). There was a high correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation (n=55, r=0.81, P<0.0001). When based on the CYP2B6 apoprotein levels, the *6 carriers had significantly higher CPA 4-hydroxylation (P<0.05). CPA 4-hydroxylation also correlated significantly with other CYP2B6-specific reactions (n=20, P<0.0001). V(max) and K(m) for CPA 4-hydroxylation in recombinant CYP2B6 enzyme were 338 nmol/min/nmol enzyme and 1.4 mM, respectively. CYP2B6 showed much higher in vitro intrinsic clearance than previously observed in recombinant CYP2C19 and CYP2C9 variants in yeast expression system. Our results demonstrate that the polymorphic CYP2B6 is a major enzyme in the bioactivation of CPA. Moreover, we identified a strong impact of CYP2B6*6 on CPA 4-hydroxylation.

  16. Recombinant human erythropoietin in sports: a review

    Directory of Open Access Journals (Sweden)

    Rafael Maia de Almeida Bento

    2003-06-01

    Full Text Available Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of oxygen to the muscle tissue. The administration of recombinant erythropoietin was prohibited by the International Olympic Committee and its use considered as doping. This review has the intention to describe the physical, biological and pharmacokinetic properties of the endogenous erythropoietin, as well as its recombinant form, describing also its use in sports and the process of searching methodologies for its detection in doping control.

  17. Marine carotenoids: Bioactivities and potential benefits to human health.

    Science.gov (United States)

    Chuyen, Hoang Van; Eun, Jong-Bang

    2017-08-13

    Among natural pigments, carotenoids play important roles in physiological functions. The characteristics of carotenoids and their effects on human health have been reported for a long time, but most studies have focused on carotenoids from vegetables, fruits, and other parts of higher plants. Few reports are available on carotenoids from marine sources, such as seaweeds, microalgae, and marine animals, which have attracted attention in recent decades. Hundreds of carotenoids have been identified and isolated from marine organisms and their beneficial physiological functions, such as anticancer, antiobesity, antidiabetic, anti-inflammatory, and cardioprotective activities have been reported. The purpose of this review is to discuss the literature on the beneficial bioactivities of some of the most abundant marine carotenoids, including fucoxanthin, astaxanthin, cantaxanthin, peridinin, fucoxanthinol, and halocynthiaxanthin.

  18. RECOMBINANT HUMAN INTERLEUKIN-3 IN CLINICAL ONCOLOGY

    NARCIS (Netherlands)

    DEVRIES, EGE; VANGAMEREN, MM; WILLEMSE, PHB

    Interleukin 3 (IL-3) is a multipotent hematopoietic growth factor which became available as a recombinant (rh) growth factor for use in the clinic a few years ago. In dose-finding studies, this hematopoietic growth factor has been evaluated without and after standard chemotherapy. Stimulatory

  19. Purification of human recombinant granulocyte colony stimulating ...

    African Journals Online (AJOL)

    In Escherichia coli, recombinant proteins were produced either as three dimensionally folded forms or as unfolded forms, inclusion body (IB). The formation of IB was a frequent consequence of high-level protein production and inadequacy of folding agents namely chaperones in the cytoplasm. The structure of the protein in ...

  20. Expression of the phycoerythrin gene of Gracilaria lemaneiformis (Rhodophyta) in E. coli and evaluation of the bioactivity of recombinant PE

    Science.gov (United States)

    Wen, Ruobing; Sui, Zhenghong; Zhang, Xuecheng; Zhang, Shuang; Qin, Song

    2007-10-01

    Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) ( P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mg mL-1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still

  1. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  2. Bioactive compounds in berries relevant to human health

    NARCIS (Netherlands)

    Battino, M.; Beekwilder, M.J.; Denoyes-Rothan, B.; Laimer, M.

    2009-01-01

    Berries contain powerful antioxidants, potential allergens, and other bioactive compounds. Genetic and environmental factors affect production and storage of such compounds. For this reason breeding and biotechnological approaches are currently used to control or to increase the content of specific

  3. Ovarian response to recombinant human follicle-stimulating hormone

    DEFF Research Database (Denmark)

    Arce, Joan-Carles; Andersen, Anders Nyboe; Fernández-Sánchez, Manuel

    2014-01-01

    OBJECTIVE: To evaluate the dose-response relationship of a novel recombinant human FSH (rhFSH; FE 999049) with respect to ovarian response in patients undergoing IVF/intracytoplasmic sperm injection treatment; and prospectively study the influence of initial antimüllerian hormone (AMH) concentrat......OBJECTIVE: To evaluate the dose-response relationship of a novel recombinant human FSH (rhFSH; FE 999049) with respect to ovarian response in patients undergoing IVF/intracytoplasmic sperm injection treatment; and prospectively study the influence of initial antimüllerian hormone (AMH...

  4. Expression, Purification and Bioactivities Analysis of Recombinant Active Peptide from Shark Liver

    Directory of Open Access Journals (Sweden)

    Boping Ye

    2009-06-01

    Full Text Available The Active Peptide from Shark Liver (APSL was expressed in E. coli BL21 cells. The cDNA encoding APSL protein was obtained from shark regenerated hepatic tissue by RT-PCR, then it was cloned in the pET-28a expression vector. The expressed fusion protein was purified by Ni-IDA affinity chromatography. SDS-PAGE and HPLC analysis showed the purity of the purified fusion protein was more than 98%. The recombinant APSL (rAPSL was tested for its biological activity both in vitro, by its ability to improve the proliferation of SMMC7721 cells, and in vivo, by its significant protective effects against acute hepatic injury induced by CCl4 and AAP (acetaminophen in mice. In addition, the rAPSL could decrease the blood glucose concentration of mice with diabetes mellitus induced by alloxan. Paraffin sections of mouse pancreas tissues showed that rAPSL (3 mg/kg could effectively protect mouse islets from lesions induced by alloxan, which indicated its potential application in theoretical research and industry.

  5. Recombination in the human Pseudoautosomal region PAR1.

    Directory of Open Access Journals (Sweden)

    Anjali G Hinch

    2014-07-01

    Full Text Available The pseudoautosomal region (PAR is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.

  6. Recombinant expression and purification of L2 domain of human ...

    African Journals Online (AJOL)

    The aim of the present study was cloning and expressing the fragment coding for L2 region of human EGFR for the production of recombinant L2 protein. The total RNA from A431 cells line was extracted and used for amplification of the sequence coding for L2 domain of EGFR by reverse transcriptase-polymerase chain ...

  7. Effects of recombinant human nerve growth factor on cervical cancer

    African Journals Online (AJOL)

    Jane

    2011-07-25

    Jul 25, 2011 ... systems. However, the roles of NGF to cervical cancer remain deeply unknown. This study investigated the effect of recombinant human nerve growth factor ... In addition, the immune abilities of thymus and spleen were improved by rhNGF. Finally ... polypeptide neurotrophin, plays a crucial role in the life of.

  8. Effect of Cytotoxicity of Pegylated Liposomal Recombinant Human ...

    African Journals Online (AJOL)

    Purpose: To evaluate the cytotoxic effect of pegylated liposomal Recombinant Human Erythropoietin- alfa (rHuEPO) nanoparticles synthesized by reverse phase evaporation technique on SH-SY5Y cell line. Methods: To prepare the nanoparticles of the drug, rHuEPO, PEG3000, cholesterol and phosphatidylcholine were ...

  9. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

    Science.gov (United States)

    Sockolosky, Jonathan T.; Szoka, Francis C.

    2012-01-01

    A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (DE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni2+ affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli. PMID:23168094

  10. Expression and characterization of recombinant human serum ...

    African Journals Online (AJOL)

    C-peptide (CP), connecting the A and B chains in proinsulin, has been considered to possess physiological effects in diabetes. In order to prolong the half-life of CP in vivo, a long acting CP analog [human serum albumin (HSA-CP)] was obtained by direct gene fusion of a single-chain CP to HSA and expressed in host ...

  11. Structural analysis of recombinant human protein QM

    International Nuclear Information System (INIS)

    Gualberto, D.C.H.; Fernandes, J.L.; Silva, F.S.; Saraiva, K.W.; Affonso, R.; Pereira, L.M.; Silva, I.D.C.G.

    2012-01-01

    Full text: The ribosomal protein QM belongs to a family of ribosomal proteins, which is highly conserved from yeast to humans. The presence of the QM protein is necessary for joining the 60S and 40S subunits in a late step of the initiation of mRNA translation. Although the exact extra-ribosomal functions of QM are not yet fully understood, it has been identified as a putative tumor suppressor. This protein was reported to interact with the transcription factor c-Jun and thereby prevent c-Jun actives genes of the cellular growth. In this study, the human QM protein was expressed in bacterial system, in the soluble form and this structure was analyzed by Circular Dichroism and Fluorescence. The results of Circular Dichroism showed that this protein has less alpha helix than beta sheet, as described in the literature. QM protein does not contain a leucine zipper region; however the ion zinc is necessary for binding of QM to c-Jun. Then we analyzed the relationship between the removal of zinc ions and folding of protein. Preliminary results obtained by the technique Fluorescence showed a gradual increase in fluorescence with the addition of increasing concentration of EDTA. This suggests that the zinc is important in the tertiary structure of the protein. More studies are being made for better understand these results. (author)

  12. Human DNA repair and recombination genes

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs

  13. Human oligoclonal recombinant antivenom against the black mamba (Dendroaspis polylepis)

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Karatt-­Vellatt, Aneesh; Slavny, Peter

    Snakebite envenoming is a major cause of death and morbidity in tropical parts of the world. Current therapies are based on animal-­derived antisera that are associated with a high degree of immunogenicity, high cost, and batch-to-batch variation. Here, we report the results of our ongoing efforts...... of developing the world’s first fully recombinant antivenom based on human IgGs targeting the key toxins from the notorious black mamba (Dendroaspis polylepis)....

  14. Phylogenetic and recombination analysis of human bocavirus 2

    Directory of Open Access Journals (Sweden)

    Li Huiying

    2011-02-01

    Full Text Available Abstract Background Human bocavirus 2(HBoV2 and other human bocavirus species (HBoV, HBoV3, and HBoV4 have been discovered recently. But the precise phylogenetic relationships among these viruses are not clear yet. Methods We collected 632 diarrhea and 162 healthy children in Lanzhou, China. Using PCR, Human bocavirus (HBoV, HBoV2, HBoV3 and HBoV4 were screened. The partial genes of NS, NP1 and VP, and two nearly complete sequences of HBoV2 were obtained. Result Phylogenetic analysis showed the different genes of HBoV2 strain were homogenous with different reference strains. HBoV3 may be a recombinant derived from HBoV and HBoV4. We also observed that the VP1 and VP2 region of HBoV3 is as similar to HBoV2 as to HBoV4. Conclusions A single genetic lineage of HBoV2 is circulating in children with and without gastroenteritis in Lanzhou, China. Current evidence in this study was not enough to support recombination between HBoV2 strains, and HBoV3 may be a recombinant between HBoV and the common ancestor of HBoV2 and HBoV4.

  15. Effects of selected bioactive food compounds on human white adipocyte function

    DEFF Research Database (Denmark)

    Björk, Christel; Wilhelm, Uta; Mandrup, Susanne

    2016-01-01

    bioactive nutrients on fat metabolism, we investigated their role on human white adipocyte function. METHODS: The influence of the omega-3-fatty acid docosahexaenoic acid (DHA), the anthocyanin (AC) cyanidin-3-glucoside and its metabolite protocatechuic acid, and the beta-glucan metabolite propionic acid...... of the compounds was found to be cytotoxic. CONCLUSION: The studied bioactive food compounds or their metabolites have beneficial effects in human primary fat cells measured as decreased basal lipolytic activity and secretion of inflammatory markers. A minor effect was also observed on insulin-stimulated glucose...... uptake albeit only with the combination of DHA and AC. Taken together, our results may link the reported health benefits of the selected bioactives on metabolic disorders such as insulin resistance, hypertension and dyslipidemia to effects on white adipocytes....

  16. Production of biologically active recombinant human factor H in Physcomitrella.

    Science.gov (United States)

    Büttner-Mainik, Annette; Parsons, Juliana; Jérôme, Hanna; Hartmann, Andrea; Lamer, Stephanie; Schaaf, Andreas; Schlosser, Andreas; Zipfel, Peter F; Reski, Ralf; Decker, Eva L

    2011-04-01

    The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  17. Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

    Directory of Open Access Journals (Sweden)

    Pierson Janice

    2008-05-01

    Full Text Available Abstract Background Human butyrylcholinesterase (huBChE has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA and characterize the fusion protein. Results Secretion level of the fusion protein produced in vitro in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h. In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. Conclusion Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.

  18. Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

    Science.gov (United States)

    Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming

    2016-02-01

    A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Human cytochrome P450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Boersma, M.G.; Bogaards, J.J.P.; Fiamegos, Y.C.; Schilter, B.; Bladeren, P.J. van; Cnubben, N.H.P.; Rietjens, I.M.C.M.

    2007-01-01

    Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1′-hydroxyestragole were identified and compared to the enzymes of importance for 1′-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that

  20. [Construction of recombinant human nerve growth factor (rh-β-NGF) eukaryotic vector and its expression in HEK293 cells].

    Science.gov (United States)

    Li, Jingchuan; Xue, Bofu; Yuan, Yuan; Ma, Mo; Zhu, Lin; Milburn, Rebecca; Le, Li; Hu, Peizhen; Ye, Jing

    2015-03-01

    Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.

  1. Recombinant human growth hormone in the treatment of Turner syndrome

    Directory of Open Access Journals (Sweden)

    Bessie E Spiliotis

    2008-12-01

    Full Text Available Bessie E SpiliotisDivision of Pediatric Endocrinology and Diabetes, Department of Pediatrics, University of Patras, School of Medicine, Patras, GreeceAbstract: Turner syndrome (TS is a common chromosomal disorder in women that is associated with the absence of one of the X chromosomes. Severe short stature and a lack of pubertal development characterize TS girls, causing psychosocial problems and reduced bone mass. The growth impairment in TS seems to be due to multiple factors including an abnormal growth hormone (GH – insulin-like growth factor (IGF – IGF binding protein axis and haploinsufficiency of the short stature homeobox-containing gene. Growth hormone and sex steroid replacement therapy has enhanced growth, pubertal development, bone mass, and the quality of life of TS girls. Recombinant human GH (hGH has improved the height potential of TS girls with varied results though, depending upon the dose of hGH and the age of induction of puberty. The best final adult height and peak bone mass achievement results seem to be achieved when hGH therapy is started early and puberty is induced at the normal age of puberty in a regimen mimicking physiologic puberty. The initiation of estradiol therapy at an age-appropriate time may also help the TS patients avoid osteoporosis during adulthood. Recombinant hGH therapy in TS seems to be safe. Studies so far show no adverse effects on cardiac function, glucose metabolism or any association with neoplasms but research is still in progress to provide conclusive data on long-term safety.Keywords: Turner syndrome, recombinant growth hormone, growth hormone deficiency, SHOX gene, hormonal replacement therapy

  2. Use of Cuban recombinant human erythropoietin in Parkinson's disease treatment.

    Science.gov (United States)

    Pedroso, Ivonne; Bringas, María Luisa; Aguiar, Anubis; Morales, Lilia; Alvarez, Mario; Valdés, Pedro A; Alvarez, Lázaro

    2012-01-01

    Recombinant human erythropoietin is used primarily to treat anemia. There is evidence of its neuroprotective capacity from preclinical studies in Parkinson's disease and other neurodegenerative diseases. Recombinant human erythropoietin produced in Cuba (ior-EPOCIM) is registered and approved for use in humans in Cuba and in a number of other countries. Assess safety and possible neuroprotective effect of ior-EPOCIM in a group of Parkinson's disease patients. A three-phase exploratory study (proof of concept) was conducted from August 2008 to April 2009: preliminary assessment, treatment (weeks 1-5), and post-treatment (weeks 6-35). Participants were 10 Parkinson's disease patients (8 men, 2 women) from the outpatient clinic at the International Neurological Restoration Center, all at least one year post onset, aged 47-65 years. The ior-EPOCIM was administered subcutaneously in a once-weekly dose (60 IU/kg body weight) for five weeks. Therapy with patients' antiparkinsonian drugs was maintained throughout the study, except during motor examination, conducted following a 12-hour withdrawal (OFF condition). Safety was evaluated primarily by recording adverse events (by intensity and causality) from start of treatment until the study's completion. Hematological parameters and blood pressure were also measured because of their direct relationship to the medication's action. To evaluate possible neuroprotective activity, variables were included related to patients' motor function and cognitive and affective status, measured using internationally recognized scales. All variables were evaluated before, during and after treatment. Data were processed using a fixed-effects linear model, with a repeated-measures design (significance level p ≤ 0.05). Three patients experienced mild adverse events (precordial discomfort and hypertension in one; leg fatigue in another; renal colic in a third), with a possible causal relationship in the first two that was neither life

  3. Comparison of bioactive aldehydes modifying action on human albumin

    OpenAIRE

    I. P. Krysiuk; A. J. Knaub; S. G. Shandrenko

    2014-01-01

    Protein’s postsynthetic modifications are a cause and a consequence of many diseases. Endogenous aldehydes are one of the main factors of these modifications formation. The human albumin’s modification under some aldehydes influence in in vitro experiment has been investigated. Human albumin (20 mM) was incubated with following aldehydes: ribose, glyoxal, methylglyoxal and formaldehyde (20 mM each) and their combinations in 0.1 M Na-phosphate buffer (pH 7.4) with 0.02% sodium azide at 37 °C i...

  4. In Vivo Bioassay of Recombinant Human Growth Hormone Synthesized in B. mori Pupae

    Directory of Open Access Journals (Sweden)

    Hanglian Lan

    2010-01-01

    Full Text Available The human growth hormone (hGH has been expressed in prokaryotic expression system with low bioactivity previously. Then the effective B. mori baculovirus system was employed to express hGH identical to mature hGH successfully in larvae, but the expression level was still limited. In this work, the hGH was expressed in B. mori pupae by baculovirus system. Quantification of recombinant hGH protein (BmrhGH showed that the expression of BmrhGH reached the level of approximately 890 μg/mL pupae supernatant solution, which was five times more than the level using larvae. Furthermore, Animals were gavaged with BmrhGH at the dose of 4.5 mg/rat.day, and the body weight gain (BWG of treated group had a significant difference (P<.01 compared with the control group. The other two parameters of liver weight and epiphyseal width were also found to be different between the two groups (P<.05. The results suggested that BmrhGH might be used as a protein drug by oral administration.

  5. Heterogeneity of commercial recombinant human growth hormone (r-hGH) preparations containing a thioether variant.

    Science.gov (United States)

    Lispi, Monica; Datola, Antonio; Bierau, Horst; Ceccarelli, Domenico; Crisci, Carmelina; Minari, Katia; Mendola, Daniele; Regine, Aniello; Ciampolillo, Cinzia; Rossi, Mara; Giartosio, Carlo Emanuele; Pezzotti, Anna Rita; Musto, Raffaella; Jone, Carl; Chiarelli, Francesco

    2009-12-01

    The objective of the present study was to assess (I) the potential presence of a recently discovered thioether variant in commercially available recombinant human growth hormone (r-hGH) preparations, and (II) the impact of the thioether modification on the in-vivo bioactivity and the receptor binding kinetics. Samples were tested employing European (EP) and US Pharmacopeia (USP) Somatropin monograph and mass spectrometry methods. None of the international standards contained this variant. All products conformed to EP specifications but six out of eight lots contained the variant. An artificially enriched thioether sample exhibited a significantly reduced in vivo biopotency and altered receptor-binding properties compared with a control. The absence of the variant in the pituitary hGH standard, and the possibility to generate it artificially suggests that it is not naturally occurring and that it may arise from an uncontrolled manufacturing process. Controlled studies may be required to assess its clinical efficacy and safety. EP and USP methods may need to be adapted to reliably detect the presence of the variant. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

  6. Comparison of Recombinant Human Haptocorrin Expressed in Human Embryonic Kidney Cells and Native Haptocorrin

    DEFF Research Database (Denmark)

    Furger, Evelyne; Fedosov, Sergey N; Lildballe, Dorte Launholt

    2012-01-01

    HC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand......Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like...... milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC...

  7. Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells

    OpenAIRE

    MESTIERI,Leticia Boldrin; GOMES-CORNÉLIO,Ana Lívia; RODRIGUES,Elisandra Márcia; SALLES,Loise Pedrosa; BOSSO-MARTELO,Roberta; GUERREIRO-TANOMARU,Juliane Maria; TANOMARU-FILHO,Mário

    2015-01-01

    Mineral Trioxide Aggregate (MTA) is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus. Objective The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs). Material and Methods The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1...

  8. Short-term effects of recombinant human growth hormone and feeding on gluconeogenesis in humans

    Science.gov (United States)

    After a short-term fast, lactating women have increased rates of glucose production but not gluconeogenesis (GNG) despite relative hypoinsulinemia. We explored the effects of non-insulin-dependent increase in glucose utilization and recombinant human growth hormone (rhGH) on glucose production, glyc...

  9. Nutritional and biochemical properties of human milk: II. Lipids, micronutrients, and bioactive factors.

    Science.gov (United States)

    Rodriguez-Palmero, M; Koletzko, B; Kunz, C; Jensen, R

    1999-06-01

    Human milk lipids contain preformed LCPUFA in considerable amounts, which serve as precursors for the formation of prostaglandins, prostacyclins, and other lipid mediators, as well as essential components in membrane-rich tissues (such as the brain and the retina), thus affecting functional outcomes. Besides a balanced nutrient composition and a number of conditionally essential nutrients, human milk provides different types and classes of bioactive factors, such as enzymes, hormones, and growth factors, many of which appear to have a role in supporting infantile growth and development. The bioactive agents include antimicrobial factors (e.g., secretory IgA, oligosaccharides, FA); anti-inflammatory agents; transporters (e.g., lactoferrin); and digestive enzymes (e.g., BSSL). Several nonpeptide hormones (thyroid hormones, cortisol, progesterone, pregnanediol, estrogens, and artificial contraceptive) and peptide hormones and growth factors (erythropoietin, hHG, gonadotropin-releasing hormone, epidermal growth factor insulin, insulin-like growth factor-I, nerve growth factor, transforming growth factor-alpha, gastrointestinal regulatory peptides and thyroid-parathyroid hormones) have been isolated and quantitated in human milk. Some of these components are also involved in the maturation of the gastrointestinal tract of the infant. In addition to the passive benefits provided by human milk, several data support the hypothesis that breastfeeding promotes the development of the infant's own immune system, which might confer long-term benefits for the newborn infant. The risk of IDDM, Crohn's disease, and atopic disease is lower in individuals who had been breastfed during infancy. Areas of major interest in human milk research include the study of human milk synthesis and the contributions of dietary composition and maternal metabolism to human milk composition, infantile utilization of human milk components, and the study of bioactive components, such as

  10. Comparison of bioactive aldehydes modifying action on human albumin

    Directory of Open Access Journals (Sweden)

    I. P. Krysiuk

    2014-04-01

    Full Text Available Protein’s postsynthetic modifications are a cause and a consequence of many diseases. Endogenous aldehydes are one of the main factors of these modifications formation. The human albumin’s modification under some aldehydes influence in in vitro experiment has been investigated. Human albumin (20 mM was incubated with following aldehydes: ribose, glyoxal, methylglyoxal and formaldehyde (20 mM each and their combinations in 0.1 M Na-phosphate buffer (pH 7.4 with 0.02% sodium azide at 37 °C in the dark for up to 30 days. We have determined the fluorescent properties of the samples, the content of protein’s carbonyl groups and the redistribution of protein’s molecular weight. The following ratings of aldehydes from the lowest to the highest effect have been obtained. Fluo­rescent albumin adducts formation: formaldehyde, methylglyoxal, ribose, glyoxal; carbonylation of the protein: ribose, formaldehyde, glyoxal, methyl­glyoxal; polymerization of albumin – the formation of intermolecular crosslinks: ribose, methylglyoxal, glyoxal, formaldehyde. The results indicate that these aldehydes have different capability for protein’s modifications. For example, formaldehyde, having the lowest ability to form fluorescent adducts, shows the highest ability to form protein’s intermolecular crosslinks. Therefore, methods and parame­ters in order to evaluate the protein postsynthetic modification intensity have to be chosen correctly according to carbonyl stress peculiarity in order to evaluate the protein’s postsynthetic modification intensity.

  11. [Comparison of bioactive aldehydes modifying action on human albumin].

    Science.gov (United States)

    Krysiuk, I P; Knaub, A Ia; Shandrenko, S H

    2014-01-01

    Protein's postsynthetic modifications are a cause and a consequence of many diseases. Endogenous aldehydes are one of the main factors of these modifications formation. The human albumin's modification under some aldehydes influence in in vitro experiment has been investigated. Human albumin (20 mM) was incubated with following aldehydes: ribose, glyoxal, methylglyoxal and formaldehyde (20 mM each) and their combinations in 0.1 M Na-phosphate buffer (pH 7.4) with 0.02% sodium azide at 37 degrees C in the dark for up to 30 days. We have determined the fluorescent properties of the samples, the content of protein's carbonyl groups and the redistribution of protein's molecular weight. The following ratings of aldehydes from the lowest to the highest effect have been obtained. Fluorescent albumin adducts formation: formaldehyde, methylglyoxal, ribose, glyoxal; carbonylation of the protein: ribose, formaldehyde, glyoxal, methylglyoxal; polymerization of albumin--the formation of intermolecular crosslinks: ribose, methylglyoxal, glyoxal, formaldehyde. The results indicate that these aldehydes have different capability for protein's modifications. For example, formaldehyde, having the lowest ability to form fluorescent adducts, shows the highest ability to form protein's intermolecular crosslinks. Therefore, methods and parameters in order to evaluate the protein postsynthetic modification intensity have to be chosen correctly according to carbonyl stress peculiarity in order to evaluate the protein's postsynthetic modification intensity.

  12. Bioactive Compounds in Some Culinary Aromatic Herbs and Their Effects on Human Health.

    Science.gov (United States)

    Guiné, Raquel P F; Gonçalves, Fernando J

    2016-01-01

    Culinary herbs are herbaceous (leafy) plants that add flavour and colour to all types of meals. There is a wide variety of herbs that are used for culinary purposes worldwide, which are also recognized for their beneficial health effects, and thus have also been used in folk medicine. Besides their nutritional value herbs are rich in many phytochemical components with bioactive effects, thus improving human health. The aim of the present work was to make a general overview of some of these herbs, including their gastronomic usage, their chemical composition in bioactive components and their reported health effects. This work showed that the health effects are very diverse and differ according to the herb in question. However, some of the most frequently citted biological activities include antioxidant, antimicrobial, and antiviral effects.

  13. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

    Science.gov (United States)

    2014-01-01

    Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of

  14. Recombinant human endostatin improves tumor vasculature and alleviates hypoxia in Lewis lung carcinoma

    International Nuclear Information System (INIS)

    Peng Fang; Wang Jin; Zou Yi; Bao Yong; Huang Wenlin; Chen Guangming; Luo Xianrong; Chen Ming

    2011-01-01

    Objective: To investigate whether recombinant human endostatin can create a time window of vascular normalization prior to vascular pruning to alleviate hypoxia in Lewis lung carcinoma in mice. Methods: Kinetic changes in morphology of tumor vasculature in response to recombinant human endostatin were detected under a confocal microscope with immunofluorescent staining in Lewis lung carcinomas in mice. The hypoxic cell fraction of different time was assessed with immunohistochemical staining . Effects on tumor growth were monitored as indicated in the growth curve of tumors . Results: Compared with the control group vascularity of the tumors was reduced over time by recombinant human endostatin treatment and significantly regressed for 9 days. During the treatment, pericyte coverage increased at day 3, increased markedly at day 5, and fell again at day 7. The vascular basement membrane was thin and closely associated with endothelial cells after recombinant human endostatin treatment, but appeared thickened, loosely associated with endothelial cells in control tumors. The decrease in hypoxic cell fraction at day 5 after treatment was also found. Tumor growth was not accelerated 5 days after recombinant human endostatin treatment. Conclusions: Recombinant human endostatin can normalize tumor vasculature within day 3 to 7, leading to improved tumor oxygenation. The results provide important experimental basis for combining recombinant human endostatin with radiation therapy in human tumors. (authors)

  15. Preoperative use of recombinant human erythropoietin before total joint arthroplasty.

    Science.gov (United States)

    Bezwada, Hari P; Nazarian, David G; Henry, David H; Booth, Robert E

    2003-09-01

    Previous reports have suggested that the use of recombinant human erythropoietin is effective for decreasing the need for perioperative allogeneic blood transfusion. The purpose of this study was to evaluate the efficacy of erythropoietin in combination with, and compared with, preoperative autologous donation for reducing allogeneic blood requirements for total joint arthroplasty. Two hundred and forty patients undergoing primary and revision total hip or knee arthroplasty were enrolled into three groups with different treatment regimens: (1) erythropoietin and preoperative autologous donation (Group 1), (2) erythropoietin alone (Group 2), and (3) preoperative autologous donation alone (Group 3). Patients were evaluated with regard to requirements for allogeneic transfusion, change from the baseline to the lowest postoperative hemoglobin value, postoperative complications, and adverse reactions. The rate of allogeneic transfusion was 11% in Group 1 (erythropoietin and preoperative autologous donation) compared with 28% in Group 2 (erythropoietin alone) and 33% in Group 3 (preoperative autologous donation alone). Within Group 1, patients who had a unilateral primary arthroplasty had an allogeneic transfusion rate of 4% and those who had a bilateral or revision arthroplasty had an allogeneic transfusion rate of 17%. In Groups 2 and 3, the allogeneic transfusion rates were 14% and 15%, respectively, for the patients who had a unilateral primary arthroplasty and 35% and 47%, respectively, for those who had a bilateral or revision arthroplasty. Preoperative use of erythropoietin in conjunction with preoperative autologous donation reduces the need for allogeneic blood transfusion associated with total joint arthroplasty more effectively than does either erythropoietin or preoperative autologous donation alone.

  16. Accelerators of Osteogenesis by Recombinant Human Bone Morphogenetic Protein-2

    Directory of Open Access Journals (Sweden)

    Yasunori Okubo

    2007-01-01

    Full Text Available Bone morphogenetic protein (BMP appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fibroblast growth factor (bFGF, FK506, elcatonin, and hyperbaric oxygenation (HBO on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2, 2 or 5 μg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. The amount of new bone in the treatment group was significantly greater than that in the control group. The alkaline phosphatase activity and calcium content in the treatment group were significantly higher than those in the control group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by rhBMP2. These results may be useful when BMP is applied clinically in near future.

  17. Accelerators of Osteogenesis by Recombinant Human Bone Morphogenetic Protein-2

    Directory of Open Access Journals (Sweden)

    Yasunori Okubo

    2007-01-01

    Full Text Available Bone morphogenetic protein (BMP appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fibroblast growth factor (bFGF, FK506, elcatonin, and hyperbaric oxygenation (HBO on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2, 2 or 5 μg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. The amount of new bone in the treatment group was signifi cantly greater than that in the control group. The alkaline phosphatase activity and calcium content in the treatment group were signifi cantly higher than those in the control group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by rhBMP2. These results may be useful when BMP is applied clinically in near future.

  18. Expression and biochemical characterization of recombinant human epididymis protein 4.

    Science.gov (United States)

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Use of recombinant human antithrombin concentrate in pregnancy

    Directory of Open Access Journals (Sweden)

    Baumann Kreuziger LM

    2013-09-01

    Full Text Available Lisa M Baumann Kreuziger,1 Tracy L Prosen,2 Mark T Reding1 1Division of Hematology, Oncology, and Transplantation, University of Minnesota, MN, USA; 2Obstetrics and Gynecology, Maternal Fetal Medicine Center, University of Minnesota, MN, USAWe read with great interest James et al’s article "Prevention and treatment of venous thromboembolism in pregnancy in patients with hereditary antithrombin deficiency." The authors reported a case series of six women with antithrombin (AT deficiency treated with plasma derived antithrombin concentrate (pdAT; Thrombate III®, Grifols Therapeutics, Clayton, NC, USA.1 In contrast to these cases, we have managed three AT-deficient women over the past two years with low-molecular-weight-heparin during pregnancy and recombinant human antithrombin concentrate (rhAT; ATryn®, GTC Biotherapeutics, Framingham, MA, USA at delivery. In the Phase III trial leading to approval of rhAT, pregnant women required frequent dose modifications, and an alternate dosing regimen is currently recommended by the manufacturer.2,3 The detailed information provided in these cases may assist future providers in managing pregnant patients with AT deficiency.View original paper by James and colleagues.

  20. Effects of Recombinant Human Prolactin on Breast Milk Composition

    Science.gov (United States)

    Powe, Camille E.; Puopolo, Karen M.; Newburg, David S.; Lönnerdal, Bo; Chen, Ceng; Allen, Maureen; Merewood, Anne; Worden, Susan

    2011-01-01

    OBJECTIVE: The objective of this study was to determine the impact of recombinant human prolactin (r-hPRL) on the nutritional and immunologic composition of breast milk. METHODS: We conducted 2 trials of r-hPRL treatment. In the first study, mothers with documented prolactin deficiency were given r-hPRL every 12 hours in a 28-day, open-label trial. In the second study, mothers with lactation insufficiency that developed while they were pumping breast milk for their preterm infants were given r-hPRL daily in a 7-day, double-blind, placebo-controlled trial. Breast milk characteristics were compared before and during 7 days of treatment. RESULTS: Among subjects treated with r-hPRL (N = 11), milk volumes (73 ± 36 to 146 ± 54 mL/day; P lactogenesis. r-hPRL also increased antimicrobially active oligosaccharide concentrations. These effects were achieved for women with both prolactin deficiency and lactation insufficiency. PMID:21262884

  1. Zinc effect on the structure of recombinant human growth hormone

    International Nuclear Information System (INIS)

    El Nabarwy, F.S.

    2002-01-01

    A number of protein substances are known to undergo chemical decomposition when stored under non-ideal conditions. Metal induced precipitation of protein is being used and further developed as a processing step in protein formulation and may have utility in protein purification bulk storage. In such processes, it is important that native protein structure is maintained and the metal complexation is reversible. Series of dialysis experiments, treatment with chelating agents and divalent metal ions showed that zinc was required for such process. In this study, the effect of zinc induced precipitation recombinant human growth hormone (rh GH) was investigated. Ethylene diamine tetra acetic acid (EDTA) was added and the precipitate was dissolved yielding complete recovery of native protein. The protein has specific metal binding sites and requires specific molar ratios of zinc to protein to initiate precipitation (zinc: rh GH=2:1). Furthermore, the secondary structure of the protein was unperturbed in soluble zinc complexes and zinc induced precipitates, as measured by infrared and spectroscopy. The soluble zinc complex of rh GH had minor tertiary structural alterations. This study indicated that metal induced precipitation provides a good method maintain proteins in their native state in precipitates, which may be useful for purification storage and formulation. In addition, formation of a Zn (2+)-rh GH dimeric complex may be important for storage of rh GH in secretory granules

  2. Genetic heterogeneity and recombination in type-3 human astroviruses.

    Science.gov (United States)

    Medici, Maria Cristina; Tummolo, Fabio; Martella, Vito; Banyai, Krisztián; Bonerba, Elisabetta; Chezzi, Carlo; Arcangeletti, Maria Cristina; De Conto, Flora; Calderaro, Adriana

    2015-06-01

    Human astroviruses (HAstVs) are important enteric pathogens and can be classified genetically and antigenically into eight types. During molecular surveillance for HAstVs in Italy, sequence analysis of the diagnostic region C (about 400 nucleotide in length), located on the capsid (ORF2) gene, identified a novel type-3 strain. Upon sequencing of the full-length ORF2, the type-3 HAstV strain was characterized as a novel ORF2 genetic lineage, designated as 3c. By converse, in the ORF1b the virus was more similar to type-1 HAstVs, rather than to type-3 strains, suggesting a recombination nature, with the crossover site being mapped to the ORF1b/ORF2 junction region. Region C sequences of similar type-3 HAstV identified from European and extra-European countries were retrieved in the databases, suggesting the global distribution of this novel type-3 lineage. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency.

    Science.gov (United States)

    Lee, Yu Nee; Frugoni, Francesco; Dobbs, Kerry; Walter, Jolan E; Giliani, Silvia; Gennery, Andrew R; Al-Herz, Waleed; Haddad, Elie; LeDeist, Francoise; Bleesing, Jack H; Henderson, Lauren A; Pai, Sung-Yun; Nelson, Robert P; El-Ghoneimy, Dalia H; El-Feky, Reem A; Reda, Shereen M; Hossny, Elham; Soler-Palacin, Pere; Fuleihan, Ramsay L; Patel, Niraj C; Massaad, Michel J; Geha, Raif S; Puck, Jennifer M; Palma, Paolo; Cancrini, Caterina; Chen, Karin; Vihinen, Mauno; Alt, Frederick W; Notarangelo, Luigi D

    2014-04-01

    The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T(-)B(-) severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes. We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations. We have developed a flow cytometry-based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1(-/-) pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology. Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity. Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  4. Investigating the Antimicrobial Bioactivity of Cyano bacterial Extracts on Some Plant and Human Pathogens

    International Nuclear Information System (INIS)

    El-Semary, N.A.; Osman, M.E.; Ahmed, A.S.; Botros, H.W.; Farag, A.T.

    2014-01-01

    The search for broad spectrum antimicrobial agents against microbial pathogens, as the available bioactive compounds, has decreasing efficacy and the multidrug resistance trait is spreading among pathogens. Accordingly, the study was carried out to investigate the antimicrobial bioactivity of extracts derived from a cyano bacterial strain from Egypt. The solvents used were diethyl ether, chloroform and methanol. The antimicrobial bioassay of the lipophilic fraction dissolved in diethyl ether of Synechococcus spp. (isolated from Wadi El-Natroun, Egypt) showed the highest broad spectrum bioactivity as it inhibited the growth of both plant and human pathogens. The extract was also effective on the filamentous plant pathogenic fungi Aspergillus flavus and Aspergillus niger. The effects of incubation periods, growth media and pH values on both growth and antimicrobial activity of Synechococcus spp. were investigated. Chu medium was the medium that gave the highest growth followed by BG11 medium then Oscillatoria medium and all these three media showed antibacterial activities but only BG11 showed both antibacterial and antifungal activities after 18 days of incubation. The pH value 10 proved to be the best for growth and antimicrobial activities of Synechococcus spp. in BG11 medium

  5. Facilitated uptake of a bioactive metabolite of maritime pine bark extract (pycnogenol into human erythrocytes.

    Directory of Open Access Journals (Sweden)

    Max Kurlbaum

    Full Text Available Many plant secondary metabolites exhibit some degree of biological activity in humans. It is a common observation that individual plant-derived compounds in vivo are present in the nanomolar concentration range at which they usually fail to display measurable activity in vitro. While it is debatable that compounds detected in plasma are not the key effectors of bioactivity, an alternative hypothesis may take into consideration that measurable concentrations also reside in compartments other than plasma. We analysed the binding of constituents and the metabolite δ-(3,4-dihydroxy-phenyl-γ-valerolactone (M1, that had been previously detected in plasma samples of human consumers of pine bark extract Pycnogenol, to human erythrocytes. We found that caffeic acid, taxifolin, and ferulic acid passively bind to red blood cells, but only the bioactive metabolite M1 revealed pronounced accumulation. The partitioning of M1 into erythrocytes was significantly diminished at higher concentrations of M1 and in the presence of glucose, suggesting a facilitated transport of M1 via GLUT-1 transporter. This concept was further supported by structural similarities between the natural substrate α-D-glucose and the S-isomer of M1. After cellular uptake, M1 underwent further metabolism by conjugation with glutathione. We present strong indication for a transporter-mediated accumulation of a flavonoid metabolite in human erythrocytes and subsequent formation of a novel glutathione adduct. The physiologic role of the adduct remains to be elucidated.

  6. The Biomineralization of a Bioactive Glass-Incorporated Light-Curable Pulp Capping Material Using Human Dental Pulp Stem Cells

    OpenAIRE

    Jun, Soo-Kyung; Lee, Jung-Hwan; Lee, Hae-Hyoung

    2017-01-01

    The aim of this study was to investigate the biomineralization of a newly introduced bioactive glass-incorporated light-curable pulp capping material using human dental pulp stem cells (hDPSCs). The product (Bioactive? [BA]) was compared with a conventional calcium hydroxide-incorporated (Dycal [DC]) and a light-curable (Theracal? [TC]) counterpart. Eluates from set specimens were used for investigating the cytotoxicity and biomineralization ability, determined by alkaline phosphatase (ALP) a...

  7. Comparative pharmacology of a new recombinant FSH expressed by a human cell line

    DEFF Research Database (Denmark)

    Koechling, Wolfgang; Plaksin, Daniel; Croston, Glenn E.

    2017-01-01

    Recombinant FSH proteins are important therapeutic agents for the treatment of infertility, including follitropin alfa expressed in Chinese Hamster Ovary (CHO) cells and, more recently, follitropin delta expressed in the human cell line PER.C6. These recombinant FSH proteins have distinct glycosy...

  8. Human Prostate Cancer in a Tissue Recombination Model

    National Research Council Canada - National Science Library

    Williams, Karin

    2002-01-01

    .... Tissue recombinations (TR) composed of hPrE and rat urogenital sinus mesenchyme (rUGM) grafted beneath the renal capsule of immunocompromised rat hosts recapitulate many key events in prostatic development and adult function...

  9. Human Prostate Cancer in a Tissue Recombination Model

    National Research Council Canada - National Science Library

    Williams, Karin

    2003-01-01

    .... Tissue recombinations (TR) composed of hPrE and rat urogenital sinus mesenchyme (rUGM) grafted beneath the renal capsule of immunocompromised rodent hosts recapitulate many key events in prostatic development and adult function...

  10. Expression of recombinant Streptokinase from local Egyptian ...

    African Journals Online (AJOL)

    Jane

    2011-08-17

    Aug 17, 2011 ... DISCUSSION. Isolation and identification of Streptococcus sp. In this study, SK isolated from local Streptococcus sp. SalMarEg was efficiently produced in a recombinant bioactive form. It is worthy to mention that the binding of plasminogen by pathogenic Group C streptococci isolated from human, horses, ...

  11. Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

    Science.gov (United States)

    Lambright, D G; Balasubramanian, S; Boxer, S G

    1989-05-05

    Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively

  12. Boundary mode lubrication of articular cartilage by recombinant human lubricin.

    Science.gov (United States)

    Gleghorn, Jason P; Jones, Aled R C; Flannery, Carl R; Bonassar, Lawrence J

    2009-06-01

    Lubrication of cartilage involves a variety of physical and chemical factors, including lubricin, a synovial glycoprotein that has been shown to be a boundary lubricant. It is unclear how lubricin boundary lubricates a wide range of bearings from tissue to artificial surfaces, and if the mechanism is the same for both soluble and bound lubricin. In the current study, experiments were conducted to investigate the hypothesis that recombinant human lubricin (rh-lubricin) lubricates cartilage in a dose-dependent manner and that soluble and bound fractions of rh-lubricin both contribute to the lubrication process. An rh-lubricin dose response was observed with maximal lubrication achieved at concentrations of rh-lubricin greater than 50 microg/mL. A concentration-response variable-slope model was fit to the data, and indicated that rh-lubricin binding to cartilage was not first order. The pattern of decrease in equilibrium friction coefficient indicated that aggregation of rh-lubricin or steric arrangement may regulate boundary lubrication. rh-lubricin localized at the cartilage surface was found to lubricate a cartilage-glass interface in boundary mode, as did soluble rh-lubricin at high concentrations (150 microg/mL); however, the most effective lubrication occurred when both soluble and bound rh-lubricin were present at the interface. These findings point to two distinct mechanisms by which rh-lubricin lubricates, one mechanism involving lubricin bound to the tissue surface and the other involving lubricin in solution. Copyright 2008 Orthopaedic Research Society

  13. Recombinant human TSH in radioiodine treatment of differentiated thyroid cancer

    International Nuclear Information System (INIS)

    Kovatcheva, R. D.; Kirilov, G. G.; Lozanov, B. S.; Hadjieva, T.D.

    2004-01-01

    Recombinant human TSH (rhTSH) has been developed to facilitate the follow-up for persistent or recurrent differentiated thyroid cancer (DTC), avoiding the hypothyroid symptoms after the withdrawal of Levothyroxine (L-T 4 ) suppressive therapy. To analyse the effect of rhTSH in providing stimulation of radioiodine uptake (RAIU) for the ablation of thyroid remnant and/or malignant thyroid tissue in patients with metastatic DTC. Ten subjects (4 women, 6 men), mean age 53 years, with DTC (7 papillary, 2 follicular and 1 Huerthle-cell), requiring radioiodine therapy (RIT) were studied. Nine of them had a positive diagnostic whole body scan (dWBS) or CT for thyroid remnant, lymph nodes and/or distant metastases. One patient with an invasive tall cell PTC had an increased serum Tg and a negative dWBS. Serum TSH was measured before and two days after the rhTSH injection. Thyroglobulin measurements were performed before the rhTSH administration, 3 and 6 months after RIT. There were no serious side effects of the rhTSH application. Serum TSH after the rhTSH injection rose to 156.5 ± 60.9 mIU/L and induced RAIU in 8 out of 10 patients. Basal serum Tg was increased in 6 patients and decreased three months later in 2 of them. The post-therapy WBS (pthWBS) showed: 1) additional metastatic lesions in 3 patients with positive dWBS, 2) lung nodular metastases in 1 patient with negative dWBS, 3) similar image as the dWBS in 4 patients, 4) negative image in 1 patient with positive dWBS. RhTSH is a safe and promising method for the stimulation of RAIU in patients with thyroid remnant and/or persistent or recurrent DTC, avoiding L-T 4 withdrawal. (author)

  14. Myelostimulatory activity of recombinant human interleukin-2 in mice

    Energy Technology Data Exchange (ETDEWEB)

    Talmadge, J.E.; Schneider, M.; Keller, J.; Ruscetti, F.; Longo, D.; Pennington, R.; Bowersox, O.; Tribble, H.

    1989-05-01

    In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.

  15. [Construction and expression of recombinant adenovirus containing human catalase gene in vitro].

    Science.gov (United States)

    Ou-Yang, Xiao-ling; Li, Ai-ling; Ning, Qi-lan; Yang, Xu-dong; Xu, Nan; Wang, Hui-lian

    2011-05-01

    To construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro. Total RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods. High expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity. Ad/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.

  16. Recombination analysis and structure prediction show correlation between breakpoint clusters and RNA hairpins in the pol gene of human immunodeficiency virus type 1 unique recombinant forms

    DEFF Research Database (Denmark)

    Galli, Andrea; Lai, Alessia; Corvasce, Stefano

    2008-01-01

    throughout the genome, leading to viral recombination. Some recombination hotspots have been identified and found to correlate with RNA structure or sequence features. The aim of this study was to evaluate the presence of recombination hotspots in the pol gene of HIV-1 and to assess their correlation......Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly...... with the underlying RNA structure. Analysis of the recombination pattern and breakpoint distribution in a group of unique recombinant forms (URFs) detected two recombination hotspots in the pol region. Two stable and conserved hairpins were consistently predicted corresponding to the identified hotspots using six...

  17. Effects of cooling and freezing storage on the stability of bioactive factors in human colostrum.

    Science.gov (United States)

    Ramírez-Santana, C; Pérez-Cano, F J; Audí, C; Castell, M; Moretones, M G; López-Sabater, M C; Castellote, C; Franch, A

    2012-05-01

    Breast milk constitutes the best form of newborn alimentation because of its nutritional and immunological properties. Banked human milk is stored at low temperature, which may produce losses of some bioactive milk components. During lactation, colostrum provides the requirements of the newborn during the first days of life. The aim of this study was to evaluate the effect of cooling storage at 4°C and freezing storage at -20°C and -80°C on bioactive factors in human colostrum. For this purpose, the content of IgA, growth factors such as epidermal growth factor, transforming growth factor (TGF)-β1 and TGF-β2, and some cytokines such as IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α, and its type I receptor TNF-RI, were quantified. Some colostrum samples were stored for 6, 12, 24, and 48 h at 4°C and others were frozen at -20°C or -80°C for 6 and 12 mo. We quantified IgA, epidermal growth factor, TGF-β1, and TGF-β2 by indirect ELISA. Concentrations of IL-6, IL-10, and TNF-α cytokines, IL-8 chemokine, and TNF-RI were measured using the BD Cytometric Bead Array (BD Biosciences, Erembodegem, Belgium). Bioactive immunological factors measured in this study were retained in colostrum after cooling storage at 4°C for at least 48h, with the exception of IL-10. None of the initial bioactive factor concentrations was modified after 6 mo of freezing storage at either -20°C or -80°C. However, freezing storage of colostrum at -20°C and -80°C for 12 mo produced a decrease in the concentrations of IgA, IL-8, and TGF-β1. In summary, colostrum can be stored at 4°C for up to 48 h or at -20°C or -80°C for at least 6 mo without losing its immunological properties. Future studies are necessary to develop quality assurance guidelines for the storage of colostrum in human milk banks, and to focus not only on the microbiological safety but also on the maintenance of the immunological properties of colostrum. Copyright © 2012 American Dairy Science Association

  18. The endogenous GABA bioactivity of camel, bovine, goat and human milks.

    Science.gov (United States)

    Limon, Agenor; Gallegos-Perez, Jose-Luis; Reyes-Ruiz, Jorge M; Aljohi, Mohammad A; Alshanqeeti, Ali S; Miledi, Ricardo

    2014-02-15

    GABA orally administered has several beneficial effects on health, including the regulation of hyperglycaemic states in humans. Those effects are similar to the effects reported for camel milk (CMk); however, it is not known whether compounds with GABAergic activity are present in milk from camels or other species. We determined CMk free-GABA concentration by LS/MS and its bioactivity on human GABA receptors. We found that camel and goat milks have significantly more bioavailable GABA than cow and human milks and are able to activate GABAρ receptors. The relationship between GABA and taurine concentrations suggests that whole camel milk may be more efficient to activate GABAρ1 receptors than goat milk. Because GABAρ receptors are normally found in enteroendocrine cells in the lumen of the digestive tract, these results suggest that GABA in camel and goat milk may participate in GABA-modulated functions of enteroendocrine cells in the GI lumen. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. 76 FR 65210 - Certain Products and Pharmaceutical Compositions Containing Recombinant Human Erythropoetin...

    Science.gov (United States)

    2011-10-20

    ... From the Federal Register Online via the Government Publishing Office INTERNATIONAL TRADE COMMISSION Certain Products and Pharmaceutical Compositions Containing Recombinant Human Erythropoetin... sale within the United States after importation of certain products and pharmaceutical compositions...

  20. Comparison of the Genetic Recombination Rates of Human Immunodeficiency Virus Type 1 in Macrophages and T Cells†

    OpenAIRE

    Chen, Jianbo; Rhodes, Terence D.; Hu, Wei-Shau

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) exhibits a high level of genetic variation generated by frequent mutation and genetic recombination during reverse transcription. We have measured HIV-1 recombination rates in T cells in one round of virus replication. It was recently proposed that HIV-1 recombines far more frequently in macrophages than in T cells. In an attempt to delineate the mechanisms that elevate recombination, we measured HIV-1 recombination rates in macrophages at three dif...

  1. Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells.

    Science.gov (United States)

    Wan, Aini; Xu, Dongsheng; Liu, Kedong; Peng, Lin; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

    2017-08-09

    Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500 µg/L) and the half-life of IGF-1 in blood circulation is only 4.5 min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA-IGF-1 reached 100 mg/L. The fusion protein HSA-IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA-IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA-IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA-IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.

  2. Is the use of recombinant human erythropoietin in anaemia of ...

    African Journals Online (AJOL)

    In a double-blind placebo-eontrolled study we showed a 3-fold decrease in blood transfusions (BTFs) given to preterm infants with anaemia of prematurity who received recombinant erythropoietin. However, only 50% of placebo recipients required a BTF. Data from the placebo group indicated that either mean daily weight ...

  3. Laboratory scale production of the human recombinant iduronate 2 ...

    African Journals Online (AJOL)

    sulfate sulfatase-Like (hIDSLike) was employed for low-scale production of the recombinant enzyme in a saline culture media without phosphate. The biological activity found was between 7.3 and 29.5 nmol h-1 mg-1 of total protein. It is about 1.73 ...

  4. Is the use of recombinant human erythropoietin in anaemia of ...

    African Journals Online (AJOL)

    1996-03-03

    Mar 3, 1996 ... In a double-blind placebo-eontrolled study we showed a. 3-fold decrease in blood transfusions (BTFs) given to preterm infants with anaemia of prematurity who received recombinant erythropoietin. However, only 50% of placebo recipients required a BTF. Data from the placebo group indicated that either ...

  5. Recombinant human CIS2 (SOCS2) protein: subcloning, expression, purification, and characterization.

    Science.gov (United States)

    Biener, Eva; Maurice, Sarah; Sandowski, Yael; Cohen, Yael; Gusakowsky, Eugene E; Hooghe, Robert; Yoshimura, Akihiko; Livnah, Oded; Gertler, Arieh

    2002-07-01

    The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS-PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2.

  6. Structure of recombinant human carboxylesterase 1 isolated from whole cabbage looper larvae

    International Nuclear Information System (INIS)

    Greenblatt, Harry M.; Otto, Tamara C.; Kirkpatrick, Melanie G.; Kovaleva, Elena; Brown, Susan; Buchman, George; Cerasoli, Douglas M.; Sussman, Joel L.

    2012-01-01

    Large quantities of recombinant human carboxylesterase 1 have been produced in an economical whole insect larvae system. The crystal structure of this enzyme is essentially identical to that produced by cell culture techniques. The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 has been produced in and isolated from whole Trichoplusia ni larvae. The recombinant protein was crystallized and its structure was solved to 2.2 Å resolution. The results indicate that the larvae-produced enzyme is essentially identical to that isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies

  7. Short-term effect of recombinant human growth hormone in patients with alcoholic cirrhosis

    DEFF Research Database (Denmark)

    Møller, S; Becker, U; Grønbaek, M

    1994-01-01

    , and liver function. Twenty consecutive patients with cirrhosis were randomized to recombinant human growth hormone (Norditropin, 4 I.U. twice daily) subcutaneously for 6 weeks (n = 10) or conventional medical treatment (n = 10). The serum concentrations of insulin-like growth factor-I in the recombinant...... patients as well as in controls, whereas no change in insulin-like growth factor binding protein-1 concentrations was found. No significant changes were seen in the area under the curve for biochemical liver function tests. We conclude that administration of recombinant human growth hormone induces......As growth hormone possesses anabolic properties that are active on protein metabolism, and thus of potential benefit to patients with chronic liver disease, we determined the metabolic effects of recombinant human growth hormone on insulin-like growth factor-I (IGF-I) its specific binding proteins...

  8. BIOACTIVATION, PROTEIN HAPTENATION, AND TOXICITY OF SULFAMETHOXAZOLE AND DAPSONE IN NORMAL HUMAN DERMAL FIBROBLASTS1

    Science.gov (United States)

    Bhaiya, Payal; Roychowdhury, Sanjoy; Vyas, Piyush M.; Doll, Mark A.; Hein, David W.; Svensson, Craig K.

    2006-01-01

    Cutaneous drug reactions (CDRs) associated with sulfonamides are believed to be mediated through the formation of reactive metabolites that result in cellular toxicity and protein haptenation. We evaluated the bioactivation and toxicity of sulfamethoxazole (SMX) and dapsone (DDS) in normal human dermal fibroblasts (NHDF). Incubation of cells with DDS or its metabolite (D-NOH) resulted in protein haptenation readily detected by confocal microscopy and ELISA. While the metabolite of SMX (S-NOH) haptenated intracellular proteins, adducts were not evident in incubations with SMX. Cells expressed abundant N-acetyltransferase-1 (NAT1) mRNA and activity, but little NAT2 mRNA or activity. Neither NAT1 nor NAT2 protein were detectable. Incubation of NHDF with S-NOH or D-NOH increased reactive oxygen species formation and reduced glutathione content. NHDF were less susceptible to the cytotoxic effect of S-NOH and D-NOH than are keratinocytes. Our studies provide the novel observation that NHDF are able to acetylate both arylamine compounds and bioactivate the sulfone, DDS, giving rise to haptenated proteins. The reactive metabolites of SMX and DDS also provoke oxidative stress in these cells in a time- and concentration-dependent fashion. Further work is needed to determine the role of the observed toxicity in mediating CDRs observed with these agents. PMID:16603214

  9. Low-density lipoprotein oxidation biomarkers in human health and disease and effects of bioactive compounds.

    Science.gov (United States)

    Winklhofer-Roob, Brigitte M; Faustmann, Gernot; Roob, Johannes M

    2017-10-01

    Based on the significance of oxidized low-density lipoprotein (LDL) in health and disease, this review focuses on human studies addressing oxidation of LDL, including three lines of biomarkers, (i) ex vivo LDL resistance to oxidation, a "challenge test" model, (ii) circulating oxidized LDL, indicating the "current in vivo status", and (iii) autoantibodies against oxidized LDL as fingerprints of an immune response to oxidized LDL, along with circulating oxysterols and 4-hydroxynonenal as biomarkers of lipid peroxidation. Lipid peroxidation and oxidized LDL are hallmarks in the development of various metabolic, cardiovascular and other diseases. Changes further occur across life stages from infancy to older age as well as in athletes and smokers. Given their responsiveness to targeted nutritional interventions, markers of LDL oxidation have been employed in a rapidly growing number of human studies for more than 2 decades. There is growing interest in foods, which, besides providing energy and nutrients, exert beneficial effects on human health, such as protection of DNA, proteins and lipids from oxidative damage. Any health claim, however, needs to be substantiated by supportive evidence derived from human studies, using reliable biomarkers to demonstrate such beneficial effects. A large body of evidence has accumulated, demonstrating protection of LDL from oxidation by bioactive food compounds, including vitamins, other micronutrients and secondary plant ingredients, which will facilitate the selection of oxidation biomarkers for future human intervention studies and health claim support. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Evidence of a recombinant wild-type human astrovirus strain from a Kenyan child with gastroenteritis.

    Science.gov (United States)

    Wolfaardt, Marianne; Kiulia, Nicholas M; Mwenda, Jason M; Taylor, Maureen B

    2011-02-01

    A human astrovirus (HAstV) strain from Kenya was characterized by nucleotide sequence analysis. Sequences from open reading frame 1a (ORF1a) clustered with genotype 6/7, those from ORF1b clustered with genotype 3, and those from ORF2 clustered with genotype 2. A recombination point in the ORF1b-ORF2 junction was identified, with a second possible recombination point within the ORF1a region.

  11. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    by recombinant technics to examine the relation between the TKl gene and the TKl protein. In the second part of this investigation a direct expression system for human TKl in E.coli was developed to produce a source of high amounts of TKl, to be able to examine the structure of TKl. The resulting recombinant TKl...... cells and that this modification can not be performed in E.coli....

  12. Human breast tissue disposition and bioactivity of limonene in women with early stage breast cancer

    Science.gov (United States)

    Miller, Jessica A.; Lang, Julie E.; Ley, Michele; Nagle, Ray; Hsu, Chiu-Hsieh; Thompson, Patricia A; Cordova, Catherine; Waer, Amy; Chow, H.-H. Sherry

    2013-01-01

    Limonene is a bioactive food component found in citrus peel oil that has demonstrated chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited forty-three women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for 2 – 6 weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean=41.3 μg/g tissue) while the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P=0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase 3 expression. No significant changes in serum leptin, adiponectin, TGF-β1, IGFBP-3 and IL-6 levels were observed following limonene intervention. There was a small but statistically significant post-intervention increase in IGF-1 levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell cycle arrest and reduced cell proliferation. Further placebo-controlled clinical trials and translational research are warranted to establish limonene’s role for breast cancer prevention or treatment. PMID:23554130

  13. Human breast tissue disposition and bioactivity of limonene in women with early-stage breast cancer.

    Science.gov (United States)

    Miller, Jessica A; Lang, Julie E; Ley, Michele; Nagle, Ray; Hsu, Chiu-Hsieh; Thompson, Patricia A; Cordova, Catherine; Waer, Amy; Chow, H-H Sherry

    2013-06-01

    Limonene is a bioactive food component found in citrus peel oil that has shown chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open-label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited 43 women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for two to six weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean = 41.3 μg/g tissue), whereas the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P = 0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase-3 expression. No significant changes in serum leptin, adiponectin, TGF-β1, insulin-like growth factor binding protein-3 (IGFBP-3), and interleukin-6 (IL-6) levels were observed following limonene intervention. There was a small but statistically significant postintervention increase in insulin-like growth factor I (IGF-I) levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell-cycle arrest and reduced cell proliferation. Furthermore, placebo-controlled clinical trials and translational research are warranted to establish limonene's role for breast cancer prevention or treatment.

  14. A modeling approach to evaluate the balance between bioactivation and detoxification of MeIQx in human hepatocytes

    Directory of Open Access Journals (Sweden)

    Victorien Delannée

    2017-09-01

    Full Text Available Background Heterocyclic aromatic amines (HAA are environmental and food contaminants that are potentially carcinogenic for humans. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx is one of the most abundant HAA formed in cooked meat. MeIQx is metabolized by cytochrome P450 1A2 in the human liver into detoxificated and bioactivated products. Once bioactivated, MeIQx metabolites can lead to DNA adduct formation responsible for further genome instability. Methods Using a computational approach, we developed a numerical model for MeIQx metabolism in the liver that predicts the MeIQx biotransformation into detoxification or bioactivation pathways according to the concentration of MeIQx. Results Our results demonstrate that (1 the detoxification pathway predominates, (2 the ratio between detoxification and bioactivation pathways is not linear and shows a maximum at 10 µM of MeIQx in hepatocyte cell models, and (3 CYP1A2 is a key enzyme in the system that regulates the balance between bioactivation and detoxification. Our analysis suggests that such a ratio could be considered as an indicator of MeIQx genotoxicity at a low concentration of MeIQx. Conclusions Our model permits the investigation of the balance between bioactivation (i.e., DNA adduct formation pathway through the prediction of potential genotoxic compounds and detoxification of MeIQx in order to predict the behaviour of this environmental contaminant in the human liver. It highlights the importance of complex regulations of enzyme competitions that should be taken into account in any further multi-organ models.

  15. Human cytochrome P450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1′-hydroxysafrole

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Bogaards, J.J.P.; Awad, H.M.; Boersma, M.G.; Brand, W.; Fiamegos, Y.C.; Beek, T.A. van; Alink, G.M.; Sudhölter, E.J.R.; Cnubben, N.H.P.; Rietjens, I.M.C.M.

    2004-01-01

    In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1′-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1′-hydroxylation of safrole was characterized in a variety of in vitro

  16. Physiologically based kinetic modeling of bioactivation and detoxification of the alkenylbenzene methyleugenol in human as compared with rat

    NARCIS (Netherlands)

    Al-Subeihi, A.A.; Spenkelink, A.; Punt, A.; Boersma, M.G.; Bladeren, van P.J.; Rietjens, I.

    2012-01-01

    This study defines a physiologically based kinetic (PBK) model for methyleugenol (ME) in human based on in vitro and in silico derived parameters. With the model obtained, bioactivation and detoxification of methyleugenol (ME) at different doses levels could be investigated. The outcomes of the

  17. Evaluation of the interindividual human variation in bioactivation of methyleugenol using physiologically based kinetic modeling and Monte Carlo simulations

    NARCIS (Netherlands)

    Al-Subeihi, A.A.; Alhusainy, W.; Kiwamoto, R.; Spenkelink, A.; Bladeren, van P.J.; Rietjens, I.M.C.M.; Punt, A.

    2015-01-01

    The present study aims at predicting the level of formation of the ultimate carcinogenic metabolite of methyleugenol, 1'-sulfooxymethyleugenol, in the human population by taking variability in key bioactivation and detoxification reactions into account using Monte Carlo simulations. Depending on the

  18. Human cytochrome P450 enzymes of importance for the bioactivation of methyleugenol to the proximate carcinogen 1′-hydroxymethyleugenol

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Bogaards, J.J.P.; Boersma, M.G.; Horst, J.P.F. ter; Awad, H.M.; Fiamegos, Y.C.; Beek, T.A. van; Alink, G.M.; Sudhölter, E.J.R.; Cnubben, N.H.P.; Rietjens, I.M.C.M.

    2006-01-01

    In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1′-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19,

  19. The in vitro metabolism and bioactivation of 1,2-dibromoethane (ethylene dibromide) by human liver.

    Science.gov (United States)

    Wiersma, D A; Schnellmann, R G; Sipes, I G

    1986-09-01

    The nematocide, grain fumigant, and gasoline additive 1,2-dibromoethane (DBE) is both a cellular and a genetic toxin that is metabolically activated in rats and mice by mixed function oxidases (MFO) as well as glutathione 5-transferases (GST). The purpose of this study was to determine whether DBE is similarly metabolized and bioactivated by human liver in vitro. Human liver microsomal and cytosolic metabolism of DBE was monitored by the production of aqueous-soluble metabolites from [14-C]-DBE. Reactive intermediates were detected as irreversibly bound adducts to protein or DNA. 1,2-Dibromoethane was metabolized by human liver cytosolic GST, microsomal GST, and microsomal MFO. Cytosolic GST activity (9 +/- 2 nmol/20 min/mg protein) was about four times greater than the other two activities. Only MFO activity resulted in adducts irreversibly bound to protein (1.5 +/- .4 nmol/20 min/mg protein) and was inhibited by the presence of glutathione. Both MFO and GST activity resulted in irreversibly bound adducts to DNA. Microsomal and cytosolic GST activity each produced about twice as many DNA adducts as microsomal MFO activity. These results suggest that human liver, like rat and mouse liver, metabolizes DBE to aqueous-soluble metabolites by both MFO and GST activity. Furthermore, each of these activities produces reactive metabolites that can irreversibly bind to cellular macromolecules.

  20. Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.

    Science.gov (United States)

    Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

    2008-10-01

    Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.

  1. [Recombinant human parathyroid hormone in the therapy of hypoparathyroidism].

    Science.gov (United States)

    Eremkina, A K; Mokrysheva, N G; Kovaleva, E V; Krupinova, Yu A

    Hypoparathyroidism is an endocrine disease that results from deficiency or complete absence of parathyroid hormone (PTH), a biologically active 84-amino acid polypeptide. Standard therapy for chronic hypoparathyroidism includes oral calcium salts and active vitamin D metabolites and is aimed at maintaining a balance between optimal near-normal serum calcium concentration and normocalcuria. Traditional treatment regimens not always lead to the compensation for calcium and phosphorus metabolism. Until recently, hypoparathyroidism is the only endocrine disorder that has not been treated with the recombinant hormone. To date, two recombinant PTH forms have been synthesized, which can be used as pathogenetic therapy for hypoparathyroidism. This review is dedicated to replacement therapy for hypoparathyroidism, by using both the full-length PTH molecule (1-84) and its shorter, but fully active, PTH form (1-34). This review considers stages in the developmental of hormone replacement therapy for hypoparathyroidism, discusses the most rational dosing regimens, and compares their efficacy and safety, as well as prospects for the development of this area.

  2. Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

    Science.gov (United States)

    Gerashchenko, O L; Zhuravel, E V; Skachkova, O V; Khranovska, N N; Filonenko, V V; Pogrebnoy, P V; Soldatkina, M A

    2013-06-01

    The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.

  3. Tratamiento con eritropoyetina humana recombinante Human recombinant erythropoietin therapy

    Directory of Open Access Journals (Sweden)

    Hugo Donato

    2006-02-01

    Full Text Available La eritropoyetina recombinante (rHuEPO se ha transformado en la citoquina más utilizada terapéuticamente en el mundo. Luego del éxito obtenido en pacientes con insuficiencia renal terminal, se pudo establecer la utilidad de la terapia con rHuEPO para mejorar otras anemias, incluso en pacientes pediátricos y neonatos. El tratamiento o la prevención de la anemia del prematuro mediante el uso de rHuEPO llevó a una significativa reducción en cantidad de transfusiones y en exposición a dadores. Aún debe establecerse una clara definición sobre cuáles niños prematuros deben recibir tratamiento rutinariamente. Otras indicaciones en período neonatal incluyen anemias hiporregenerativas y hemolíticas. La eficacia de la rHuEPO en niños mayores, con excepción de la insuficiencia renal crónica, no ha sido tan exhaustivamente evaluada como en adultos. Mientras que durante los últimos años se han realizado gran cantidad de estudios en adultos con anemia asociada al cáncer o a infección por HIV, permitiendo establecer conclusiones claras sobre su eficacia, sólo escasa cantidad de estudios con pequeño número de pacientes han sido realizados en niños. Hasta la fecha, los resultados sugieren que la terapia con rHuEPO en niños es tan útil como en adultos, pero la realización de estudios aleatorizados prospectivos incluyendo gran número de pacientes es esencial para alcanzar conclusiones definitivas. Los resultados de estudios dirigidos a evaluar la eficacia de la rHuEpo para mantener una dosis adecuada de ribavirina en pacientes en tratamiento por hepatitis C son alentadores. La utilización potencial de los efectos no hemopoyéticos de la rHuEPO en neonatos es un terreno novedoso y apasionante. El rol de la Epo como citoprotector para sistema nervioso central y mucosa intestinal está bajo investigación exhaustiva.Recombinant human erythropoietin (rHuEpo has become the most widely used cytokine in the world. Following the success of

  4. Recombinant human endostatin normalizes tumor vasculature and enhances radiation response in xenografted human nasopharyngeal carcinoma models.

    Directory of Open Access Journals (Sweden)

    Fang Peng

    Full Text Available BACKGROUND: Hypoxic tumor cells can reduce the efficacy of radiation. Antiangiogenic therapy may transiently "normalize" the tumor vasculature to make it more efficient for oxygen delivery. The aim of this study is to investigate whether the recombinant human endostatin (endostar can create a "vascular normalization window" to alleviate hypoxia and enhance the inhibitory effects of radiation therapy in human nasopharyngeal carcinoma (NPC in mice. METHODOLOGY/PRINCIPAL FINDINGS: Transient changes in morphology of tumor vasculature and hypoxic tumor cell fraction in response to endostar were detected in mice bearing CNE-2 and 5-8F human NPC xenografts. Various treatment schedules were tested to assess the influence of endostar on the effect of radiation therapy. Several important factors relevant to the angiogenesis were identified through immunohistochemical staining. During endostar treatment, tumor vascularity decreased, while the basement membrane and pericyte coverage associated with endothelial cells increased, which supported the idea of vessel normalization. Hypoxic tumor cell fraction also decreased after the treatment. The transient modulation of tumor physiology caused by endostar improved the effect of radiation treatment compared with other treatment schedules. The expressions of vascular endothelial growth factor (VEGF, matrix metalloproteinase-2 (MMP-2, MMP-9, and MMP-14 decreased, while the level of pigment epithelium-derived factor (PEDF increased. CONCLUSIONS: Endostar normalized tumor vasculature, which alleviated hypoxia and significantly sensitized the function of radiation in anti-tumor in human NPC. The results provide an important experimental basis for combining endostar with radiation therapy in human NPC.

  5. Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells.

    Science.gov (United States)

    Mestieri, Leticia Boldrin; Gomes-Cornélio, Ana Lívia; Rodrigues, Elisandra Márcia; Salles, Loise Pedrosa; Bosso-Martelo, Roberta; Guerreiro-Tanomaru, Juliane Maria; Tanomaru-Filho, Mário

    2015-10-01

    Mineral Trioxide Aggregate (MTA) is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus. The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs). The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3 and kept in supplemented medium to obtain hDPCs adherent cultures. Cell characterization assays were performed to prove the osteogenic potential. The evaluated materials were: MTA Plus (MTAP); MTA Fillapex (MTAF) and FillCanal (FC). Biocompatibility was evaluated with MTT and Neutral Red (NR) assays, after hDPCs exposure for 24 h to different dilutions of each sealer extract (1:2, 1:3 and 1:4). Unexposed cells were the positive control (CT). Bioactivity was assessed by alkaline phosphatase (ALP) enzymatic assay in cells exposed for one and three days to sealer extracts (1:4 dilution). All data were analyzed by ANOVA and Tukey post-test (p≤0.05%). MTT and NR results showed suitable cell viability rates for MTAP at all dilutions (90-135%). Cells exposed to MTAF and FC (1:2 and 1:4 dilutions) showed significant low viability rate when compared to CT in MTT. The NR results demonstrated cell viability for all materials tested. In MTAP group, the cells ALP activity was similar to CT in one and three days of exposure to the material. MTAF and FC groups demonstrated a decrease in ALP activity when compared to CT at both periods of cell exposure. The hDPCs were suitable for the evaluation of new endodontic materialsin vitro. MTAP may be considered a promising material for endodontic treatments.

  6. Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells

    Directory of Open Access Journals (Sweden)

    Leticia Boldrin MESTIERI

    2015-10-01

    Full Text Available Mineral Trioxide Aggregate (MTA is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus.Objective The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs.Material and Methods The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3 and kept in supplemented medium to obtain hDPCs adherent cultures. Cell characterization assays were performed to prove the osteogenic potential. The evaluated materials were: MTA Plus (MTAP; MTA Fillapex (MTAF and FillCanal (FC. Biocompatibility was evaluated with MTT and Neutral Red (NR assays, after hDPCs exposure for 24 h to different dilutions of each sealer extract (1:2, 1:3 and 1:4. Unexposed cells were the positive control (CT. Bioactivity was assessed by alkaline phosphatase (ALP enzymatic assay in cells exposed for one and three days to sealer extracts (1:4 dilution. All data were analyzed by ANOVA and Tukey post-test (p≤0.05%.Results MTT and NR results showed suitable cell viability rates for MTAP at all dilutions (90-135%. Cells exposed to MTAF and FC (1:2 and 1:4 dilutions showed significant low viability rate when compared to CT in MTT. The NR results demonstrated cell viability for all materials tested. In MTAP group, the cells ALP activity was similar to CT in one and three days of exposure to the material. MTAF and FC groups demonstrated a decrease in ALP activity when compared to CT at both periods of cell exposure.Conclusions The hDPCs were suitable for the evaluation of new endodontic materialsin vitro. MTAP may be considered a promising material for endodontic treatments.

  7. Use of Recombinant Human Erythropoietin in Renal Anemia in Children

    Directory of Open Access Journals (Sweden)

    Habibur Rahman

    2009-11-01

    Full Text Available Erythropoietin is a hormone highly effective as like as natural erythropoietin to maintain target hemoglobin and hematocrit level in renal anemia. Its advantage over blood transfusion has been proved by improving the quality of life and decreasing morbidity and mortality in ESRD patients. Effectiveness of r-erythropoietin depends on absences of infection, inflammation and vitamin deficiency and iron status. Iron supplementation is needed before r-erythropoietin administration and sub-cutaneous rout is better in renal anemia because of slow and sustained releases of r-erythropoietin from the site of administration. Target hemoglobin level is 11-12.5 gm/dl and hematocrit is 33% which can be achieved by this hormone therapy. Key words- Recombinant erythropoietin, renal anemia, end stage renal disease.DOI: 10.3329/bsmmuj.v2i1.3713 BSMMU J 2009; 2(1: 50-53  

  8. Expression and purification of biologically active recombinant human paraoxonase 1 from a Drosophila S2 stable cell line.

    Science.gov (United States)

    Yun, Hyeongseok; Yu, Jiyeon; Kim, Sumi; Lee, Nari; Lee, Jinhee; Lee, Sungrae; Kim, Nam Doo; Yu, Chiho; Rho, Jaerang

    2017-03-01

    Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 10 6  M -1  min -1 ) and paraoxon hydrolysis (0.015 × 10 6  M -1  min -1 ) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Conjugation of gold nanoparticles and recombinant human endostatin modulates vascular normalization via interruption of anterior gradient 2-mediated angiogenesis.

    Science.gov (United States)

    Pan, Fan; Yang, Wende; Li, Wei; Yang, Xiao-Yan; Liu, Shuhao; Li, Xin; Zhao, Xiaoxu; Ding, Hui; Qin, Li; Pan, Yunlong

    2017-07-01

    Several studies have revealed the potential of normalizing tumor vessels in anti-angiogenic treatment. Recombinant human endostatin is an anti-angiogenic agent which has been applied in clinical tumor treatment. Our previous research indicated that gold nanoparticles could be a nanoparticle carrier for recombinant human endostatin delivery. The recombinant human endostatin-gold nanoparticle conjugates normalized vessels, which improved chemotherapy. However, the mechanism of recombinant human endostatin-gold nanoparticle-induced vascular normalization has not been explored. Anterior gradient 2 has been reported to be over-expressed in many malignant tumors and involved in tumor angiogenesis. To date, the precise efficacy of recombinant human endostatin-gold nanoparticles on anterior gradient 2-mediated angiogenesis or anterior gradient 2-related signaling cohort remained unknown. In this study, we aimed to explore whether recombinant human endostatin-gold nanoparticles could normalize vessels in metastatic colorectal cancer xenografts, and we further elucidated whether recombinant human endostatin-gold nanoparticles could interrupt anterior gradient 2-induced angiogenesis. In vivo, it was indicated that recombinant human endostatin-gold nanoparticles increased pericyte expression while inhibit vascular endothelial growth factor receptor 2 and anterior gradient 2 expression in metastatic colorectal cancer xenografts. In vitro, we uncovered that recombinant human endostatin-gold nanoparticles reduced cell migration and tube formation induced by anterior gradient 2 in human umbilical vein endothelial cells. Treatment with recombinant human endostatin-gold nanoparticles attenuated anterior gradient 2-mediated activation of MMP2, cMyc, VE-cadherin, phosphorylation of p38, and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human umbilical vein endothelial cells. Our findings demonstrated recombinant human endostatin-gold nanoparticles might normalize

  10. Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

    Science.gov (United States)

    Fu, Kai; Cheng, Qin; Liu, Zhenwei; Chen, Zhen; Wang, Yan; Ruan, Honggang; Zhou, Lu; Xiong, Jie; Xiao, Ruijing; Liu, Shengwu; Zhang, Qiuping; Yang, Daichang

    2014-01-01

    Human serum albumin (HSA) is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA) before a First-in-human (FIH) trial, we compared OsrHSA and plasma-derived HSA (pHSA), evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs). The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+) and CD8(+) T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

  11. Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Kai Fu

    Full Text Available Human serum albumin (HSA is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA before a First-in-human (FIH trial, we compared OsrHSA and plasma-derived HSA (pHSA, evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs. The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+ and CD8(+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ, tumor necrosis factor-alpha (TNF-α, interleukin (IL-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

  12. Recombination affects accumulation of damaging and disease-associated mutations in human populations.

    Science.gov (United States)

    Hussin, Julie G; Hodgkinson, Alan; Idaghdour, Youssef; Grenier, Jean-Christophe; Goulet, Jean-Philippe; Gbeha, Elias; Hip-Ki, Elodie; Awadalla, Philip

    2015-04-01

    Many decades of theory have demonstrated that, in non-recombining systems, slightly deleterious mutations accumulate non-reversibly, potentially driving the extinction of many asexual species. Non-recombining chromosomes in sexual organisms are thought to have degenerated in a similar fashion; however, it is not clear the extent to which damaging mutations accumulate along chromosomes with highly variable rates of crossing over. Using high-coverage sequencing data from over 1,400 individuals in the 1000 Genomes and CARTaGENE projects, we show that recombination rate modulates the distribution of putatively deleterious variants across the entire human genome. Exons in regions of low recombination are significantly enriched for deleterious and disease-associated variants, a signature varying in strength across worldwide human populations with different demographic histories. Regions with low recombination rates are enriched for highly conserved genes with essential cellular functions and show an excess of mutations with demonstrated effects on health, a phenomenon likely affecting disease susceptibility in humans.

  13. Effects of co-medicated drugs on cyclophosphamide bioactivation in human liver microsomes

    NARCIS (Netherlands)

    de Jonge, Milly E.; Huitema, Alwin D. R.; van Dam, Selma M.; Rodenhuis, Sjoerd; Beijnen, Jos H.

    2005-01-01

    The alkylating agent cyclophosphamide (CP) is a prodrug requiring cytochrome P-450-mediated bioactivation to form the active 4-hydroxycyclophosphamide (4OHCP). Modifications in the rate of CP bioactivation may have implications for the effectiveness of CP therapy, especially in high-dose regimens.

  14. Strawberry Achenes Are an Important Source of Bioactive Compounds for Human Health

    Directory of Open Access Journals (Sweden)

    María Teresa Ariza

    2016-07-01

    Full Text Available Strawberries are highly appreciated for their taste, nutritional value and antioxidant compounds, mainly phenolics. Fruit antioxidants derive from achenes and flesh, but achene contribution to the total fruit antioxidant capacity and to the bioaccessibility after intake is still unknown. In this work, the content of total phenolic compounds, flavonoids, anthocyanins and antioxidant capacity (TEAC, FRAP and DPPH of achenes and flesh were compared in non-digested as well as in gastric and intestinal extracts after in vitro digestion. Results showed that, despite strawberry achenes represent a small fraction of the fruit, their contribution to total fruit antioxidant content was more than 41% and accounted for 81% of antioxidant capacity (TEAC. Achenes have higher quantity and different quality of antioxidants in non-digested and digested extracts. Antioxidant release was higher in the in vitro gastric digested extracts, but digestion conditions did not only affect quantity but quality, resulting in differences in antioxidant capacity and highlighting the importance of simulating physiological-like extraction conditions for assessing fruit antioxidant properties on human health. These results give new insights into the use of strawberry achenes as a source of bioactive compounds to be considered in strawberry breeding programs for improving human health.

  15. Strawberry Achenes Are an Important Source of Bioactive Compounds for Human Health

    Science.gov (United States)

    Ariza, María Teresa; Reboredo-Rodríguez, Patricia; Mazzoni, Luca; Forbes-Hernández, Tamara Yuliett; Giampieri, Francesca; Afrin, Sadia; Gasparrini, Massimiliano; Soria, Carmen; Martínez-Ferri, Elsa; Battino, Maurizio; Mezzetti, Bruno

    2016-01-01

    Strawberries are highly appreciated for their taste, nutritional value and antioxidant compounds, mainly phenolics. Fruit antioxidants derive from achenes and flesh, but achene contribution to the total fruit antioxidant capacity and to the bioaccessibility after intake is still unknown. In this work, the content of total phenolic compounds, flavonoids, anthocyanins and antioxidant capacity (TEAC, FRAP and DPPH) of achenes and flesh were compared in non-digested as well as in gastric and intestinal extracts after in vitro digestion. Results showed that, despite strawberry achenes represent a small fraction of the fruit, their contribution to total fruit antioxidant content was more than 41% and accounted for 81% of antioxidant capacity (TEAC). Achenes have higher quantity and different quality of antioxidants in non-digested and digested extracts. Antioxidant release was higher in the in vitro gastric digested extracts, but digestion conditions did not only affect quantity but quality, resulting in differences in antioxidant capacity and highlighting the importance of simulating physiological-like extraction conditions for assessing fruit antioxidant properties on human health. These results give new insights into the use of strawberry achenes as a source of bioactive compounds to be considered in strawberry breeding programs for improving human health. PMID:27409612

  16. Increased bioactive lipids content in human subcutaneous and epicardial fat tissue correlates with insulin resistance.

    Science.gov (United States)

    Błachnio-Zabielska, Agnieszka U; Baranowski, Marcin; Hirnle, Tomasz; Zabielski, Piotr; Lewczuk, Anna; Dmitruk, Iwona; Górski, Jan

    2012-12-01

    Obesity is a risk factor for metabolic diseases. Intramuscular lipid accumulation of ceramides, diacylglycerols, and long chain acyl-CoA is responsible for the induction of insulin resistance. These lipids are probably implicated in obesity-associated insulin resistance not only in skeletal muscle but also in fat tissue. Only few data are available about ceramide content in human subcutaneous adipose tissue. However, there are no data on DAG and LCACoA content in adipose tissue. The aim of our study was to measure the lipids content in human SAT and epicardial adipose tissue we sought to determine the bioactive lipids content by LC/MS/MS in fat tissue from lean non-diabetic, obese non-diabetic, and obese diabetic subjects and test whether the lipids correlate with HOMA-IR. We found, that total content of measured lipids was markedly higher in OND and OD subjects in both types of fat tissue (for all p lipids content is greater in subcutaneous and epicardial fat tissue and the particular lipids content positively correlates with HOMA-IR.

  17. Humanizing recombinant glycoproteins from Chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Hansen, Anders Holmgaard; Amann, Thomas; Kol, Stefan

    hamster ovary (CHO) cells are making a very heterogeneous mixture of NGlycans. We speculate that the CHO pattern of N-Glycans would affect half-life and/or efficacy of the glycoprotein in the bloodstream making it unsuitable for human intravenous use, whereas our humanized version would be identical...

  18. Effects of recombinant human collagen VI from Escherichia coli on ...

    African Journals Online (AJOL)

    irradiated human skin fibroblasts cells. M Wu, Z Xun, G Wang, Y Sun, G Chen. Abstract. In this study, we reported the cloning and over expression of a gene coding for human collagen peptide (CP6) in Escherichia coli and investigated the protective ...

  19. Effects of recombinant human collagen VI from Escherichia coli on ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... In this study, we reported the cloning and over expression of a gene coding for human collagen peptide. (CP6) in Escherichia coli and investigated the protective effects of CP6 on UVA-irradiated human skin fibroblasts cells. The collagen peptide (CP6) was highly soluble and the expression level was.

  20. Expression of functional recombinant human factor IX in milk of mice.

    Science.gov (United States)

    Lisauskas, Sharon F C; Cunha, Nicolau B; Vianna, Giovanni R; Mendes, Erica A; Ramos, Gustavo L; Maranhão, Andréia Q; Brígido, Marcelo M; Almeida, Jussara O S C; Baptista, Heloisa A; Motta, Fabiana L T; Pesquero, João B; Aragão, Francisco J L; Rech, Elíbio L

    2008-12-01

    Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk beta-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.

  1. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins

    DEFF Research Database (Denmark)

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J.; Shojaosadati, Seyed Abbas

    2016-01-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins...... native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better...... produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address...

  2. Experimental chemonucleolysis with recombinant human matrix metalloproteinase 7 in human herniated discs and dogs.

    Science.gov (United States)

    Haro, Hirotaka; Nishiga, Miyuki; Ishii, Daisuke; Shimomoto, Takasumi; Kato, Tsuyoshi; Takenouchi, Osami; Koyanagi, Satoshi; Ohba, Tetsuro; Komori, Hiromichi

    2014-07-01

    Chemonucleolysis has been proposed as a less invasive technique than surgery for patients with lumbar disc herniation. Once chymopapain had been approved as a chemonucleolysis drug, it was withdrawn because of serious complications. A novel agent with fewer complications would be desirable. The purpose of this study was to investigate the effects of recombinant human matrix metalloproteinase 7 (rhMMP-7) in experimental chemonucleolysis in vitro and in vivo and examine its effects on tissue damage. The study design is the experimental study using human herniated discs and enzyme substrates in vitro and dogs in vivo. The effects of rhMMP-7 on the degradation of human herniated discs were examined by measuring the wet weight in vitro. The correlations between the decrease in wet weight by rhMMP-7 and the conditions associated with herniated discs were also analyzed. The effects of rhMMP-7 on the proteoglycan and water contents were respectively examined with alcian blue staining and T2-weighted magnetic resonance imaging at 7 days after intradiscal injection in dogs. The distribution of [125I]-labeled rhMMP-7 was investigated by autoradioluminography at 7 days after intradiscal injection in dogs. An epidural injection study with rhMMP-7 was performed to evaluate the effects on the tissue damage around the discs at 1 and 13 weeks after the treatment in dogs. The Type 1 and 2 collagen cleavage rates were measured and compared with those of aggrecan in vitro. Recombinant human matrix metalloproteinase 7 concentration dependently decreased the wet weight of herniated discs in vitro. The decrease in wet weight of the discs by rhMMP-7 did not significantly correlate with the conditions associated with herniated discs. Intradiscal injection of rhMMP-7 reduced the proteoglycan and water contents, with an increase in the serum keratan sulfate levels. Radioactivity of [125I]-labeled rhMMP-7 was detected in the nucleus pulposus and annulus fibrosus but not in the muscle

  3. Formulation and process development of (recombinant human) deoxyribonuclease I as a powder for inhalation

    NARCIS (Netherlands)

    Zijlstra, Gerrit S; Ponsioen, Bart J; Hummel, Sylvia A; Sanders, Niek; Hinrichs, Wouter L J; de Boer, Anne H; Frijlink, Henderik W

    2009-01-01

    A formulation and process development study was performed to formulate recombinant human deoxyribonuclease I as a powder for inhalation. First, excipient compatibility (with bovine DNase as a model substance) was examined with a stability study at stressed conditions (60 and 85 degrees C) while

  4. Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli

    NARCIS (Netherlands)

    G.P. Patrinos (George); M. de Krom (Mariken); S. Bottardi; R.J. Janssens; E. Katsantoni (Eleni); A.W. Wai; D.J. Sherratt; F.G. Grosveld (Frank); A.M.A. Imam (Ali)

    2000-01-01

    textabstractWe report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for

  5. Bioconversion of Mono- and Sesquiterpenoids by Recombinant Human Cytochrome P450 Monooxygenases

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Fichera, Mario A.; Malz, Frank; Ebbelaar, Monique; Bos, Rein; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver

    2008-01-01

    Cytochrome P450 monooxygenases play an important role in the biosynthesis and metabolism of terpenoids. We explored the potential of recombinant human liver cytochrome P450 monooxygenases CYP1A2, CYP2C9, and CYP3A4, heterologously expressed in Escherichia coli, to convert mono- and sesquiterpenoids

  6. Immunoglobulin A-specific serodiagnosis of acute human cytomegalovirus infection by using recombinant viral antigens

    NARCIS (Netherlands)

    Vornhagen, R; Hinderer, W; Sonneborn, HH; Bein, G; Matter, L; The, TH; Jahn, G; Plachter, B

    The immunoglobulin A-specific reactivities of recombinant viral proteins from nine different reading frames of human cytomegalovirus were evaluated in enzyme-linked immunosorbent assay experiments. Antigen fragments of reading frames pUL32, pUL44, and pUL57 were identified as preferable antigens for

  7. Human recombinant protein C for severe sepsis and septic shock in adult and paediatric patients

    DEFF Research Database (Denmark)

    Martí-Carvajal, Arturo J; Solà, Ivan; Gluud, Christian

    2012-01-01

    Sepsis is a common and frequently fatal condition. Human recombinant activated protein C (APC) has been introduced to reduce the high risk of death associated with severe sepsis or septic shock. This systematic review is an update of a Cochrane review originally published in 2007....

  8. Recombinant human C1-inhibitor in the treatment of acute angioedema attacks

    NARCIS (Netherlands)

    Choi, Goda; Soeters, Maarten R.; Farkas, Henriette; Varga, Lilian; Obtulowicz, Krystyna; Bilo, Barbara; Porebski, Greg; Hack, C. Erik; Verdonk, Rene; Nuijens, Jan; Levi, Marcel

    2007-01-01

    BACKGROUND: Patients with hereditary C1-inhibitor deficiency have recurrent attacks of angioedema, preferably treated with C1-inhibitor concentrate. A recombinant human C1-inhibitor (rHuC1INH) was developed, derived from milk from transgenic rabbits. This study was undertaken to investigate the

  9. Recombinant human activated protein C: current insights into its mechanism of action

    NARCIS (Netherlands)

    Levi, Marcel; van der Poll, Tom

    2007-01-01

    Impairment of the protein C pathway plays a central role in the pathogenesis of sepsis. Administration of recombinant human activated protein C (rhAPC) may correct the dysregulated anticoagulant mechanism and prevent propagation of thrombin generation and formation of microvascular thrombosis.

  10. Intraarticular Sprifermin (Recombinant Human Fibroblast Growth Factor 18) in Knee Osteoarthritis

    DEFF Research Database (Denmark)

    Lohmander, L. S.; Hellot, S.; Dreher, D.

    2014-01-01

    Objective. To evaluate the efficacy and safety of intraarticular sprifermin (recombinant human fibroblast growth factor 18) in the treatment of symptomatic knee osteoarthritis (OA). Methods. The study was a randomized, double-blind, placebo-controlled, proof-of-concept trial. Intraarticular...

  11. Short-term effect of recombinant human growth hormone in patients with alcoholic cirrhosis

    DEFF Research Database (Denmark)

    Møller, S; Becker, U; Grønbaek, M

    1994-01-01

    As growth hormone possesses anabolic properties that are active on protein metabolism, and thus of potential benefit to patients with chronic liver disease, we determined the metabolic effects of recombinant human growth hormone on insulin-like growth factor-I (IGF-I) its specific binding proteins...

  12. Structural Evolution of Human Recombinant alfaB-Crystallin under UV Irradiation

    DEFF Research Database (Denmark)

    Sugiyama, Masaaki; Fujii, Noriko; Morimoto, Yukio

    2008-01-01

    External stresses cause certain proteins to lose their regular structure and aggregate. In order to clarify this abnormal aggregation process, a structural evolution of human recombinant aB-crystallin under UV irradiation was observed with in situ small-angle neutron scattering. The abnormal...

  13. Screening of formulation parameters for stabilizing recombinant human serum albumin (rHSA) in liquid formulations

    DEFF Research Database (Denmark)

    Baldursdottir, Stefania; Tauhaybeche, Mariam; Pekka Pajander, Jari

    2016-01-01

    In the present study, the stabilizing effect of excipients present in recombinant Human Serum Albumin (rHSA) solutions are studied using rheological analysis. The study was conducted using Design of Experiment (DoE) principles and multivariate analysis approach. It is observed that the excipients...

  14. Radioiodine therapy of benign non-toxic goitre. Potential role of recombinant human TSH

    DEFF Research Database (Denmark)

    Fast, S; Bonnema, S J; Hegedüs, L

    2011-01-01

    This review provides an update on recombinant human TSH (rh-TSH) augmented radioiodine (¹³¹I) therapy and outlines its potential role in the treatment of symptomatic benign multinodular non-toxic goitre. In some countries, ¹³¹I has been used for three decades to reduce the size of nodular goitres...

  15. How bio-questionable are the different recombinant human erythropoietin copy products in Thailand?

    NARCIS (Netherlands)

    Halim, Liem Andhyk|info:eu-repo/dai/nl/412608294; Brinks, Vera|info:eu-repo/dai/nl/31395979X; Jiskoot, Wim; Romeijn, Stefan; Praditpornsilpa, Kearkiat; Assawamakin, Anunchai; Schellekens, Huub|info:eu-repo/dai/nl/068406762

    2014-01-01

    PURPOSE: The high prevalence of pure red cell aplasia in Thailand has been associated with the sharp increase in number of recombinant human erythropoietin (rhEPO) copy products, based on a classical generic regulatory pathway, which have entered the market. This study aims to assess the quality of

  16. RECOMBINANT HUMAN INTERLEUKIN-6 INDUCES A RAPID AND REVERSIBLE ANEMIA IN CANCER-PATIENTS

    NARCIS (Netherlands)

    NIEKEN, J; MULDER, NH; VELLENGA, E; LIMBURG, PC; PIERS, DA; DEVRIES, EGE

    1995-01-01

    Initial studies have shown that recombinant human interleukin-6 (rhIL-6) induces anemia. Until now, the pathophysiologic mechanism of this induced anemia has been unknown. To unravel the underlying mechanism, we examined 15 cancer patients receiving rhIL-6 as an antitumor immunotherapy in a phase II

  17. Recombinant human DNase nebulisation in children with cystic fibrosis: Before bedtime or after waking up?

    NARCIS (Netherlands)

    L.J. van der Giessen (Lianne); R.A.A.M. Gosselink (Rik); W.C.J. Hop (Wim); H.A.W.M. Tiddens (Harm)

    2007-01-01

    textabstractThe present study focused on patients with cystic fibrosis (CF), who were on maintenance therapy with recombinant human deoxyribonuclease (rhDNase), with the aim of comparing efficacy and possible side effects of nebulisation of rhDNase when taken before bedtime with efficacy and side

  18. A Child with Local Lipohypertrophy following Recombinant Human Growth Hormone Administration

    NARCIS (Netherlands)

    Koppen, Ilan J. N.; Bakx, Roel; de Kruiff, Chris C.; van Trotsenburg, A. S. Paul

    2016-01-01

    Local lipohypertrophy due to recombinant human growth hormone (rhGH) administration is a rare phenomenon. Here, we report a case of an 11-year-old girl who presented with a paraumbilical swelling, approximately one year after the start of rhGH treatment for short stature due to the presumed

  19. Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds without supplemental osteogenic growth factors.

    Science.gov (United States)

    Polini, Alessandro; Pisignano, Dario; Parodi, Manuela; Quarto, Rodolfo; Scaglione, Silvia

    2011-01-01

    The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.

  20. Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds without supplemental osteogenic growth factors.

    Directory of Open Access Journals (Sweden)

    Alessandro Polini

    Full Text Available The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL and nano-sized hydroxyapatite (HA or beta-tricalcium phosphate (TCP, which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP, an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2 and bone sialoprotein (BSP, in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.

  1. Human Coronary Artery Smooth Muscle Cell Responses to Bioactive Polyelectrolyte Multilayer Interfaces

    Directory of Open Access Journals (Sweden)

    Robert G. Newcomer

    2011-01-01

    Full Text Available Under normal physiological conditions, mature human coronary artery smooth muscle cells (hCASMCs exhibit a “contractile” phenotype marked by low rates of proliferation and protein synthesis, but these cells possess the remarkable ability to dedifferentiate into a “synthetic” phenotype when stimulated by conditions of pathologic stress. A variety of polyelectrolyte multilayer (PEMU films are shown here to exhibit bioactive properties that induce distinct responses from cultured hCASMCs. Surfaces terminated with Nafion or poly(styrenesulfonic acid (PSS induce changes in the expression and organization of intracellular proteins, while a hydrophilic, zwitterionic copolymer of acrylic acid and 3-[2-(acrylamido-ethyl dimethylammonio] propane sulfonate (PAA-co-PAEDAPS is resistant to cell attachment and suppresses the formation of key cytoskeletal components. Differential expression of heat shock protein 90 and actin is observed, in terms of both their magnitude and cellular localization, and distinct cytoplasmic patterns of vimentin are seen. The ionophore A23187 induces contraction in confluent hCASMC cultures on Nafion-terminated surfaces. These results demonstrate that PEMU coatings exert direct effects on the cytoskeletal organization of attaching hCASMCs, impeding growth in some cases, inducing changes consistent with phenotypic modulation in others, and suggesting potential utility for PEMU surfaces as a coating for coronary artery stents and other implantable medical devices.

  2. Crystallization and preliminary crystallographic analysis of recombinant human galectin-1

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Stacy A. [Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222 (Australia); Scott, Ken [School of Biological Sciences, University of Auckland, Auckland (New Zealand); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222 (Australia)

    2007-11-01

    Human galectin-1 has been cloned, expressed in E. coli, purified and crystallized in the presence of both lactose (ligand) and β-mercaptoethanol under six different conditions. The X-ray diffraction data obtained have enabled the assignment of unit-cell parameters for two novel crystal forms of human galectin-1. Galectin-1 is considered to be a regulator protein as it is ubiquitously expressed throughout the adult body and is responsible for a broad range of cellular regulatory functions. Interest in galectin-1 from a drug-design perspective is founded on evidence of its overexpression by many cancers and its immunomodulatory properties. The development of galectin-1-specific inhibitors is a rational approach to the fight against cancer because although galectin-1 induces a plethora of effects, null mice appear normal. X-ray crystallographic structure determination will aid the structure-based design of galectin-1 inhibitors. Here, the crystallization and preliminary diffraction analysis of human galectin-1 crystals generated under six different conditions is reported. X-ray diffraction data enabled the assignment of unit-cell parameters for crystals grown under two conditions, one belongs to a tetragonal crystal system and the other was determined as monoclinic P2{sub 1}, representing two new crystal forms of human galectin-1.

  3. Limited infection upon human exposure to a recombinant raccoon pox vaccine vector.

    Science.gov (United States)

    Rocke, Tonie E; Dein, F Joshua; Fuchsberger, Martina; Fox, Barry C; Stinchcomb, Dan T; Osorio, Jorge E

    2004-07-29

    A laboratory accident resulted in human exposure to a recombinant raccoon poxvirus (RCN) developed as a vaccine vector for antigens of Yersinia pestis for protection of wild rodents (and other animals) against plague. Within 9 days, the patient developed a small blister that healed within 4 weeks. Raccoon poxvirus was cultured from the lesion, and the patient developed antibody to plague antigen (F1) and RCN. This is the first documented case of human exposure to RCN.

  4. Comparison of pituitary and recombinant human thyrotropin standards in an immunoradiometric system

    International Nuclear Information System (INIS)

    Blanca Fernandez, Silvia; Rodriguez Gonzalez, Julio Cesar; Nisembaum Alas, Amaparo; Sevy Gonzalez, O.

    1998-01-01

    Results of two standards of human thyrotropin of pituitaries (B) and recombinant (C) origen supplied by the Instituto of pesquisas Energeticas y Nucleares, Brazil, were compared in our immunoradiometric reference system that use an human thyrotropin pituitary standard of local production (A). This work was supported by the International Atomic Energy Agency for an inter-regional comparison and set up of a reference standard

  5. Expression of Recombinant Streptokinase from Streptococcus Pyogenes and its Reaction with Infected Human and Murine Sera

    Science.gov (United States)

    Molaee, Neda; Abtahi, Hamid; Mosayebi, Ghasem

    2013-01-01

    Objective(s): Streptokinase (SKa) is an antigenic protein which is secreted by Streptococcus pyogenes. Streptokinase induces inflammation by complement activation, which may play a role in post infectious diseases. In the present study, recombinant streptokinase from S. pyogenes was produced and showed that recombinant SKa protein was recognized by infected human sera using Western blot analysis. Materials and Methods: In this study, the ska gene from S. pyogenes was amplified and cloned into pET32a which is a prokaryotic expression vector. pET32a-ska was transformed to Escherichia coli BL21 (DE3) pLysS and gene expression was induced by IPTG. Protein production was improved by modification of composition of the bacterial culture media and altering the induction time by IPTG. The expressed protein was purified by affinity chromatography using the Ni-NTA resin. The integrity of the product was confirmed by Westernblot analysis using infected mice. Serum reactivity of five infected individuals was further analyzed against the recombinant SKa protein. Results: Data indicated that recombinant SKa protein from S. pyogenes can be recognized by patient and mice sera. The concentration of the purified recombinant protein was 3.2 mg/L of initial culture. The highest amount of the expressed protein after addition of IPTG was obtained in a bacterial culture without glucose with the culture optical density of 0.8 (OD600 = 0.8). Conclusion : Present data shows, recombinant SKa protein has same epitopes with natural form of this antigen. Recombinant SKa also seemed to be a promising antigen for the serologic diagnosis of S. pyogenes infections. PMID:24171077

  6. Expression of Recombinant Streptokinase from Streptococcus Pyogenes and Its Reaction with Infected Human and Murine Sera

    Directory of Open Access Journals (Sweden)

    Neda Molaee

    2013-09-01

    Full Text Available   Objective(s: Streptokinase (SKa is an antigenic protein which is secreted by Streptococcus pyogenes. Streptokinase induces inflammation by complement activation, which may play a role in post infectious diseases. In the present study, recombinant streptokinase from S. pyogenes was produced and showed that recombinant SKa protein was recognized by infected human sera using Western blot analysis.   Materials and Methods: In this study, the ska gene from S. pyogenes was amplified and cloned into pET32a which is a prokaryotic expression vector. pET32a-ska was transformed to Escherichia coli BL21 (DE3 pLysS and gene expression was induced by IPTG. Protein production was improved by modification of composition of the bacterial culture media and altering the induction time by IPTG. The expressed protein was purified by affinity chromatography using the Ni-NTA resin. The integrity of the product was confirmed by Westernblot analysis using infected mice. Serum reactivity of five infected individuals was further analyzed against the recombinant SKa protein. Results: Data indicated that recombinant SKa protein from S. pyogenes can be recognized by patient and mice sera. The concentration of the purified recombinant protein was 3.2 mg/L of initial culture. The highest amount of the expressed protein after addition of IPTG was obtained in a bacterial culture without glucose with the culture optical density of 0.8 (OD600 = 0.8. Conclusion : Present data shows, recombinant SKa protein has same epitopes with natural form of this antigen. Recombinant SKa also seemed to be a promising antigen for the serologic diagnosis of S. pyogenes infections.

  7. Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Julie Bomholt

    Full Text Available In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

  8. Anti-VDAC3 recombinant antibody decreased human sperm motility and membrane integrity: A potential spermicide for contraception

    Directory of Open Access Journals (Sweden)

    Asmarinah

    2017-11-01

    Full Text Available Objective: To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3 protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded. Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly. Conclusions: Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

  9. [How safe is the recombinant human growth hormone?

    Science.gov (United States)

    Calzada-León, Raúl

    2017-01-01

    In this paper, several aspects related to the safety of the use of biosynthetic human growth hormone are reviewed. For example, its classification as a biosynthetic drug, the phases that need to be performed in Mexico to verify its safety (obtaining, purification, preclinical studies, clinical trials, and finally observational clinical studies), as well as the evidence that exists in relation to the association of intracranial hypertension, muscular events, scoliosis, slipped capital femoral epiphysis, obstructive sleep apnea, pancreatitis, alterations in cortisol, thyroid hormones alterations, cardiovascular disease, metabolic risk, mortality and cancer, adverse events not related to its use, and finally dosing and safety.

  10. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Hélix-Nielsen, Claus; Scharff-Poulsen, Peter

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction...... and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes....

  11. [Construction and expression of recombinant human serum albumin-EPO fusion protein].

    Science.gov (United States)

    Huang, Ying-Chun; Gou, Xing-Hua; Han, Lei; Li, De-Hua; Zhao, Lan-Ying; Wu, Qia-Qing

    2011-05-01

    OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

  12. A novel multi-epitope recombined protein for diagnosis of human brucellosis.

    Science.gov (United States)

    Yin, Dehui; Li, Li; Song, Xiuling; Li, Han; Wang, Juan; Ju, Wen; Qu, Xiaofeng; Song, Dandan; Liu, Yushen; Meng, Xiangjun; Cao, Hongqian; Song, Weiyi; Meng, Rizeng; Liu, Jinhua; Li, Juan; Xu, Kun

    2016-05-21

    In epidemic regions of the world, brucellosis is a reemerging zoonosis with minimal mortality but is a serious public hygiene problem. Currently, there are various methods for brucellosis diagnosis, however few of them are available to be used to diagnose, especially for serious cross-reaction with other bacteria. To overcome this disadvantage, we explored a novel multi-epitope recombinant protein as human brucellosis diagnostic antigen. We established an indirect enzyme-linked immunosorbent assay (ELISA) based on this recombinant protein. 248 sera obtained from three different groups including patients with brucellosis (146 samples), non-brucellosis patients (82 samples), and healthy individuals (20 samples) were tested by indirect ELISA. To evaluate the assay, a receiver-operating characteristic (ROC) analysis and immunoblotting were carried out using these characterized serum samples. For this test, the area under the ROC curve was 0.9409 (95 % confidence interval, 0.9108 to 0.9709), and a sensitivity of 88.89 % and a specificity of 85.54 % was given with a cutoff value of 0.3865 from this ROC analysis. The Western blot results indicate that it is feasible to differentiate human brucellosis and non-brucellosis with the newly established method based on this recombinant protein. Our results obtained high diagnostic accuracy of the ELISA assay which encourage the use of this novel recombinant protein as diagnostic antigen to implement serological diagnosis of brucellosis.

  13. Recombinant expression of homodimeric 660 kDa human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin.

    Science.gov (United States)

    Powell, Rebecca; Hudson, Laura C; Lambirth, Kevin C; Luth, Diane; Wang, Kan; Bost, Kenneth L; Piller, Kenneth J

    2011-07-01

    Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex proteins that are currently difficult or impossible to express using traditional plant and non-plant-based host systems. To test this hypothesis, we transformed soybeans with a synthetic gene encoding human thyroglobulin (hTG)-a 660 kDa homodimeric protein that is widely used in the diagnostic industry for screening and detection of thyroid disease. In the absence of a recombinant system that can produce recombinant hTG, research and diagnostic grade hTG continues to be purified from cadaver and surgically removed thyroid tissue. These less-than-ideal tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified hTG is used in sensitive ELISA screens. In this study, we report the successful expression of recombinant hTG in soybean seeds. Authenticity of the soy-derived protein was demonstrated using commercial ELISA kits developed specifically for the detection of hTG in patient sera. Western analyses and gel filtration chromatography demonstrated that recombinant hTG and thyroid-purified hTG are biologically similar with respect to size, mass, charge and subunit interaction. The recombinant protein was stable over three generations and accumulated to ~1.5% of total soluble seed protein. These results support our hypothesis that soybeans represent a practical alternative to traditional host systems for the expression of large and complex proteins.

  14. Bioactive Ti metal analogous to human cancellous bone: Fabrication by selective laser melting and chemical treatments.

    Science.gov (United States)

    Pattanayak, Deepak K; Fukuda, A; Matsushita, T; Takemoto, M; Fujibayashi, S; Sasaki, K; Nishida, N; Nakamura, T; Kokubo, T

    2011-03-01

    Selective laser melting (SLM) is a useful technique for preparing three-dimensional porous bodies with complicated internal structures directly from titanium (Ti) powders without any intermediate processing steps, with the products being expected to be useful as a bone substitute. In this study the necessary SLM processing conditions to obtain a dense product, such as the laser power, scanning speed, and hatching pattern, were investigated using a Ti powder of less than 45 μm particle size. The results show that a fully dense plate thinner than 1.8 mm was obtained when the laser power to scanning speed ratio was greater than 0.5 and the hatch spacing was less than the laser diameter, with a 30 μm thick powder layer. Porous Ti metals with structures analogous to human cancellous bone were fabricated and the compressive strength measured. The compressive strength was in the range 35-120 MPa when the porosity was in the range 75-55%. Porous Ti metals fabricated by SLM were heat-treated at 1300 °C for 1h in an argon gas atmosphere to smooth the surface. Such prepared specimens were subjected to NaOH, HCl, and heat treatment to provide bioactivity. Field emission scanning electron micrographs showed that fine networks of titanium oxide were formed over the whole surface of the porous body. These treated porous bodies formed bone-like apatite on their surfaces in a simulated body fluid within 3 days. In vivo studies showed that new bone penetrated into the pores and directly bonded to the walls within 12 weeks after implantation into the femur of Japanese white rabbits. The percentage bone affinity indices of the chemical- and heat-treated porous bodies were significantly higher than that of untreated implants. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. Complete genome sequence analysis of novel human bocavirus reveals genetic recombination between human bocavirus 2 and human bocavirus 4.

    Science.gov (United States)

    Khamrin, Pattara; Okitsu, Shoko; Ushijima, Hiroshi; Maneekarn, Niwat

    2013-07-01

    Epidemiological surveillance of human bocavirus (HBoV) was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. By partial sequence analysis of VP1 gene, an unusual strain of HBoV (CMH-S011-11), was initially identified as HBoV4. The complete genome sequence of CMH-S011-11 was performed and analyzed further to clarify whether it was a recombinant strain or a new HBoV variant. Analysis of complete genome sequence revealed that the coding sequence starting from NS1, NP1 to VP1/VP2 was 4795 nucleotides long. Interestingly, the nucleotide sequence of NS1 gene of CMH-S011-11 was most closely related to the HBoV2 reference strains detected in Pakistan, which contradicted to the initial genotyping result of the partial VP1 region in the previous study. In addition, comparison of NP1 nucleotide sequence of CMH-S011-11 with those of other HBoV1-4 reference strains also revealed a high level of sequence identity with HBoV2. On the other hand, nucleotide sequence of VP1/VP2 gene of CMH-S011-11 was most closely related to those of HBoV4 reference strains detected in Nigeria. The overall full-length sequence analysis revealed that this CMH-S011-11 was grouped within HBoV4 species, but located in a separate branch from other HBoV4 prototype strains. Recombination analysis revealed that CMH-S011-11 was the result of recombination between HBoV2 and HBoV4 strains with the break point located near the start codon of VP2. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

    Science.gov (United States)

    Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano

    2017-10-01

    Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. [Safety of human papillomavirus 6, 11, 16 and 18 (recombinant): systematic review and meta-analysis].

    Science.gov (United States)

    Coelho, Pedro Luiz Spinelli; da Silva Calestini, Gustavo Lacerda; Alvo, Fernando Salgueiro; de Moura Freitas, Jefferson Michel; Castro, Paula Marcela Vilela; Konstantyner, Tulio

    2015-12-01

    To identify and quantify the adverse effects associated with the recombinant human papillomavirus (types 6, 11, 16 and 18) vaccine in adolescents. Systematic review of randomized clinical trials from PubMed, SciELO and Lilacs databases. Articles investigating the safety of the vaccine in subjects under 18 years and comparing the recombinant human papillomavirus types 6, 11, 16 and 18 vaccine with a control group were included. Meta-analyses were performed for the outcomes of pain, erythema, swelling and fever, using clinical trials with maximum Jadad score. Fourteen studies were included. The most commons adverse effects related to the human papillomavirus vaccine were effects with no severity (pain, erythema, edema, and fever). Five studies were used for the meta-analyses: Pain-Risk Difference (RD)=11% (p<0.001); edema-RD=8% (p<0.001); erythema-RD=5% (p<0.001); fever-RD=2% (p<0.003). The recombinant human papillomavirus types 6, 11, 16 and 18 vaccine was safe and well tolerated. The main adverse effects related to vaccination were pain, erythema, edema and fever. The low frequency of severe adverse effects encourages the administration of the vaccine in the population at risk. Copyright © 2015 Sociedade de Pediatria de São Paulo. Publicado por Elsevier Editora Ltda. All rights reserved.

  18. Protection of non-human primates against rabies with an adenovirus recombinant vaccine.

    Science.gov (United States)

    Xiang, Z Q; Greenberg, L; Ertl, H C; Rupprecht, C E

    2014-02-01

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Protection of Non-Human Primates against Rabies with an Adenovirus Recombinant Vaccine

    Science.gov (United States)

    Xiang, Z.Q.; Greenberg, L.; Ertl, H. C.; Rupprecht, C.E.

    2014-01-01

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. PMID:24503087

  20. The interaction of recombinant tissue type plasminogen activator and recombinant plasminogen activator (r-PA/BM 06.022) with human endothelial cells

    NARCIS (Netherlands)

    Mulder, M.; Kohnert, U.; Fischer, S.; Hinsbergh, V.W.M. van; Verheijen, J.H.

    1997-01-01

    The Escherichia coli-expressed recombinant plasminogen activator (r-PA) comprising the kringle 2 and protease domains of human tissue-type plasminogen activator (t-PA) has a four-fold longer half-life time in the circulation than t-PA, possibly resulting in an increased opportunity for r-PA to

  1. Expression of human β-defensin-1 in recombinant Escherichia coli ...

    African Journals Online (AJOL)

    Escherichia coli BL21(DE3) was transformed with a pHCD1 plasmid harboring the human β-defensin-1 (hBD1) gene fused in frame behind a disulfide bond isomerase (DsbC), a His-tag, and an enterokinase cleavage site. After induction, the DsbC-hBD1 was expressed as a ~36 kDa soluble fusion protein in recombinant E.

  2. Mechanical Property Characterization of Electrospun Recombinant Human Tropoelastin for Vascular Graft Biomaterials

    OpenAIRE

    McKenna, Kathryn A.; Hinds, Monica T.; Sarao, Rebecca C.; Wu, Ping-Cheng; Maslen, Cheryl L.; Glanville, Robert W.; Babcock, Darcie; Gregory, Kenton W.

    2011-01-01

    The development of vascular grafts has focused on finding a biomaterial that is non-thrombogenic, minimizes intimal hyperplasia, matches the mechanical properties of native vessels and allows for regeneration of arterial tissue. In this study, the structural and mechanical properties and the vascular cell compatibility of electrospun recombinant human tropoelastin (rTE) were evaluated as a potential vascular graft support matrix. Disuccinimidyl suberate (DSS) was used to cross-link electrospu...

  3. Bioactive substances

    Digital Repository Service at National Institute of Oceanography (India)

    Wahidullah, S.

    Chemistry related to certain bioactive molecules, from Indian Ocean Region, developed into drugs or which served as models for the synthesis of more effective bioactive substances or in use in fundamental studies of physiological and biochemical...

  4. Refined genetic maps reveal sexual dimorphism in human meiotic recombination at multiple scales

    Science.gov (United States)

    Bhérer, Claude; Campbell, Christopher L.; Auton, Adam

    2017-04-01

    In humans, males have lower recombination rates than females over the majority of the genome, but the opposite is usually true near the telomeres. These broad-scale differences have been known for decades, yet little is known about differences at the fine scale. By combining data sets, we have collected recombination events from over 100,000 meioses and have constructed sex-specific genetic maps at a previously unachievable resolution. Here we show that, although a substantial fraction of the genome shows some degree of sexually dimorphic recombination, the vast majority of hotspots are shared between the sexes, with only a small number of putative sex-specific hotspots. Wavelet analysis indicates that most of the differences can be attributed to the fine scale, and that variation in rate between the sexes can mostly be explained by differences in hotspot magnitude, rather than location. Nonetheless, known recombination-associated genomic features, such as THE1B repeat elements, show systematic differences between the sexes.

  5. [Recombinant human gapM1 expressed in Pichia pastoris and its anti-diabetic effect].

    Science.gov (United States)

    Mei, Xiang; Du, Renqian; Li, Xi; Huang, Haiyan; Yu, Min; Tang, Qiqun

    2009-08-01

    Adiponectin is an adipokine predominantly synthesized and secreted by adipocytes in the white adipose tissue, and it can lower the blood glucose level and increase free fatty acid oxidation. In the current study, we developed the globular domain of adiponectin (gapM1) to treat type II diabetes. In both flask and fermentor, we cultivated Pichia pastoris expressing recombinant gapM1 and established the purification procedure by using gel filtration and anion exchange chromatography. To evaluate the biological activity of recombinant gapM1, we used rat type II diabetes model fed high-fat diet in combination with low-dose STZ (Streptozocin) induction. We purified 200 mg gapM1 with purity of 96% from 10 liters of supernatant. The recombinant gapM1 significantly lowered blood glucose (34.2%), serum triglyceride (79.6%) and total cholesterol (62.1%) in type II diabetes induced rat. Therefore, the recombinant human gapM1 is successfully expressed in Pichia pastoris and effectively treated type II diabetes in rat models.

  6. Genetic recombination within the human T-cell receptor α-chain gene complex

    International Nuclear Information System (INIS)

    Robinson, M.A.; Kindt, T.J.

    1987-01-01

    Genetic analyses of the human T-cell receptor (TCR) α-chain genes indicate that recombination events may occur frequently within this gene complex. Examination of the inheritance of restriction fragment length polymorphisms (RFLP) detected by using probes for constant or variable region gene segments made it possible to assign TCRα haplotypes to the 16 parents and 43 offspring of eight families studied. A total of six RFLP, three for the constant region and three for variable region segments, were examined in the present studies. Most enzyme and probe combinations tested revealed no polymorphism and those finally selected for the study showed limited polymorphism in that only two or, in one case, three allelic forms of the gene were seen. In spite of limited variability at this level, extensive heterogeneity was observed for the combinations of markers present in haplotypes, suggesting that frequent recombination events have occurred. Most strikingly, multiple combinations of RFLP occurring in close proximity of the TCRα constant region gene were observed in this study. A high recombination frequency for the TCRα gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCRα haplotypes

  7. Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in alloimmunized baboons.

    Science.gov (United States)

    Tillou, Xavier; Poirier, Nicolas; Le Bas-Bernardet, Stéphanie; Hervouet, Jeremy; Minault, David; Renaudin, Karine; Vistoli, Fabio; Karam, Georges; Daha, Mohamed; Soulillou, Jean Paul; Blancho, Gilles

    2010-07-01

    Acute antibody-mediated rejection is an unsolved issue in transplantation, especially in the context of pretransplant immunization. The deleterious effect of preformed cytotoxic anti-HLA antibodies through complement activation is well proven, but very little is known concerning complement blockade to prevent/cure this rejection. Here, we used a baboon model of preimmunization to explore the prevention of acute antibody-mediated rejection by an early inhibition of the classical complement pathway using human recombinant C1-inhibitor. Baboons were immunized against peripheral blood mononuclear cells from allogeneic donors and, once a specific and stable immunization had been established, they received a kidney from the same donor. Rejection occurred at day 2 posttransplant in untreated presensitized recipients, with characteristic histological lesions and complement deposition. As recombinant human C1-inhibitor blocks in vitro cytotoxicity induced by donor-specific antibodies, other alloimmunized baboons received the drug thrice daily intravenously during the first 5 days after transplant. Rejection was prevented during this treatment but occurred after discontinuation of treatment. We show here that early blockade of complement activation by recombinant human C1-inhibitor can prevent acute antibody-mediated rejection in presensitized recipients. This treatment could also be useful in other forms of acute antibody-mediated rejection caused by induced antibodies.

  8. Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

    Science.gov (United States)

    Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A

    1998-06-01

    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

  9. High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration.

    Science.gov (United States)

    Wang, Qi; Liu, Yongdong; Zhang, Chun; Guo, Fangxia; Feng, Cui; Li, Xiunan; Shi, Hong; Su, Zhiguo

    2017-05-01

    Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5-4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Milk derived bioactive peptides and their impact on human health – A review

    Directory of Open Access Journals (Sweden)

    D.P. Mohanty

    2016-09-01

    Full Text Available Milk-derived bioactive peptides have been identified as potential ingredients of health-promoting functional foods. These bioactive peptides are targeted at diet-related chronic diseases especially the non-communicable diseases viz., obesity, cardiovascular diseases and diabetes. Peptides derived from the milk of cow, goat, sheep, buffalo and camel exert multifunctional properties, including anti-microbial, immune modulatory, anti-oxidant, inhibitory effect on enzymes, anti-thrombotic, and antagonistic activities against various toxic agents. Majority of those regulate immunological, gastrointestinal, hormonal and neurological responses, thereby playing a vital role in the prevention of cancer, osteoporosis, hypertension and other disorders as discussed in this review. For the commercial production of such novel bioactive peptides large scale technologies based on membrane separation and ion exchange chromatography methods have been developed. Separation and identification of those peptides and their pharmacodynamic parameters are necessary to transfer their potent functional properties into food applications. The present review summarizes the preliminary classes of bioactive milk-derived peptides along with their physiological functions, general characteristics and potential applications in health-care.

  11. Optimization of production of recombinant human growth hormone in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Marzieh Rezaei

    2012-01-01

    Full Text Available Background: Human growth hormone (hGH is a single-chain polypeptide that participates in a wide range of biological functions such as metabolism of proteins, carbohydrates and lipids as well as in growth, development and immunity. Growth hormone deficiency in human occurs both in children and adults. The routine treatment for this condition is administration of recombinant human growth hormone (rhGH made by prokaryotes. Since nonglycosylated human growth hormone is a biologically active protein, prokaryotic expression systems are preferred for its production. Materials and Methods: Different strains of E.coli were transformed by plasmid containing human growth hormone gene and cultured in different conditions. After induction by IPTG, recombinant human growth hormone production was assessed using ELISA, dot blotting and western blotting techniques. Results: High levels of rhGH were produced using E.coli prokaryotic protein production system. Conclusion: This simple and cost effective production process could be recruited for large scale production of rhGH.

  12. Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink.

    Science.gov (United States)

    Babavalian, H; Latifi, A M; Shokrgozar, M A; Bonakdar, S; Tebyanian, H; Shakeri, F

    2016-07-31

    The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 μg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.

  13. Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

    Energy Technology Data Exchange (ETDEWEB)

    Crow, J. Allen; Bittles, Victoria; Herring, Katye L.; Borazjani, Abdolsamad [Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762 (United States); Potter, Philip M. [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105 (United States); Ross, Matthew K., E-mail: mross@cvm.msstate.edu [Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762 (United States)

    2012-01-01

    Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC{sub 50} values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k{sub inact}/K{sub I}) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC{sub 50} values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1

  14. Recombinant Human Plasminogen Activator Inhibitor-1 Accelerates Odontoblastic Differentiation of Human Stem Cells from Apical Papilla.

    Science.gov (United States)

    Jin, Bin; Choung, Pill-Hoon

    2016-05-01

    Dental caries, the most prevalent oral disease in dental patients, involves the phases of demineralization and destruction of tooth hard tissues like enamel, dentin, and cementum. Dentin is a major component of the root and is also the innermost layer that protects the tooth nerve, exposure of which results in pain. In this study, we used human stem cells from apical papilla (hSCAP), which are early progenitor cells, to examine the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on odontogenic differentiation in vitro and in vivo. We demonstrated that rhPAI-1 promoted the proliferation and odontogenic differentiation of hSCAP and increased the expression levels of odontoblast-associated markers. We also observed that rhPAI-1 upregulated the expression of Smad4, nuclear factor I-C (NFI-C), Runx2, and osterix (OSX) during odontogenic differentiation. Notably, transplantation of rhPAI-1-treated hSCAP effectively induced odontoblastic differentiation and dentinal formation. And the differentiated odontoblast-like cells showed numerous odontoblast processes inserted in dentin tubules and arranged collagen fibers. Furthermore, odontoblast-associated markers were more highly expressed in the rhPAI-1-induced differentiated odontoblast-like cells compared with the control group. These markers were also more highly expressed in the newly formed dentin-like tissue of the rhPAI-1-treated group compared with the control group. Consistent with our in vitro results, the expression levels of Smad4, NFI-C, and OSX were also increased in the rhPAI-1-treated group compared with the control group. Taken together, these results suggest that rhPAI-1 promotes odontoblast differentiation and dentin formation of hSCAP, and Smad4/NFI-C/OSX may play critical roles in the rhPAI-1-induced odontogenic differentiation. Thus, dental stem cells from apical papilla combined with rhPAI-1 could lead to dentin regeneration in clinical implications.

  15. Virulence evolution of the human pathogen Neisseria meningitidis by recombination in the core and accessory genome.

    Directory of Open Access Journals (Sweden)

    Biju Joseph

    Full Text Available BACKGROUND: Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH and multilocus sequence typing (MLST of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences. PRINCIPAL FINDINGS: We found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins. CONCLUSIONS: Recombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence.

  16. Construction of pPIC9 Recombinant Vector Containing Human Stem Cell Factor

    Directory of Open Access Journals (Sweden)

    Behrooz Farhadi

    2013-08-01

    Full Text Available Purpose: Various cytokine regulates hematopoesis; they promote number of stages in stem cells biology such as proliferation, differentiation and endurance. Biological effects of SCF, as a hematopoietic cytokine; is triggered by binding to its ligand c-kit. Potential therapeutic applications of SCF include hematopoietic stem cell mobilization, exvivo stem/progenitor cell expansion, gene therapy, and immunotherapy. In this study we tried to construct of pPIC9 recombinant vector containing human SCF. Methods: hSCF cDNA was amplified by PCR and both hSCF cDNA and pPIC9 as yeast expression vector (shuttle vector digested by EcoR I and Xho I restriction enzymes. Subsequent the digestion reaction, ligation reaction was carried out. In order to verifying of pPIC9 recombinant vector containing hSCF, PCR and sequence analysis was performed. Results: The construction of recombinant expression vector of pPIC9 containing hSCF cDNA was confirmed by sequencing method successfully. Conclusion: rhSCF/pPIC9 vector can be transformed into the Picha pastoris yeast as a eukaryotic host in order to produce human SCF at industrial scale.

  17. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

    Science.gov (United States)

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

    2016-05-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

  18. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    OpenAIRE

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

  19. Engineering the oxygen sensing regulation results in an enhanced recombinant human hemoglobin production by Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Martinez Ruiz, José Luis; Liu, Lifang; Petranovic, Dina

    2015-01-01

    Efficient production of appropriate oxygen carriers for transfusions (blood substitutes or artificial blood) has been pursued for many decades, and to date several strategies have been used, from synthetic polymers to cell-free hemoglobin carriers. The recent advances in the field of metabolic...... engineering also allowed the generation of different genetically modified organisms for the production of recombinant human hemoglobin. Several studies have showed very promising results using the bacterium Escherichia coli as a production platform, reporting hemoglobin titers above 5% of the total cell...... protein content. However, there are still certain limitations regarding the protein stability and functionality of the recombinant hemoglobin produced in bacterial systems. In order to overcome these limitations, yeast systems have been proposed as the eukaryal alternative. We recently reported...

  20. Recombinant Newcastle disease virus-vectored vaccines against human and animal infectious diseases.

    Science.gov (United States)

    Duan, Zhiqiang; Xu, Houqiang; Ji, Xinqin; Zhao, Jiafu

    2015-01-01

    Recent advances in recombinant genetic engineering techniques have brought forward a leap in designing new vaccines in modern medicine. One attractive strategy is the application of reverse genetics technology to make recombinant Newcastle disease virus (rNDV) deliver protective antigens of pathogens. In recent years, numerous studies have demonstrated that rNDV-vectored vaccines can induce quicker and better humoral and mucosal immune responses than conventional vaccines and are protective against pathogen challenges. With deeper understanding of NDV molecular biology, it is feasible to develop gene-modified rNDV vaccines accompanied by good safety, high efficacy, low toxicity and better immunogenicity. This review summarizes the development of reverse genetics technology in using NDV as a promising vaccine vector to design new vaccines for human and animal use.

  1. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step...... at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 micro...

  2. Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    Directory of Open Access Journals (Sweden)

    Adam Bajinting

    2017-06-01

    Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

  3. Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk.

    Science.gov (United States)

    Baldassarre, Hernan; Hockley, Duncan K; Doré, Monique; Brochu, Eric; Hakier, Bernard; Zhao, Xin; Bordignon, Vilceu

    2008-02-01

    The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1-5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.

  4. Safety of recombinant human platelet-derived growth factor-BB in Augment® Bone Graft

    Directory of Open Access Journals (Sweden)

    Luis A Solchaga

    2012-12-01

    Full Text Available This article discusses nonclinical and clinical data regarding the safety of recombinant human platelet-derived growth factor-BB as a component of the Augment® Bone Graft (Augment. Augment is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Nonclinical studies included assessment of the pharmacokinetic profile of intravenously administered recombinant human platelet-derived growth factor-BB in rat and dog, effects of intravenous administration of recombinant human platelet-derived growth factor-BB in a reproductive and development toxicity study in rats, and chronic toxicity and carcinogenicity of Augment in a 12-month implantation model. These studies showed that systemic exposure was brief and clearance was rapid. No signs of toxicity, carcinogenicity, or tumor promotion were observed even with doses far exceeding the maximum clinical dose. Results of clinical trials (605 participants and commercial use of recombinant human platelet-derived growth factor-BB containing products indicate that these products are not associated with increased incidence of adverse events or cancer. The safety data presented provide evidence that recombinant human platelet-derived growth factor-BB is a safe therapeutic when used in combination products as a single administration during surgical procedures for bone repair and fusion. There is no evidence associating use of recombinant human platelet-derived growth factor-BB in Augment with chronic toxicity, carcinogenicity, or tumor promotion.

  5. Soy Phytochemicals and Tea Bioactive Components Synergistically Inhibit Androgen-Sensitive Human Prostate Tumors in Mice

    OpenAIRE

    Zhou, Jin-Rong; Yu, Lunyin; Zhong, Ying; Blackburn, George L.

    2003-01-01

    Although high doses of single bioactive agents may have potent anticancer effects, the chemopreventive properties of the Asian diet may result from interactions among several components that potentiate the activities of any single constituent. In Asia, where intake of soy products and tea consumption are very high, aggressive prostate cancer is significantly less prevalent in Asian men. The objective of the present study was to identify possible synergistic effects between soy and tea compone...

  6. Protection of non-human primates against rabies with an adenovirus recombinant vaccine

    International Nuclear Information System (INIS)

    Xiang, Z.Q.; Greenberg, L.; Ertl, H.C.; Rupprecht, C.E.

    2014-01-01

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus

  7. Protection of non-human primates against rabies with an adenovirus recombinant vaccine

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Z.Q. [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Greenberg, L. [Centers for Disease Control and Prevention, Atlanta, GA (United States); Ertl, H.C., E-mail: ertl@wistar.upenn.edu [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Rupprecht, C.E. [The Global Alliance for Rabies Control, Manhattan, KS (United States); Ross University School of Veterinary Medicine, Basseterre (Saint Kitts and Nevis)

    2014-02-15

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.

  8. In Vitro Assessment of Bioactivities of Lactobacillus Strains as Potential Probiotics for Humans and Chickens.

    Science.gov (United States)

    Shokryazdan, P; Jahromi, M F; Liang, J B; Sieo, C C; Kalavathy, R; Idrus, Z; Ho, Y W

    2017-11-01

    Twelve previously isolated Lactobacillus strains were investigated for their in vitro bioactivities, including bile salt hydrolase (BSH), cholesterol-reducing and antioxidant activities, cytotoxic effects against cancer cells, enzyme activity, and biogenic amine production. Among them, only 4 strains showed relatively high BSH activity, whereas the rest exhibited low BSH activity. All 12 strains showed cholesterol-reducing and antioxidant activities, especially in their intact cells, which in most of the cases, the isolated strains were stronger in these activities than the tested commercial reference strains. None of the tested strains produced harmful enzymes (β-glucosidase and β-glucuronidase) or biogenic amines. Among the 12 strains, 3 strains were tested for their cytotoxic effects against 3 cancer cell lines, which exhibited strong cytotoxic effects, and they also showed selectivity in killing cancer cells when compared to normal cells. Hence, all 12 Lactobacillus strains could be considered good potential probiotic candidates because of their beneficial functional bioactivities. The Lactobacillus strains tested in this study could be considered good potential probiotic candidates for food/feed industry because of their beneficial functional bioactivities such as good cholesterol-reducing ability, high antioxidant activity, and good and selective cytotoxic effect against cancer cells. © 2017 Institute of Food Technologists®.

  9. Potency of full-length MGF to induce maximal activation of the IGF-I R Is similar to recombinant human IGF-I at high equimolar concentrations

    NARCIS (Netherlands)

    J.A.M.J.L. Janssen (Joseph); L.J. Hofland (Leo); C.J. Strasburger; E.S.R.D. Van Dungen (Elisabeth S.R. Den); M. Thevis (Mario)

    2016-01-01

    textabstractAims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin

  10. Evaluation of the interindividual human variation in bioactivation of methyleugenol using physiologically based kinetic modeling and Monte Carlo simulations

    Energy Technology Data Exchange (ETDEWEB)

    Al-Subeihi, Ala' A.A., E-mail: subeihi@yahoo.com [Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen (Netherlands); BEN-HAYYAN-Aqaba International Laboratories, Aqaba Special Economic Zone Authority (ASEZA), P. O. Box 2565, Aqaba 77110 (Jordan); Alhusainy, Wasma; Kiwamoto, Reiko; Spenkelink, Bert [Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen (Netherlands); Bladeren, Peter J. van [Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen (Netherlands); Nestec S.A., Avenue Nestlé 55, 1800 Vevey (Switzerland); Rietjens, Ivonne M.C.M.; Punt, Ans [Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen (Netherlands)

    2015-03-01

    The present study aims at predicting the level of formation of the ultimate carcinogenic metabolite of methyleugenol, 1′-sulfooxymethyleugenol, in the human population by taking variability in key bioactivation and detoxification reactions into account using Monte Carlo simulations. Depending on the metabolic route, variation was simulated based on kinetic constants obtained from incubations with a range of individual human liver fractions or by combining kinetic constants obtained for specific isoenzymes with literature reported human variation in the activity of these enzymes. The results of the study indicate that formation of 1′-sulfooxymethyleugenol is predominantly affected by variation in i) P450 1A2-catalyzed bioactivation of methyleugenol to 1′-hydroxymethyleugenol, ii) P450 2B6-catalyzed epoxidation of methyleugenol, iii) the apparent kinetic constants for oxidation of 1′-hydroxymethyleugenol, and iv) the apparent kinetic constants for sulfation of 1′-hydroxymethyleugenol. Based on the Monte Carlo simulations a so-called chemical-specific adjustment factor (CSAF) for intraspecies variation could be derived by dividing different percentiles by the 50th percentile of the predicted population distribution for 1′-sulfooxymethyleugenol formation. The obtained CSAF value at the 90th percentile was 3.2, indicating that the default uncertainty factor of 3.16 for human variability in kinetics may adequately cover the variation within 90% of the population. Covering 99% of the population requires a larger uncertainty factor of 6.4. In conclusion, the results showed that adequate predictions on interindividual human variation can be made with Monte Carlo-based PBK modeling. For methyleugenol this variation was observed to be in line with the default variation generally assumed in risk assessment. - Highlights: • Interindividual human differences in methyleugenol bioactivation were simulated. • This was done using in vitro incubations, PBK modeling

  11. PRODUCTION OF A HUMAN RECOMBINANT ANTIBODY AGAINST SEROTYPE A CANDIDA ALBICANS

    Directory of Open Access Journals (Sweden)

    A A. Jafari

    2005-07-01

    Full Text Available After using 3 different generations of antibodies including human and non-human hyperimmune sera, monoclonal antibodies and chimeric antibodies, more recently a newer approach has been developed in which the antibody genes are cloned directly from a patient peripheral B-lymphocytes and expressed in a host like E. coli. In this study the Candida albicans serotype A (NCTC 3153 mannan was purified using a modified Fehling method and used for selection of human recombinant antibody from a C. albicans phage antibody library. After four rounds of affinity selecting (panning, 2 predominant clones were chosen by DNA fingerprinting and ELISA. A 248 amino acid DNA fragment coding for anti-C. albicans mannan scFv was sequenced and cloned in a pBAD-TOPO cloning vector to produce a soluble and phage free antibody. The analysis of antibody sequences by V base Index (DNAPLOT confirmed the human antibody origin with the VH4 family in V segment of heavy variable chain and VL3 (Lambda 3 in J segment of the light variable chain. This antibody fragment was purified using immobilized metal affinity chromatography and inmmunoblotted as a 31kDa recombinant protein.

  12. The Development of Novel Recombinant Human Gelatins as Replacements for Animal-Derived Gelatin in Pharmaceutical Applications

    Science.gov (United States)

    Olsen, David; Chang, Robert; Williams, Kim E.; Polarek, James W.

    We have developed a recombinant expression system to produce a series of novel recombinant human gelatins that can substitute for animal sourced gelatin preparations currently used in pharmaceutical and nutraceutical applications. This system allows the production of human sequence gelatins, or, if desired, gelatins from any other species depending on the availability of the cloned gene. The gelatins produced with this recombinant system are of defined molecular weight, unlike the animal-sourced gelatins, which consist of numerous polypeptides of varying size. The fermentation and purification process used to prepare these recombinant gelatins does not use any human- or animal-derived components and thus this recombinant material should be free from viruses and agents that cause transmissible spongiform encephalopathies. The recombinant gelatins exhibit lot-to-lot reproducibility and we have performed extensive analytical testing on them. We have demonstrated the utility of these novel gelatins as biological stabilizers and plasma expanders, and we have shown they possess qualities that are important in applications where gel formation is critical. Finally, we provide examples of how our system allows the engineering of these recombinant gelatins to optimize the production process.

  13. A comparative study of recombinant and native frutalin binding to human prostate tissues

    Directory of Open Access Journals (Sweden)

    Domingues Lucília

    2009-09-01

    Full Text Available Abstract Background Numerous studies indicate that cancer cells present an aberrant glycosylation pattern that can be detected by lectin histochemistry. Lectins have shown the ability to recognise these modifications in several carcinomas, namely in the prostate carcinoma, one of the most lethal diseases in man. Thus, the aim of this work was to investigate if the α-D-galactose-binding plant lectin frutalin is able to detect such changes in the referred carcinoma. Frutalin was obtained from different sources namely, its natural source (plant origin and a recombinant source (Pichia expression system. Finally, the results obtained with the two lectins were compared and their potential use as prostate tumour biomarkers was discussed. Results The binding of recombinant and native frutalin to specific glycoconjugates expressed in human prostate tissues was assessed by using an immuhistochemical technique. A total of 20 cases of prostate carcinoma and 25 cases of benign prostate hyperplasia were studied. Lectins bound directly to the tissues and anti-frutalin polyclonal antibody was used as the bridge to react with the complex biotinilated anti-rabbit IgG plus streptavidin-conjugated peroxidase. DAB was used as visual indicator to specifically localise the binding of the lectins to the tissues. Both lectins bound to the cells cytoplasm of the prostate carcinoma glands. The binding intensity of native frutalin was stronger in the neoplasic cells than in hyperplasic cells; however no significant statistical correlation could be found (P = 0.051. On the other hand, recombinant frutalin bound exclusively to the neoplasic cells and a significant positive statistical correlation was obtained (P Conclusion Native and recombinant frutalin yielded different binding responses in the prostate tissues due to their differences in carbohydrate-binding affinities. Also, this study shows that both lectins may be used as histochemical biomarkers for the prostate

  14. Recombination gives a new insight in the effective population size and the history of the old world human populations.

    Science.gov (United States)

    Melé, Marta; Javed, Asif; Pybus, Marc; Zalloua, Pierre; Haber, Marc; Comas, David; Netea, Mihai G; Balanovsky, Oleg; Balanovska, Elena; Jin, Li; Yang, Yajun; Pitchappan, R M; Arunkumar, G; Parida, Laxmi; Calafell, Francesc; Bertranpetit, Jaume

    2012-01-01

    The information left by recombination in our genomes can be used to make inferences on our recent evolutionary history. Specifically, the number of past recombination events in a population sample is a function of its effective population size (Ne). We have applied a method, Identifying Recombination in Sequences (IRiS), to detect specific past recombination events in 30 Old World populations to infer their Ne. We have found that sub-Saharan African populations have an Ne that is approximately four times greater than those of non-African populations and that outside of Africa, South Asian populations had the largest Ne. We also observe that the patterns of recombinational diversity of these populations correlate with distance out of Africa if that distance is measured along a path crossing South Arabia. No such correlation is found through a Sinai route, suggesting that anatomically modern humans first left Africa through the Bab-el-Mandeb strait rather than through present Egypt.

  15. Recombinant human nerve growth factor with a marked activity in vitro and in vivo

    Science.gov (United States)

    Colangelo, Anna M.; Finotti, Nicoletta; Ceriani, Michela; Alberghina, Lilia; Martegani, Enzo; Aloe, Luigi; Lenzi, Laura; Levi-Montalcini, Rita

    2005-01-01

    Recombinant human nerve growth factor (rhNGF) is regarded as the most promising therapy for neurodegeneration of the central and peripheral nervous systems as well as for several other pathological conditions involving the immune system. However, rhNGF is not commercially available as a drug. In this work, we provide data about the production on a laboratory scale of large amounts of a rhNGF that was shown to possess in vivo biochemical, morphological, and pharmacological effects that are comparable with the murine NGF (mNGF), with no apparent side effects, such as allodynia. Our rhNGF was produced by using conventional recombinant DNA technologies combined with a biotechnological approach for high-density culture of mammalian cells, which yielded a production of ≈21.5 ± 2.9 mg/liter recombinant protein. The rhNGF-producing cells were thoroughly characterized, and the purified rhNGF was shown to possess a specific activity comparable with that of the 2.5S mNGF by means of biochemical, immunological, and morphological in vitro studies. This work describes the production on a laboratory scale of high levels of a rhNGF with in vitro and, more important, in vivo biological activity equivalent to the native murine protein. PMID:16339317

  16. Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins.

    Science.gov (United States)

    Souza, Ana Paula; Soto, Manuel; Costa, Jackson M L; Boaventura, Viviane S; de Oliveira, Camila I; Cristal, Juqueline R; Barral-Netto, Manoel; Barral, Aldina

    2013-01-01

    Exposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure. Herein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL) against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11). Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA. Our results raise possibility of using rHSP70 for the serodiagnosis of TL.

  17. Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland.

    Science.gov (United States)

    Baldassarre, Hernan; Hockley, Duncan K; Olaniyan, Benjamen; Brochu, Eric; Zhao, Xin; Mustafa, Arif; Bordignon, Vilceu

    2008-10-01

    The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P milk secretion observed in these transgenic goats.

  18. Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins.

    Directory of Open Access Journals (Sweden)

    Ana Paula Souza

    Full Text Available BACKGROUND: Exposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure. METHODOLOGY/PRINCIPAL FINDINGS: Herein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11. Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA. CONCLUSION: Our results raise possibility of using rHSP70 for the serodiagnosis of TL.

  19. Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

    Directory of Open Access Journals (Sweden)

    Pappu Kameshwari M

    2011-06-01

    Full Text Available Abstract Background Collagens require the hydroxylation of proline (Pro residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H as a posttranslational processing step. Results A recombinant human collagen type I α-1 (rCIα1 with high percentage of hydroxylated prolines (Hyp was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H. Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47% and the native human CIa1 (14.59%, respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1. Conclusions This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.

  20. Characterization of the oligosaccharide structure of human glycosylated prolactin (G-hPRL) native and recombinant

    International Nuclear Information System (INIS)

    Marcos Vinicius Nucci Capone

    2013-01-01

    Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the N ational Hormone & Peptide Program (NHPPU. S.) . The composition of N-glycans present

  1. Recombinant human erythropoietin increases survival and reduces neuronal apoptosis in a murine model of cerebral malaria

    DEFF Research Database (Denmark)

    Wiese, Lothar; Hempel, Casper; Penkowa, Milena

    2008-01-01

    BACKGROUND: Cerebral malaria (CM) is an acute encephalopathy with increased pro-inflammatory cytokines, sequestration of parasitized erythrocytes and localized ischaemia. In children CM induces cognitive impairment in about 10% of the survivors. Erythropoietin (Epo) has - besides of its well known...... with recombinant human Epo (rhEpo; 50-5000 U/kg/OD, i.p.) at different time points. The effect on survival was measured. Brain pathology was investigated by TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labelling), as a marker of apoptosis. Gene...

  2. Brief study about the distribution of recombinant human Epidermic Growth Factor (rh-EGF)

    International Nuclear Information System (INIS)

    Rodriguez Garcia, J.C.; De Dios D Espaux, R.; Bello Garciga, J.L.

    1997-01-01

    This report describes results of the study about biodistribution of I-125 recombinant human Epidermic Growth Factor (rhEGF). The radiolabelled product was administrated to Sprague Dawley rats in three different ways: intramuscular, subcutaneous and epidermic; the highest concentration of EGF in blood was found 4 hours after rhEGF administration, with a greater distribution in the plasma with regard to cellular pellet. The slowest plasma clearance corresponded to the intramuscular administration. The highest concentration of radiolabelled rhEGF was found in liver, kidney and intestine. It was found that radiolabelled EGF is excreted mainly throughout urine and faeces although other excretion pathways could exist

  3. Enhancement of bone formation in rabbits by recombinant human growth hormone

    International Nuclear Information System (INIS)

    Ehrnberg, A.; Brosjoe, O.; Laaftman, P.; Nilsson, O.; Stroemberg, L.

    1993-01-01

    We studied the effect of human recombinant growth hormone on diaphyseal bone in 40 adult rabbits. The diaphyseal periosteum of one femur in each animal was mechanically stimulated by a nylon cerclage band. The bands induced an increase in bone formation, bone mineral content, and maximum torque capacity of the diaphyseal bone at 1 and 2 months. Growth hormone enhanced the anabolic effect of the cerclage bands on bone metabolism, evidenced by a further increase in torsional strength of the femurs. (au) (32 refs.)

  4. Prolonged administration of recombinant human erythropoietin increases submaximal performance more than maximal aerobic capacity

    DEFF Research Database (Denmark)

    Thomsen, J J; Rentsch, R L; Robach, P

    2007-01-01

    The effects of recombinant human erythropoietin (rHuEpo) treatment on aerobic power (VO2max) are well documented, but little is known about the effects of rHuEpo on submaximal exercise performance. The present study investigated the effect on performance (ergometer cycling, 20-30 min at 80...... week 11), TTE was decreased by 26.8% as compared to pre rHuEpo administration. In conclusion, in healthy non-athlete subjects rHuEpo administration prolongs submaximal exercise performance by about 54% independently of the approximately 12% increase in VO2max....

  5. Effects of bioactive compounds from carrots (Daucus carota L.), polyacetylenes, beta-carotene and lutein on human lymphoid leukaemia cells.

    Science.gov (United States)

    Zaini, Rana G; Brandt, Kirsten; Clench, Malcolm R; Le Maitre, Christine L

    2012-07-01

    New therapies for leukaemia are urgently needed. Carrots have been suggested as a potential treatment for leukaemia in traditional medicine and have previously been studied in other contexts as potential sources of anticancer agents. Indicating that carrots may contain bioactive compounds, which may show potential in leukaemia therapies. This study investigated the effects of five fractions from carrot juice extract (CJE) on human lymphoid leukaemia cell lines, together with five purified bioactive compounds found in Daucus carota L, including: three polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) and two carotenoids (beta-carotene and lutein). Their effects on induction of apoptosis using Annexin V/PI and Caspase 3 activity assays analysed via flow cytometry and inhibition of cellular proliferation using Cell Titer Glo assay and cell cycle analysis were investigated. Treatment of all three lymphoid leukaemia cell lines with the fraction from carrot extracts which contained polyacetylenes and carotenoids was significantly more cytotoxic than the 4 other fractions. Treatments with purified polyacetylenes also induced apoptosis in a dose and time responsive manner. Moreover, falcarinol and falcarindiol-3-acetate isolated from Daucus carota L were more cytotoxic than falcarindiol. In contrast, the carotenoids showed no significant effect on either apoptosis or cell proliferation in any of the cells investigated. This suggests that polyacetylenes rather than beta-carotene or lutein are the bioactive components found in Daucus carota L and could be useful in the development of new leukemic therapies. Here, for the first time, the cytotoxic effects of polyacetylenes have been shown to be exerted via induction of apoptosis and arrest of cell cycle.

  6. Effect of recombinant adenovirus encoding human p53 tumor suppressor gene combined with radiation therapy on human lymphoma cells lines

    International Nuclear Information System (INIS)

    Yu Zeyang; Fan Wo; Li Dongqing; Zhu Ran; Wan Jianmei; Wang Yongqing; Wu Jinchang

    2008-01-01

    This paper analyzes the inhibitory effect and radiation sensitization of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on human lymphoma cell lines. Human lymphoma cell lines were treated with rAd-p53, radiation therapy and combined treatment, respectively. The cell growth inhibition was assessed by MTF. The cell cycle and apoptosis were detected by flow cytometry, and the p53 protein expression was detected by Western blotting. The results showed that extrinsic p53 gene have expressed to some degree, but not at high level. The role of inhibition and radiation sensitivity of rAd-p53 was not significant to human lymphoma cell lines. (authors)

  7. Fucoxanthin, a Marine Carotenoid Present in Brown Seaweeds and Diatoms: Metabolism and Bioactivities Relevant to Human Health

    Directory of Open Access Journals (Sweden)

    Jiang-Hai Wang

    2011-10-01

    Full Text Available The marine carotenoid fucoxanthin can be found in marine brown seaweeds, the macroalgae, and diatoms, the microalgae, and has remarkable biological properties. Numerous studies have shown that fucoxanthin has considerable potential and promising applications in human health. In this article, we review the current available scientific literature regarding the metabolism, safety, and bioactivities of fucoxanthin, including its antioxidant, anti-inflammatory, anticancer, anti-obese, antidiabetic, antiangiogenic and antimalarial activities, and its protective effects on the liver, blood vessels of the brain, bones, skin, and eyes. Although some studies have shown the bioavailability of fucoxanthin in brown seaweeds to be low in humans, many studies have suggested that a dietary combination of fucoxanthin and edible oil or lipid could increase the absorption rate of fucoxanthin, and thus it might be a promising marine drug.

  8. Compensatory rebalancing of rice prolamins by production of recombinant prolamin/bioactive peptide fusion proteins within ER-derived protein bodies.

    Science.gov (United States)

    Takaiwa, Fumio; Yang, Lijun; Wakasa, Yuhya; Ozawa, Kenjiro

    2018-02-01

    Bioactive peptide was produced by fusion to rice prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between prolamins within PB-Is. Peptide immunotherapy using analogue peptide ligands (APLs) is one of promising treatments against autoimmune diseases. Use of seed storage protein as a fusion carrier is reasonable strategy for production of such small size bioactive peptides. In this study, to examine the efficacy of various rice prolamins deposited in ER-derived protein bodies (PB-Is), the APL12 from the Glucose-6-phosphate isomerase (GPI325-339) was expressed by fusion to four types of representative prolamins under the control of the individual native promoters. When the 14 and 16 kDa Cys-rich prolamins, which were localized in middle layer of PB-Is, were used for production of the APL12, they highly accumulated in transgenic rice seeds (~ 200 µg/grain). By contrast, fusion to the 10 and 13 kDa prolamins, which were localized in the core and outermost layer of PB-Is, resulted in lower levels of accumulation (~ 40 µg/grain). These results suggest that accumulation levels were highly affected by their deposition sites. Next, when different prolamin/APL12 fusion proteins were co-expressed to increase accumulation levels, they could not be increased so much as their expected additive levels. High accumulation of one type prolamin/APL12 led to reduction of other type(s) prolamin/APL12 to maintain the limited amounts of prolamins that can be deposited in PB-Is. Moreover, suppression of endogenous seed proteins by RNA interference also did not significantly enhance the accumulation levels of prolamin/APL12. These findings suggest that there may be compensatory rebalancing mechanism that controls the accumulation levels of prolamins deposited within PB-Is.

  9. Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems

    Directory of Open Access Journals (Sweden)

    Daryl G.S. Smith

    2015-09-01

    Full Text Available Human serum albumin (HSA is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3 (2012 209–290. Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA (Chen et al., Biochim. Biophys. Acta (BBA—Gen. Subj. 1830(12 (2013 5515–5525; Kobayashi, Biologicals 34(1 (2006 55–59. Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15 (2012 4661–4670, both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9 (2014 e109893. We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.

  10. Relation between chloroguanide bioactivation to cycloguanil and the genetically determined metabolism of mephenytoin in humans.

    Science.gov (United States)

    Funck-Brentano, C; Bosco, O; Jacqz-Aigrain, E; Keundjian, A; Jaillon, P

    1992-05-01

    It has been suggested recently that the bioactivation of chloroguanide hydrochloride (proguanil) to its active antimalarial metabolite cycloguanil cosegregates with the genetically determined polymorphism of mephenytoin hydroxylation. We determined the chloroguanide to cycloguanil ratio in urine after oral administration of a single dose of 200 mg proguanil either alone or together with 100 mg racemic mephenytoin or 40 mg dextromethorphan in a randomized crossover study performed in 24 healthy subjects. The mephenytoin hydroxylation index was also determined after administration of 100 mg racemic mephenytoin either alone or together with 200 mg proguanil. Two subjects were poor metabolizers and one subject was an intermediate metabolizer of mephenytoin. These three subjects had chloroguanide to cycloguanil ratios of more than 50. The 21 subjects with the extensive metabolizer phenotype for mephenytoin hydroxylation had chloroguanide to cycloguanil ratios of less than 10. The chloroguanide to cycloguanil ratio was not significantly altered by mephenytoin or dextromethorphan coadministration. The trend toward a correlation between chloroguanide/cycloguanil ratio and log mephenytoin hydroxylation index did not reach statistical significance. Inclusion of the dextromethorphan metabolic ratio into the model did not improve the relationship. These findings confirm that the bioactivation of chloroguanide to cycloguanil cosegregates with the genetically determined activity of the CYP2C family. However, the chloroguanide to cycloguanil ratio and the mephenytoin hydroxylation index do not similarly reflect the variable activity of CYP2C.

  11. Hydroxylated phenylacetamides derived from bioactive benzoxazinoids are bioavailable in humans after habitual consumption of whole grain sourdough rye bread.

    Science.gov (United States)

    Beckmann, Manfred; Lloyd, Amanda J; Haldar, Sumanto; Seal, Chris; Brandt, Kirsten; Draper, John

    2013-10-01

    Understanding relationships between dietary whole grain and health is hindered by incomplete knowledge of potentially bioactive metabolites derived from these foods. We aimed to discover compounds in urine correlated with changes in amounts of whole grain rye consumption. After a wash-out period, volunteers consumed 48-g whole grain rye foods per day for 4 wk and then doubled their intake for a further 4 wk. Samples of 24-h urines were analyzed by flow infusion electrospray MS followed by supervised multivariate data analysis. Urine samples from participants who reported high intakes of rye flakes, rye pasta, or total whole grain rye products could not be discriminated adequately from their wash-out samples. However, discrimination was seen in urine samples from participants who reported high whole grain sourdough rye bread consumption. Accurate mass analysis of explanatory signals followed by fragmentation identified conjugates of the benzoxazinoid lactam 2-hydroxy-1,4-benzoxazin-3-one and hydroxylated phenyl acetamide derivatives. Statistical validation showed sensitivities of 84-96% and specificities of 70-81% (p values bread consumption. Several potentially bioactive alkaloids have been identified in humans consuming fermented whole grain sourdough rye bread. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. The Biomineralization of a Bioactive Glass-Incorporated Light-Curable Pulp Capping Material Using Human Dental Pulp Stem Cells.

    Science.gov (United States)

    Jun, Soo-Kyung; Lee, Jung-Hwan; Lee, Hae-Hyoung

    2017-01-01

    The aim of this study was to investigate the biomineralization of a newly introduced bioactive glass-incorporated light-curable pulp capping material using human dental pulp stem cells (hDPSCs). The product (Bioactive® [BA]) was compared with a conventional calcium hydroxide-incorporated (Dycal [DC]) and a light-curable (Theracal® [TC]) counterpart. Eluates from set specimens were used for investigating the cytotoxicity and biomineralization ability, determined by alkaline phosphatase (ALP) activity and alizarin red staining (ARS). Cations and hydroxide ions in the extracts were measured. An hDPSC viability of less than 70% was observed with 50% diluted extract in all groups and with 25% diluted extract in the DC. Culturing with 12.5% diluted BA extract statistically lowered ALP activity and biomineralization compared to DC ( p 0.05). Ca (~110 ppm) and hydroxide ions (pH 11) were only detected in DC and TC. Ionic supplement-added BA, which contained similar ion concentrations as TC, showed similar ARS mineralization compared to TC. In conclusion, the BA was similar to, yet more cytotoxic to hDPSCs than, its DC and TC. The BA was considered to stimulate biomineralization similar to DC and TC only when it released a similar amount of Ca and hydroxide ions.

  13. The Biomineralization of a Bioactive Glass-Incorporated Light-Curable Pulp Capping Material Using Human Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Soo-Kyung Jun

    2017-01-01

    Full Text Available The aim of this study was to investigate the biomineralization of a newly introduced bioactive glass-incorporated light-curable pulp capping material using human dental pulp stem cells (hDPSCs. The product (Bioactive® [BA] was compared with a conventional calcium hydroxide-incorporated (Dycal [DC] and a light-curable (Theracal® [TC] counterpart. Eluates from set specimens were used for investigating the cytotoxicity and biomineralization ability, determined by alkaline phosphatase (ALP activity and alizarin red staining (ARS. Cations and hydroxide ions in the extracts were measured. An hDPSC viability of less than 70% was observed with 50% diluted extract in all groups and with 25% diluted extract in the DC. Culturing with 12.5% diluted BA extract statistically lowered ALP activity and biomineralization compared to DC (p0.05. Ca (~110 ppm and hydroxide ions (pH 11 were only detected in DC and TC. Ionic supplement-added BA, which contained similar ion concentrations as TC, showed similar ARS mineralization compared to TC. In conclusion, the BA was similar to, yet more cytotoxic to hDPSCs than, its DC and TC. The BA was considered to stimulate biomineralization similar to DC and TC only when it released a similar amount of Ca and hydroxide ions.

  14. Refolded Recombinant Human Paraoxonase 1 Variant Exhibits Prophylactic Activity Against Organophosphate Poisoning.

    Science.gov (United States)

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Datusalia, Ashok K; Sharma, Shyam S; Pande, Abhay H

    2016-09-01

    Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-poisoning are considered as unsatisfactory and inadequate. There is an urgent need for the development of more effective treatment(s) for OP-poisoning. Human paraoxonase 1 (h-PON1) is known to hydrolyze a variety of OP-compounds and is a leading candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. Non-availability of effective system(s) for the production of recombinant h-PON1 (rh-PON1) makes it hard to produce improved variant(s) of this enzyme and analyze their in vivo efficacy in animal models. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop variant(s) of h-PON1. Recently, we have developed a procedure to produce active rh-PON1 enzymes by using E. coli expression system. In this study, we have characterized the OP-hydrolyzing properties of refolded rh-PON1(wt) and rh-PON1(H115W;R192K) variant. Our results show that refolded rh-PON1(H115W;R192K) variant exhibit enhanced OP-hydrolyzing activity in in vitro and ex vivo assays and exhibited prophylactic activity in mouse model of OP-poisoning, suggesting that refolded rh-PON1 can be developed as a therapeutic candidate.

  15. Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide

    International Nuclear Information System (INIS)

    Lai Yuping; Peng Yifei; Zuo Yi; Li Jun; Huang Jing; Wang Linfa; Wu Zirong

    2005-01-01

    Antimicrobial peptides from human skin are an important component of the innate immune response and play a key role as a first line of defense against infections. One such peptide is the recently discovered dermcidin-1L. To better understand its mechanism and to further investigate its antimicrobial spectrum, recombinant dermcidin-1L was expressed in Escherichia coli as a fusion protein and purified by affinity chromatography. The fusion protein was cleaved by factor Xa protease to produce recombinant dermcidin-1L. Antimicrobial and hemolytic assays demonstrated that dermcidin-1L displayed microbicidal activity against several opportunistic nosocomial pathogens, but no hemolytic activity against human erythrocytes even at concentrations up to 100 μM. Structural studies performed by circular dichroism spectroscopy indicated that the secondary structure of dermcidin-1L was very flexible, and both α-helix and β-sheet structures might be required for the antimicrobial activity. Our results confirmed previous findings indicating that dermcidin-1L could have promising therapeutic potentials and shed new light on the structure-function relationship of dermcidin-1L

  16. A Butter Aroma Recombinate Activates Human Class-I Odorant Receptors.

    Science.gov (United States)

    Geithe, Christiane; Andersen, Gaby; Malki, Agne; Krautwurst, Dietmar

    2015-11-04

    With ∼400 olfactory G protein-coupled receptors (GPCR), humans sensitively perceive ∼230 key aroma compounds as best natural agonists of ∼10000 food volatiles. An understanding of odorant coding, thus, critically depends on the knowledge about interactions of key food aroma chemicals and their mixtures with their cognate receptors. Genetically designed test cell systems enable the screening, deorphaning, and characterization of single odorant receptors (OR). This study shows for the food aroma-specific and quantitative butter aroma recombinate, and its single components, specific in vitro class-I OR activity patterns, as well as the activation of selected OR in a concentration-dependent manner. Recently, chemosensory receptors, especially class-I OR, were demonstrated to be expressed on blood leukocytes, which may encounter foodborne aroma compounds postprandially. This study shows that butter aroma recombinate induced chemotaxis of isolated human neutrophils in a defined gradient, and in a concentration-dependent and pertussis toxin-sensitive manner, suggesting at least a GPCR-mediated activation of blood leukocytes by key food odorants.

  17. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

    Science.gov (United States)

    Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

  18. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

    Directory of Open Access Journals (Sweden)

    Hanyu Wu

    Full Text Available Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

  19. Physical Characterization of human centromeric regions using transformation-associated recombination cloning technology

    Energy Technology Data Exchange (ETDEWEB)

    Vladimir Larionov, Ph D

    2007-06-05

    A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that can be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii

  20. Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10

    DEFF Research Database (Denmark)

    de Lemos Rieper, Carina; Galle, Pia; Svenson, Morten

    2009-01-01

    Radio iodinated recombinant human IL-10 was prepared and validated for the measurement of natural human anti-IL-10 antibodies. Iodination of IL-10 was accomplished by means of the chloramine-T method. The crude tracer was purified by size chromatography as homo-dimeric IL-10 with a specific...... activity of 75 cpm/pg. Validation of the tracer confirmed preserved antibody epitopes and receptor binding ability. A robust Radio Immuno Assay (RIA) was developed and validated to detect natural human anti-IL-10 antibodies based on the formation of (125)I-labeled IL-10-IgG complexes in solution...... and separation of the complexes by chromatography on mini-columns. The RIA was applied to 3360 plasma samples derived from normal Danish blood donors. Generally, IL-10 did not bind to plasma factors other than natural anti-IL-10 IgG antibodies. The prevalence of donors high positive for antibodies against IL-10...

  1. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces Cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter

    2014-01-01

    transcription factor and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30oC was due to in vivo mal-folding. Reduction of the expression temperature to 15oC almost completely...... of solutes. Aquaporins constitute a family of physiologically very important integral membrane proteins that are found in all three kingdoms, eubacteria, archaea and eukaryotes. As protein channels, they facilitate passive transport of water across cell membranes. In the present study the yeast Saccharomyces...... cerevisiae was exploited as a host for heterologous expression of human aquaporins. Aquaporin cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human aquaporin was C-terminally tagged with yeast-enhanced GFP to quantify functional expression...

  2. Characteristics of recombinantly expressed rat and human histamine H3 receptors.

    Science.gov (United States)

    Wulff, Birgitte S; Hastrup, Sven; Rimvall, Karin

    2002-10-18

    Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor. Copyright 2002 Elsevier Science B.V.

  3. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available Tyrosinase (TYR catalyzes the rate-limiting, first step in melanin production and its gene (TYR is mutated in many cases of oculocutaneous albinism (OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.The intra-melanosomal domain of human tyrosinase (residues 19-469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  4. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    Science.gov (United States)

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  5. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Science.gov (United States)

    Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2014-01-01

    Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  6. Expression of recombinant human lysozyme in egg whites of transgenic hens.

    Directory of Open Access Journals (Sweden)

    Dainan Cao

    Full Text Available Chicken egg lysozyme (cLY is an enzyme with 129 amino acid (AA residue enzyme. This enzyme is present not only in chicken egg white but also in mucosal secretions such as saliva and tears. The antibacterial properties of egg white can be attributed to the presence of lysozyme, which is used as an anti-cancer drug and for the treatment of human immunodeficiency virus (HIV infection. In this study, we constructed a lentiviral vector containing a synthetic cLY signal peptide and a 447 bp synthetic human lysozyme (hLY cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. The transgene inserted in the chicken chromosomes directs the synthesis and secretion of hLY which has three times higher specific activity than cLY. Three G1 transgenic chickens were identified, the only female of which expressed recombinant human lysozyme (rhLY at 57.66 ± 4.10 μg/ml in the egg white and the G2 transgenic hens of the G1 transgenic cock A011 expressed rhLY at 48.72 ± 1.54 μg/ml. This experiment demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results of this research could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems, such as cow, sheep and goats, for the production of therapeutic proteins and other applications.

  7. Expression of recombinant human lysozyme in egg whites of transgenic hens.

    Science.gov (United States)

    Cao, Dainan; Wu, Hanyu; Li, Qingyuan; Sun, Yingmin; Liu, Tongxin; Fei, Jing; Zhao, Yaofeng; Wu, Sen; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Chicken egg lysozyme (cLY) is an enzyme with 129 amino acid (AA) residue enzyme. This enzyme is present not only in chicken egg white but also in mucosal secretions such as saliva and tears. The antibacterial properties of egg white can be attributed to the presence of lysozyme, which is used as an anti-cancer drug and for the treatment of human immunodeficiency virus (HIV) infection. In this study, we constructed a lentiviral vector containing a synthetic cLY signal peptide and a 447 bp synthetic human lysozyme (hLY) cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. The transgene inserted in the chicken chromosomes directs the synthesis and secretion of hLY which has three times higher specific activity than cLY. Three G1 transgenic chickens were identified, the only female of which expressed recombinant human lysozyme (rhLY) at 57.66 ± 4.10 μg/ml in the egg white and the G2 transgenic hens of the G1 transgenic cock A011 expressed rhLY at 48.72 ± 1.54 μg/ml. This experiment demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results of this research could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems, such as cow, sheep and goats, for the production of therapeutic proteins and other applications.

  8. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Directory of Open Access Journals (Sweden)

    Sepehrizadeh Z

    2008-05-01

    Full Text Available Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by flow cytometry and also for the RNA extraction. Real time RT-PCR amplification was carried out for β-globin. Results and conclusion: There was a significant correlation between the total RNA amounts (R2= 0.9995, relative quantity of β-globin mRNA (R2= 0.984 and reticulocyte counts (R2= 0.9742 with rhEpo concentrations. Total RNA and quantitative RT-PCR showed significant dose dependent results as well the reticulocyte counts by flow cytometry for the biological activity assay of rhEpo and so these methods could be considered as alternatives for flow cytometry.

  9. Emerging Anti-Mitotic Activities and Other Bioactivities of Sesquiterpene Compounds upon Human Cells

    Directory of Open Access Journals (Sweden)

    Alessandra Bosco

    2017-03-01

    Full Text Available We review the bio-activities of natural product sesquiterpenes and present the first description of their effects upon mitosis. This type of biological effect upon cells is unexpected because sesquiterpenes are believed to inactivate proteins through Michael-type additions that cause non-specific cytotoxicity. Yet, certain types of sesquiterpenes can arrest cells in mitosis as measured by cell biology, biochemical and imaging techniques. We have listed the sesquiterpenes that arrest cells in mitosis and analyzed the biological data that support those observations. In view of the biochemical complexity of mitosis, we propose that a subset of sesquiterpenes have a unique chemical structure that can target a precise protein(s required for mitosis. Since the process of mitotic arrest precedes that of cell death, it is possible that some sesquiterpenes that are currently classified as cytotoxic might also induce a mitotic arrest. Our analysis provides a new perspective of sesquiterpene chemical biology

  10. Ag-loaded mesoporous bioactive glasses against Enterococcus faecalis biofilm in root canal of human teeth.

    Science.gov (United States)

    Fan, Wei; Wu, Daming; Ma, Tengjiao; Fan, Bing

    2015-01-01

    Ag-loaded mesoporous bioactive glass (Ag-MBG) powders were synthesized and characterized. The ions release of Ag-MBGs in Tris-HCl and the pH stability of simulated body fluids after immersing Ag-MBGs were tested. Root canals were inoculated with Enterococcus faecalis for 4 weeks, and the antibacterial activity of MBGs, Ag-MBGs and calcium hydroxide against E. faecalis biofilm were evaluated. Results showed that Ag-MBGs possessed highly ordered mesoporous structure with silver nanoparticles deposited in the mesopores, which enabled a sustained Ag ions released. The biofilms treated with Ag-MBGs showed a significant structural disruption compared with MBGs. These results indicated that Ag-MBGs possess a potent antibacterial effect against E.faecalis biofilm in root canal, and the antibacterial activity was induced by the release of Ag ions from Ag-MBGs.

  11. Recombinant human proinsulin from transgenic corn endosperm: solvent screening and extraction studies

    Directory of Open Access Journals (Sweden)

    C. S. Farinas

    2007-09-01

    Full Text Available Recombinant pharmaceutical proteins are being produced in different systems such as bacteria and mammalian cell cultures. The use of transgenic plants as bioreactors has recently arisen as an alternative system offering many practical and economic advantages. However, finding an optimum strategy for the downstream processing (DSP of recombinant proteins from plants still remains a challenge. In this work, we studied the extraction of recombinant human proinsulin (rhProinsulin produced in the endosperm of transgenic corn seeds. An efficient extraction solvent was selected and the effects of temperature, solvent-to-solid ratio, time, and impeller rotational speed on the extraction were evaluated using an experimental design. After an extraction kinetics study, temperature was further evaluated to maximize rhProinsulin concentration in the extracts and to minimize the native corn components carbohydrates, phenolic compounds, and proteins. A high efficiency condition for extracting rhProinsulin with the selected solvent - 50 mM sodium bicarbonate buffer pH 10.0 and 5 mM DTT - was an extraction time of 2 h at a solvent-to-solid ratio of 10:1 and 25º C. The maximum rhProinsulin concentration in the extracts at that condition was 18.87 mg l-1 or 0.42% of the total soluble protein. These values are within the range in which the production of pharmaceutical proteins in plants can be competitive with other expression systems. The results presented provide information for the development of an additional production platform for the hormone insulin.

  12. Distribution of recombination hotspots in the human genome--a comparison of computer simulations with real data.

    Directory of Open Access Journals (Sweden)

    Dorota Mackiewicz

    Full Text Available Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar.

  13. Apatite formation on bioactive calcium-silicate cements for dentistry affects surface topography and human marrow stromal cells proliferation.

    Science.gov (United States)

    Gandolfi, Maria Giovanna; Ciapetti, Gabriela; Taddei, Paola; Perut, Francesca; Tinti, Anna; Cardoso, Marcio Vivan; Van Meerbeek, Bart; Prati, Carlo

    2010-10-01

    The effect of ageing in phosphate-containing solution of bioactive calcium-silicate cements on the chemistry, morphology and topography of the surface, as well as on in vitro human marrow stromal cells viability and proliferation was investigated. A calcium-silicate cement (wTC) mainly based on dicalcium-silicate and tricalcium-silicate was prepared. Alpha-TCP was added to wTC to obtain wTC-TCP. Bismuth oxide was inserted in wTC to prepare a radiopaque cement (wTC-Bi). A commercial calcium-silicate cement (ProRoot MTA) was tested as control. Cement disks were aged in DPBS for 5 h ('fresh samples'), 14 and 28 days, and analyzed by ESEM/EDX, SEM/EDX, ATR-FTIR, micro-Raman techniques and scanning white-light interferometry. Proliferation, LDH release, ALP activity and collagen production of human marrow stromal cells (MSC) seeded for 1-28 days on the cements were evaluated. Fresh samples exposed a surface mainly composed of calcium-silicate hydrates CSH (from the hydration of belite and alite), calcium hydroxide, calcium carbonate, and ettringite. Apatite nano-spherulites rapidly precipitated on cement surfaces within 5 h. On wTC-TCP the Ca-P deposits appeared thicker than on the other cements. Aged cements showed an irregular porous calcium-phosphate (Ca-P) coating, formed by aggregated apatite spherulites with interspersed calcite crystals. All the experimental cements exerted no acute toxicity in the cell assay system and allowed cell growth. Using biochemical results, the scores were: fresh cements>aged cements for cell proliferation and ALP activity (except for wTC-Bi), whereas fresh cementssynthesis. Summarizing (1) non-aged cements showed higher cell proliferation than aged cements, probably favoured by the presence of Si-OH gel and the early formation of apatite nano-spherulites; (2) the alpha-TCP doped cement aged for 28 days displayed the highest bioactivity and cell proliferation; (3) the deleterious effect of bismuth on cell proliferation was reduced by

  14. Development of recombinant human IgA for anticardiolipin antibodies assay standardization.

    Science.gov (United States)

    Knappik, Achim; Capuano, Francesco; Frisch, Christian; Ylera, Francisco; Bonelli, Fabrizio

    2009-09-01

    Controls and calibrators in autoimmune assays are typically developed from patient sera. However, the use of sera is accompanied by a number of disadvantages, such as lack of monospecificity, lack of assay comparability, and supply limitations. Ideally, the control reagent would be an antigen-specific human monoclonal antibody preparation that is defined and pure, easy to produce without any supply limitations, and of defined isotype (IgG, IgM, or IgA). The generation of antigen-specific human monoclonal antibodies has been complicated, but recent advances in development of fully human antibodies by means of in vitro antibody gene library selection has opened a way for the isolation of human antibodies to virtually any antigen, including self-antigens. Such antibodies can be converted to any isotype by gene cloning. Here we developed a set of human monoclonal IgA antibodies specific for the cardiolipin-beta2-glycoprotein 1 complex, using the HuCAL technology. We evaluated the IgA variants of those antibodies for their use as standards in IgA anticardiolipin antibody assays and compared these reagents with serum controls. Such recombinant antibodies may ultimately replace patient sera as assay control and calibration reagents.

  15. Involvement of recombination in x-ray mutagenesis of human cells

    Energy Technology Data Exchange (ETDEWEB)

    Amundson, S.A. (Los Alamos National Lab., NM (United States)); Xia, F.; Liber, H.L. (Harvard School of Public Health, Boston, MA (United States))

    1993-01-01

    Closely related human lymphoblastoid cell lines derived from WI-L2 differ greatly in their responses to X-irradiation. Compared with TK6 (ATCC CRL 8015), WI-L2-NS (ATCC CRL 8155) has an enhanced X-ray survival. The induction of mutation by X-rays is also markedly different. The hemizygous hprt locus is slightly more mutable in WI-L2-NS than in TK6, and the dose response fits best to a linear-quadratic curve rather than the linear fit of TK6X-ray induced mutation at the autosomal tk locus in heterozygotes derived from WI-L2-NS is 20-50 fold higher than in heterozygotes derived from TK6. A larger proportion of WI-L2-NS mutants had lost heterozygosity compared with mutants of TK6. , Fluorescence in situ hybridization indicated that loss of heterozygosity was due almost uniformly to deletion of an allele in mutants of TK6, and to recombination or gene conversion in mutants of WI-L2-NS. These results indicate that recombinational repair contributes to both cell survival and mutation following exposure to ionizing radiation.

  16. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Science.gov (United States)

    Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

    2012-01-01

    Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

  17. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Directory of Open Access Journals (Sweden)

    Juliana Branco Novo

    2012-01-01

    Full Text Available Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr− cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa and secreted (63–69 kDa form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

  18. The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin.

    Directory of Open Access Journals (Sweden)

    Begoña O Alén

    Full Text Available The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD, nuclear magnetic resonance (NMR spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1. Other analogues, including human non-amidated obestatin (2 and the fragment peptides (6-23-obestatin (3, (11-23-obestatin (4, and (16-23-obestatin (5 have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS. Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation. Our findings emphasize the importance of both the primary structure (composition and size and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively at both the structural and bioactivity levels.

  19. The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin.

    Science.gov (United States)

    Alén, Begoña O; Nieto, Lidia; Gurriarán-Rodríguez, Uxía; Mosteiro, Carlos S; Álvarez-Pérez, Juan C; Otero-Alén, María; Camiña, Jesús P; Gallego, Rosalía; García-Caballero, Tomás; Martín-Pastor, Manuel; Casanueva, Felipe F; Jiménez-Barbero, Jesús; Pazos, Yolanda

    2012-01-01

    The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1). Other analogues, including human non-amidated obestatin (2) and the fragment peptides (6-23)-obestatin (3), (11-23)-obestatin (4), and (16-23)-obestatin (5) have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS). Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation). Our findings emphasize the importance of both the primary structure (composition and size) and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively) at both the structural and bioactivity levels.

  20. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

    DEFF Research Database (Denmark)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani

    2015-01-01

    on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins......Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge...... such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure...

  1. Recombinant human thyrotropin in veterinary medicine: current use and future perspectives.

    Science.gov (United States)

    Campos, M; van Hoek, I; Peremans, K; Daminet, S

    2012-01-01

    Recombinant human thyrotropin (rhTSH) was developed after bovine thyrotropin (bTSH) was no longer commercially available. It was approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) as an aid to diagnostic follow-up of differentiated thyroid carcinoma in humans and for thyroid remnant ablation with radioiodine. In addition, rhTSH is used in human medicine to evaluate thyroid reserve capacity and to enhance radioiodine uptake in patients with metastatic thyroid cancer and multinodular goiter. Likewise, rhTSH has been used in veterinary medicine over the last decade. The most important veterinary use of rhTSH is thyroidal functional reserve testing for the diagnosis of canine hypothyroidism. Recent pilot studies performed at Ghent University in Belgium have investigated the use of rhTSH to optimize radioiodine treatment of canine thyroid carcinoma and feline hyperthyroidism. Radioiodine treatment optimization may allow a decreased therapeutic dosage of radioiodine and thus may improve radioprotection. This review outlines the current uses of rhTSH in human and veterinary medicine, emphasizing research performed in dogs and cats, as well as potential future applications. Copyright © 2012 by the American College of Veterinary Internal Medicine.

  2. Update on human health concerns of recombinant bovine somatotropin use in dairy cows.

    Science.gov (United States)

    Collier, R J; Bauman, D E

    2014-04-01

    The 20 yr of commercial use of recombinant bovine somatotropin (rbST) in the United States provide the backdrop for reviewing the outcome of use on human health issues by the upcoming 78th meeting of the Joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Expert Committee on Food Additives. These results and further advancements in scientific knowledge indicate there are no new human health issues related to the use of rbST by the dairy industry. Use of rbST has no effect on the micro- and macrocomposition of milk. Also, no evidence exists that rbST use has increased human exposure to antibiotic residues in milk. Concerns that IGF-I present in milk could have biological effects on humans have been allayed by studies showing that oral consumption of IGF-I by humans has little or no biological activity. Additionally, concentrations of IGF-I in digestive tract fluids of humans far exceed any IGF-I consumed when drinking milk. Furthermore, chronic supplementation of cows with rbST does not increase concentrations of milk IGF-I outside the range typically observed for effects of farm, parity, or stage of lactation. Use of rbST has not affected expression of retroviruses in cattle or posed an increased risk to human health from retroviruses in cattle. Furthermore, risk for development of type 1 or type 2 diabetes has not increased in children or adults consuming milk and dairy products from rbST-supplemented cows. Overall, milk and dairy products provide essential nutrients and related benefits in health maintenance and the prevention of chronic diseases.

  3. Enhanced sialylation and in vivo efficacy of recombinant human α-galactosidase through in vitro glycosylation

    Directory of Open Access Journals (Sweden)

    Youngsoo Sohn

    2013-03-01

    Full Text Available Human α-galactosidase A (GLA has been used in enzymereplacement therapy for patients with Fabry disease. Weexpressed recombinant GLA from Chinese hamster ovary cellswith very high productivity. When compared to an approvedGLA (agalsidase beta, its size and charge were found to besmaller and more neutral. These differences resulted from thelack of terminal sialic acids playing essential roles in the serumhalf-life and proper tissue targeting. Because a simplesialylation reaction was not enough to increase the sialic acidcontent, a combined reaction using galactosyltransferase,sialyltransferase, and their sugar substrates at the same timewas developed and optimized to reduce the incubation time.The product generated by this reaction had nearly the samesize, isoelectric points, and sialic acid content as agalsidasebeta. Furthermore, it had better in vivo efficacy to degrade theaccumulated globotriaosylceramide in target organs of Fabrymice compared to an unmodified version. [BMB Reports 2013;46(3: 157-162

  4. Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes

    DEFF Research Database (Denmark)

    Juel, C; Thomsen, J J; Rentsch, R L

    2007-01-01

    on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects...... performance by approximately 54%. Membrane transport systems and carbonic anhydrases involved in pH regulation remained unchanged. Of the Na(+), K(+)-pump isoforms only the density of the alpha2 subunit was decreased (by 22%) after treatment. The marker enzymes cytochrom c and hexokinase remained unchanged......Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment...

  5. DNA fragmentation and cytotoxicity by recombinant human tumor necrosis factor in L929 fibroblast cells

    International Nuclear Information System (INIS)

    Kosaka, T.; Kuwabara, M.; Koide, F.

    1992-01-01

    Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor alpha (rhTNF alpha) was examined by using mouse L929 cells derived from mouse fibroblast cells. The amount of DNA fragments derived from rhTNF alpha-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNF alpha caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNF alpha treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNF alpha-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNF alpha causes DNA-fragmentation but not DNA-protein cross-linking

  6. Application of recombinant human thyrotropin and redifferentiation drugs in differentiated thyroid cancer

    International Nuclear Information System (INIS)

    Meng Zhaowei; Tan Jian

    2006-01-01

    In the management of differentiated thyroid cancer, radioiodine ( 131 I) ablation of thyroid remnants and 131 I treatment of metastatic lesions are very important. To prepare for ablation or treatment, optimal level of TSH has to be achieved. Recombinant human thyrotropin (rhTSH) has the same structure and biological activity as endogenous TSH. It can effectively elevate serum TSH level and it has little side effects. rhTSH can also avoid hypothyroid symptoms which are sometimes very serious if withdrawal of thyroid hormone therapy is carried out. However, in a number of cases, dedifferentiation is observed, giving rise to poor prognostic thyroid cancers that are refractory to conventional treatment. Several novel approaches for the treatment of dedifferentiated thyroid cancers have made great breakthroughs. For instance, isotretinoin redifferentiation therapy has entered Phase II clinical trials. (authors)

  7. Homologous Recombination and Human Health: The Roles of BRCA1, BRCA2, and Associated Proteins

    Science.gov (United States)

    Prakash, Rohit; Zhang, Yu; Feng, Weiran; Jasin, Maria

    2015-01-01

    Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks in mammalian cells, the defining step of which is homologous strand exchange directed by the RAD51 protein. The physiological importance of HR is underscored by the observation of genomic instability in HR-deficient cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HR genes. The tumor suppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutated in familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51 paralogs, have also been identified as tumor suppressors. This review summarizes recent findings on BRCA1, BRCA2, and associated proteins involved in human disease with an emphasis on their molecular roles and interactions. PMID:25833843

  8. Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector.

    Science.gov (United States)

    Dolinska, Monika B; Wingfield, Paul T; Sergeev, Yuri V

    2017-08-01

    The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  9. Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes

    DEFF Research Database (Denmark)

    Ottolini, Christian S; Capalbo, Antonio; Newnham, Louise

    2016-01-01

    We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially...... activated by exposure to calcium ionophore, after which PB2 is biopsied and collected with the corresponding oocyte. The whole genomes of the polar bodies and oocytes are amplified by multiple displacement amplification and, together with maternal genomic DNA, genotyped for ∼300,000 single......-nucleotide polymorphisms (SNPs) genome-wide by microarray. Informative maternal heterozygous SNPs are phased using a haploid PB2 or oocyte as a reference. A simple algorithm is then used to identify the maternal haplotypes for each chromosome, in all of the products of meiosis for each oocyte. This allows mapping...

  10. PRELIMINARY MODELING OF AN INDUSTRIAL RECOMBINANT HUMAN ERYTHROPOIETIN PURIFICATION PROCESS BY ARTIFICIAL NEURAL NETWORKS

    Directory of Open Access Journals (Sweden)

    R. H. R. Garcel1

    2015-09-01

    Full Text Available AbstractIn the present study a preliminary neural network modelling to improve our understanding of Recombinant Human Erythropoietin purification process in a plant was explored. A three layer feed-forward back propagation neural network was constructed for predicting the efficiency of the purification section comprising four chromatographic steps as a function of eleven operational variables. The neural network model performed very well in the training and validation phases. Using the connection weight method the predictor variables were ranked based on their estimated explanatory importance in the neural network and five input variables were found to be predominant over the others. These results provided useful information showing that the first chromatographic step and the third chromatographic step are decisive to achieve high efficiencies in the purification section, thus enriching the control strategy of the plant.

  11. Molecular mechanisms of recombination restriction in the envelope gene of the human immunodeficiency virus.

    Directory of Open Access Journals (Sweden)

    Etienne Simon-Loriere

    2009-05-01

    Full Text Available The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology.

  12. Automated production of recombinant human proteins as resource for proteome research

    Directory of Open Access Journals (Sweden)

    Poustka Annemarie

    2008-01-01

    Full Text Available Abstract Background An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment. Results A robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25°C, whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead. Conclusion Soluble fusion proteins can be produced in E. coli in sufficient qualities and μg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.

  13. Expression and purification of recombinant truncated human keratinocyte growth factor-1

    International Nuclear Information System (INIS)

    Deng Lin; Ma Jisheng; Liu Xiaoju; Wang Xiaojie; Li Xiaokun; Gong Shouliang; Wang Huiyan; Tian Haishan

    2010-01-01

    Objective: To construct the genetic engineering bacteria highly expressing 23 amino acids human keratinocyte growth factor-1 (rhKGF1 dest23 ) missing N terminal, and provide experimental data for development of new drug for treatment of oral mucositis after radiotherapy and chemotherapy. Methods: PCR was used to synthese 23 amino acids rhKGF1 dest23 missing N terminal and sumo gene fragments, and construct four kinds of recombinant prokaryotic expression vectors: pET22b-rhKGF1 dest23 , pET22b-sumo-rhKGF1 dest23 , pET3c-rhKGF1 dest23 and pET3c-sumo-rhKGF1 dest23 , then they were transformed into prokaryotic expression host bacteria: Rosetta (DE3) plysS, BL21 (DE3), BL21 (DE3) Star plysS, origima(DE3) and BL21AI, the best expression combination of plasmid and host strain of rhKGF1 dest23 protein was screened and purified by CM ion-exchange and heparin affinity chromatography and identified with Western blotting. Results: pET22b-rhKGF1 dest23 plasmid and the BL21AI host bacteria was the best combination of expression, after induced by IPTG and arabinose, the majority of recombinant protein was expressed in soluble form, accounting for about 12% of the total bacterial proteins. Its purity reached to more than 95% of the protein after two steps chromatography, then conformed with Western blotting. Conclusion: Human genetic engineering bacteria of KGF1 dest23 is successfully constructed and induced by IPTG and arabinose, then after CM weak cation exchange and heparin affinity chromatography, the purified rhKGF1 dest23 protein is obtained. (authors)

  14. Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

    Directory of Open Access Journals (Sweden)

    Elizaveta O Boldinova

    Full Text Available Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

  15. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    Science.gov (United States)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  16. Identifying recombinants in human and primate immunodeficiency virus sequence alignments using quartet scanning

    Directory of Open Access Journals (Sweden)

    Martin Darren P

    2009-04-01

    Full Text Available Abstract Background Recombination has a profound impact on the evolution of viruses, but characterizing recombination patterns in molecular sequences remains a challenging endeavor. Despite its importance in molecular evolutionary studies, identifying the sequences that exhibit such patterns has received comparatively less attention in the recombination detection framework. Here, we extend a quartet-mapping based recombination detection method to enable identification of recombinant sequences without prior specifications of either query and reference sequences. Through simulations we evaluate different recombinant identification statistics and significance tests. We compare the quartet approach with triplet-based methods that employ additional heuristic tests to identify parental and recombinant sequences. Results Analysis of phylogenetic simulations reveal that identifying the descendents of relatively old recombination events is a challenging task for all methods available, and that quartet scanning performs relatively well compared to the triplet based methods. The use of quartet scanning is further demonstrated by analyzing both well-established and putative HIV-1 recombinant strains. In agreement with recent findings, we provide evidence that the presumed circulating recombinant CRF02_AG is a 'pure' lineage, whereas the presumed parental lineage subtype G has a recombinant origin. We also demonstrate HIV-1 intrasubtype recombination, confirm the hybrid origin of SIV in chimpanzees and further disentangle the recombinant history of SIV lineages in a primate immunodeficiency virus data set. Conclusion Quartet scanning makes a valuable addition to triplet-based methods for identifying recombinant sequences without prior specifications of either query and reference sequences. The new method is available in the VisRD v.3.0 package http://www.cmp.uea.ac.uk/~vlm/visrd.

  17. The pharmacokinetics, distribution and degradation of human recombinant interleukin 1 beta in normal rats

    DEFF Research Database (Denmark)

    Reimers, J; Wogensen, L D; Welinder, B

    1991-01-01

    Based upon in vivo rat experiments it was recently suggested that interleukin 1 in the circulation may be implicated in the initial events of beta-cell destruction leading to insulin-dependent diabetes mellitus (IDDM) in humans. The aim of the present study was to estimate half-lives of distribut......Based upon in vivo rat experiments it was recently suggested that interleukin 1 in the circulation may be implicated in the initial events of beta-cell destruction leading to insulin-dependent diabetes mellitus (IDDM) in humans. The aim of the present study was to estimate half......-lives of distribution (T1/2 alpha) and elimination phases (T1/2 beta) of human recombinant interleukin 1 beta (rIL-1 beta), and its tissue distribution and cellular localization by means of mono-labelled, biologically active 125I-rIL-1 beta. After intravenous (i.v.) injection, 125I-rIL-1 beta was eliminated from...... of administration was of importance for the biological effects of rIL-1 beta, as demonstrated by a reduced food intake, increased rectal temperature and blood glucose after s.c. injection of rIL-1 beta compared with i.p. The present demonstration of intact rIL-1 beta in the circulation and the islets of Langerhans...

  18. Large-Scale Recombinant Expression and Purification of Human Tyrosinase Suitable for Structural Studies.

    Directory of Open Access Journals (Sweden)

    Xuelei Lai

    Full Text Available Human tyrosinase (TYR is a glycoprotein that initiates the first two reactions in the melanin biosynthesis pathway. Mutations in its encoding gene cause Oculocutaneous Albinism type I (OCA1, the most severe form of albinism, which is a group of autosomal recessive disorders characterized by reduced or absent production of melanin in skin, hair and eyes. Despite extensive structural and characterization studies of its homologues in lower eukaryotic organisms, the catalytic mechanism of human TYR and the molecular basis of OCA1 are largely unknown. In this work, we have carried out a large-scale recombinant expression of TYR that has enabled us to obtain high yields of pure and active protein, required for crystallization trials and screening of skin whitening agents, which is highly demanded in the cosmetic industry. Addition of an N-terminal honeybee melittin signal peptide for secretion of the produced protein into the (protein-free medium, as well as a cleavable His-tag at the C-terminus, was crucial for increasing the yield of pure protein. We have successfully crystallized two TYR variants, in both glycosylated and deglycosylated forms, showing preliminary X-ray diffraction patterns at 3.5 Å resolution. Hence, we have established an expression and purification protocol suitable for the crystal structure determination of human TYR, which will give unique atomic insight into the nature and conformation of the residues that shape the substrate binding pocket that will ultimately lead to efficient compound design.

  19. Production of recombinant human von Willebrand factor in the milk of transgenic pigs.

    Science.gov (United States)

    Lee, Hyun-Gi; Lee, Hwi-Cheul; Kim, Sung Woo; Lee, Poongyeon; Chung, Hak-Jae; Lee, Yun-Keun; Han, Joo-Hee; Hwang, In-Sul; Yoo, Jong-Il; Kim, Yong-Kook; Kim, Hun-Taek; Lee, Hoon-Taek; Chang, Won-Kyong; Park, Jin-Ki

    2009-10-01

    Von Willebrand factor (vWF), a large multimeric glycoprotein present in blood plasma, is a blood protein of the coagulation system. It is defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and heyde's syndrome. We have developed a line of transgenic swine harboring recombinant human von Willebrand factor (rhvWF) cDNA through microinjection of fertilized one-cell pig zygotes. Expression of rhvWF in the mammary gland and secretion of rhvWF into the milk of the transgenic swine were confirmed by immunohistochemical and western blot analyses, respectively, and rhvWF proteins were detected in milk from all lactating founder females at concentrations that were 28- to 56-folds greater than that in circulating human plasma. The amino acid sequence of rhvWF protein in the transgenic pig milk matched that of vWF produced from human blood plasma. This study provides evidence that production of rhvWF from transgenic pig milk is a potentially valuable technology and can be used as a cost-effective alternative in clinical applications.

  20. Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

    Science.gov (United States)

    Kaiser, Germán G; Mucci, Nicolás C; González, Vega; Sánchez, Lourdes; Parrón, José A; Pérez, María D; Calvo, Miguel; Aller, Juan F; Hozbor, Federico A; Mutto, Adrián A

    2017-03-01

    Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for

  1. Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R

    Energy Technology Data Exchange (ETDEWEB)

    Atsmon, Jacob [Clinical Research Center, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University (Israel); Brill-Almon, Einat; Nadri-Shay, Carmit; Chertkoff, Raul; Alon, Sari [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Shaikevich, Dimitri; Volokhov, Inna; Haim, Kirsten Y. [Clinical Research Center, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University (Israel); Bartfeld, Daniel [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Shulman, Avidor, E-mail: avidors@protalix.com [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Ruderfer, Ilya; Ben-Moshe, Tehila; Shilovitzky, Orit [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Soreq, Hermona [Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem (Israel); Shaaltiel, Yoseph [Protalix Biotherapeutics, Science Park, Carmiel (Israel)

    2015-09-15

    PRX-105 is a plant-derived recombinant version of the human ‘read-through’ acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50 nmol/kg PRX-105, 2 min before being exposed to 1.33 × LD{sub 50} and 1.5 × LD{sub 50} of toxin and 10 min after exposure to 1.5 × LD{sub 50} survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200 mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t{sub ½}) in mice was 994 (± 173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t{sub ½} in humans was substantially longer than in mice (average 26.7 h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation. - Highlights: • PRX-105 is a PEGylated plant-derived recombinant human acetylcholinesterase-R. • PRX-105 is a promising bio-scavenger for organophosphorous toxins at lethal doses. • PRX-105 was shown to protect animals both prophylactically and post-poisoning. • First-in-human study

  2. Methylation, Glucuronidation, and Sulfonation of Daphnetin in Human Hepatic Preparations In Vitro: Metabolic Profiling, Pathway Comparison, and Bioactivity Analysis.

    Science.gov (United States)

    Liang, Si-Cheng; Xia, Yang-Liu; Hou, Jie; Ge, Guang-Bo; Zhang, Jiang-Wei; He, Yu-Qi; Wang, Jia-Yue; Qi, Xiao-Yi; Yang, Ling

    2016-02-01

    Our previous study demonstrated that daphnetin is subject to glucuronidation in vitro. However, daphnetin metabolism is still poorly documented. This study aimed to investigate daphnetin metabolism and its consequent effect on the bioactivity. Metabolic profiles obtained by human liver S9 fractions and human hepatocytes showed that daphnetin was metabolized by glucuronidation, sulfonation, and methylation to form 6 conjugates which were synthesized and identified as 7-O-glucuronide, 8-O-glucuronide, 7-O-sulfate and 8-O-sulfate, 8-O-methylate, and 7-O-suflo-8-O-methylate. Regioselective 8-O-methylation of daphnetin was investigated using in silico docking calculations, and the results suggested that a close proximity (2.03 Å) of 8-OH to the critical residue Lysine 144 might be the responsible mechanism. Compared with glucuronidation and sulfonation pathways, the methylation of daphnetin had a high clearance rate (470 μL/min/mg) in human liver S9 fractions and contributed to a large amount (37.3%) of the methyl-derived metabolites in human hepatocyte. Reaction phenotyping studies showed the major role of SULT1A1, -1A2, and -1A3 in daphnetin sulfonation, and soluble COMT in daphnetin 8-O-methylation. Of the metabolites, only 8-O-methyldaphnetin exhibited an inhibitory activity on lymphocyte proliferation comparable to that of daphnetin. In conclusion, methylation is a crucial pathway for daphnetin clearance and might be involved in pharmacologic actions of daphnetin in humans. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  3. The ergogenic effect of recombinant human erythropoietin on VO2max depends on the severity of arterial hypoxemia

    DEFF Research Database (Denmark)

    Robach, Paul; Calbet, Jose A L; Thomsen, Jonas J

    2008-01-01

    Treatment with recombinant human erythropoietin (rhEpo) induces a rise in blood oxygen-carrying capacity (CaO(2)) that unequivocally enhances maximal oxygen uptake (VO(2)max) during exercise in normoxia, but not when exercise is carried out in severe acute hypoxia. This implies that there should ...

  4. Activation of human T cells by a tumor vaccine infected with recombinant Newcastle disease virus producing IL-2

    NARCIS (Netherlands)

    Janke, M.; Peeters, B.; Zhao, H.; Leeuw, O.; Moormann, R.J.M.; Arnold, A.; Ziouta, Y.; Fournier, P.; Schirrmacher, V.

    2008-01-01

    A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2) as foreign therapeutic gene [rec(IL-2)] will be described. The foreign gene in rec(IL-2) did not affect the main features of NDV replication nor its tumor selectivity. Biologically active IL-2 was

  5. GH safety workshop position paper: A critical appraisal of recombinant human GH therapy in children and adults

    Science.gov (United States)

    Recombinant human Growth Hormone (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, t...

  6. Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antimicrobial effects in murine pneumococcal pneumonia

    NARCIS (Netherlands)

    van den Boogaard, F. E.; Brands, X.; Schultz, M. J.; Levi, M. [=Marcel M.; Roelofs, J. J. T. H.; van 't Veer, C.; van der Poll, T.

    2011-01-01

    Background: Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia and a major cause of sepsis. Recombinant human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis-induced coagulation and has been evaluated in clinical trials involving patients

  7. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit the chemotaxis and enhance the oxidative burst response of human neutrophils in vitro. The present study describes the effect of recombinant GM-CSF on the neutrophil and monocyte function in patients with lymphoma...

  8. Safety and efficacy of recombinant human interleukin 10 in chronic active Crohn's disease. Crohn's Disease IL-10 Cooperative Study Group

    NARCIS (Netherlands)

    Schreiber, S.; Fedorak, R. N.; Nielsen, O. H.; Wild, G.; Williams, C. N.; Nikolaus, S.; Jacyna, M.; Lashner, B. A.; Gangl, A.; Rutgeerts, P.; Isaacs, K.; van Deventer, S. J.; Koningsberger, J. C.; Cohard, M.; LeBeaut, A.; Hanauer, S. B.

    2000-01-01

    Interleukin (IL)-10 is a cytokine with potent anti-inflammatory properties. We investigated the safety and efficacy of different doses of human recombinant (rhu)IL-10 in patients with Crohn's disease (CD). A prospective, multicenter, double-blind, placebo-controlled study was conducted in 329

  9. Immunogenicity assessment of recombinant human c1-inhibitor: an integrated analysis of clinical studies.

    Science.gov (United States)

    Hack, C Erik; Mannesse, Maurice; Baboeram, Aartie; Oortwijn, Beatrijs; Relan, Anurag

    2012-10-01

    Recombinant human C1-inhibitor (rhC1INH) is used to treat acute angioedema attacks in hereditary angioedema (HAE) due to a genetic C1INH deficiency. Recombinant proteins in general may induce antibody responses and therefore evaluation of such responses in the target population is an essential step in the clinical development program of a recombinant protein. Here we report the assessment of the immunogenicity of rhC1INH in symptomatic HAE patients. Blood samples collected before and after administration of rhC1INH were tested for antibodies against plasma-derived (pd) or rhC1INH, or against host-related impurities (HRI). Above cut-off screening results were confirmed with displacement assays, and also tested for neutralizing anti-C1INH antibodies. Finally, the relation of antibodies to clinical efficacy and safety of rhC1INH was analyzed. Data from 155 HAE patients who received 424 treatments with rhC1INH were analyzed. 1.5% of all pre-exposure tests and 1.3% of all post-exposure tests were above the cut-off level in the screening assay for anti-C1INH antibodies. Six patients (3.9%) had anti-rhC1INH antibodies positive in the confirmatory assay. In two patients, confirmed antibodies were pre-existing with no increase post-exposure; in three patients, the antibodies occurred on a single occasion post-exposure; and in one patient, on subsequent occasions post-exposure. Neutralizing anti-pdC1INH antibodies were not found. Anti-HRI antibodies in the screening assay occurred in <0.7% of the tests before exposure to rhC1INH, in <1.9% after first exposure and in <3.1% after repeat treatment with rhC1INH. Five patients had anti-HRI antibodies positive in the confirmatory assay. In one patient, the antibodies were pre-existing, whereas in three of the 155 rhC1INH-treated patients (1.9%), confirmed anti-HRI antibodies occurred at more time points. Antibody findings were not associated with altered efficacy of rhC1INH or adverse events. These results indicate a reassuring

  10. Mechanical property characterization of electrospun recombinant human tropoelastin for vascular graft biomaterials.

    Science.gov (United States)

    McKenna, Kathryn A; Hinds, Monica T; Sarao, Rebecca C; Wu, Ping-Cheng; Maslen, Cheryl L; Glanville, Robert W; Babcock, Darcie; Gregory, Kenton W

    2012-01-01

    The development of vascular grafts has focused on finding a biomaterial that is non-thrombogenic, minimizes intimal hyperplasia, matches the mechanical properties of native vessels and allows for regeneration of arterial tissue. In this study, the structural and mechanical properties and the vascular cell compatibility of electrospun recombinant human tropoelastin (rTE) were evaluated as a potential vascular graft support matrix. Disuccinimidyl suberate (DSS) was used to cross-link electrospun rTE fibers to produce a polymeric recombinant tropoelastin (prTE) matrix that is stable in aqueous environments. Tubular 1cm diameter prTE samples were constructed for uniaxial tensile testing and 4mm small-diameter prTE tubular scaffolds were produced for burst pressure and cell compatibility evaluations from 15 wt.% rTE solutions. Uniaxial tensile tests demonstrated an average ultimate tensile strength (UTS) of 0.36±0.05 MPa and elastic moduli of 0.15±0.04 and 0.91±0.16 MPa, which were comparable to extracted native elastin. Burst pressures of 485±25 mm Hg were obtained from 4mm internal diameter scaffolds with 453±74 μm average wall thickness. prTE supported endothelial cell growth with typical endothelial cell cobblestone morphology after 48 h in culture. Cross-linked electrospun rTE has promising properties for utilization as a vascular graft biomaterial with customizable dimensions, a compliant matrix and vascular cell compatibility. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  11. Two immunosuppressive compounds from the mushroom Rubinoboletus ballouii using human peripheral blood mononuclear cells by bioactivity-guided fractionation.

    Science.gov (United States)

    Li, Long-Fei; Chan, Ben Chung-Lap; Yue, Grace Gar-Lee; Lau, Clara Bik-San; Han, Quan-Bin; Leung, Ping-Chung; Liu, Ji-Kai; Fung, Kwok-Pui

    2013-10-15

    Rubinoboletus ballouii is an edible mushroom wildly grown in Yunnan province, China. Up till now, little was known about the chemical and biological properties of this mushroom. The aim of this study was to investigate the immunomodulatory effects of the ethanolic extract of Rubinoboletus ballouii and its fractions on human peripheral blood mononuclear cells (PBMCs) using bioactivity-guided fractionation. The crude extract of the fruiting bodies of RB was fractionated by high-speed counter current chromatography (HSCCC). Twelve fractions were obtained and the third fraction (Fraction C) exerted the most potent anti-inflammatory activities in mitogen-activated PBMCs. Further fractionation of fraction C led to the isolation of two single compounds which were elucidated as 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin, respectively. The results showed that both 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin exhibited significant immunosuppressive effects on phytohemagglutinin (PHA)-stimulated human PBMCs by inhibiting [methyl-(3)H]-thymidine uptake and inflammatory cytokines productions such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, interferon (IFN)-γ and IL-1β. Besides, 1-ribofuranosyl-s-triazin-2(1H)-one was firstly found in natural resources, and pistillarin was also isolated from the family Boletaceae for the first time. They exhibited great potential in developing as anti-inflammatory reagents. Copyright © 2013 Elsevier GmbH. All rights reserved.

  12. Bioactive glass 45S5 powders: effect of synthesis route and resultant surface chemistry and crystallinity on protein adsorption from human plasma.

    Science.gov (United States)

    Bahniuk, Markian S; Pirayesh, Hamidreza; Singh, Harsh D; Nychka, John A; Unsworth, Larry D

    2012-12-01

    Despite its medical applications, the mechanisms responsible for the osseointegration of bioactive glass (45S5) have yet to be fully understood. Evidence suggests that the strongest predictor for osseointegration of bioactive glasses, and ceramics, with bone tissue as the formation of an apatitic calcium phosphate layer atop the implanted material, with osteoblasts being the main mediator for new bone formation. Most have tried to understand the formation of this apatitic calcium phosphate layer, and other bioresponses between the host and bioactive glass 45S5 using Simulated Body Fluid; a solution containing ion concentrations similar to that found in human plasma without the presence of proteins. However, it is likely that cell attachment is probably largely mediated via the adsorbed protein layer. Plasma protein adsorption at the tissue bioactive glass interface has been largely overlooked. Herein, we compare crystalline and amorphous bioactive glass 45S5, in both melt-derived as well as sol-gel forms. Thus, allowing for a detailed understanding of both the role of crystallinity and powder morphology on surface ions, and plasma protein adsorption. It was found that sol-gel 45S5 powders, regardless of crystallinity, adsorbed 3-5 times as much protein as the crystalline melt-derived counterpart, as well as a greater variety of plasma proteins. The devitrification of melt-cast 45S5 resulted in only small differences in the amount and variety of the adsorbed proteome. Surface properties, and not material crystallinity, play a role in directing protein adsorption phenomena for bioactive glasses given the differences found between crystalline melt-cast 45S5 and sol-gel derived 45S5.

  13. Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.

    Science.gov (United States)

    van Veen, Harrie A; Koiter, Jaco; Vogelezang, Carla J M; van Wessel, Noucha; van Dam, Tijtje; Velterop, Ingeborg; van Houdt, Kristina; Kupers, Luc; Horbach, Danielle; Salaheddine, Mourad; Nuijens, Jan H; Mannesse, Maurice L M

    2012-12-31

    C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Human Recombinant Peptide Sponge Enables Novel, Less Invasive Cell Therapy for Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Michiyuki Miyamoto

    2018-01-01

    Full Text Available Bone marrow stromal cell (BMSC transplantation has the therapeutic potential for ischemic stroke. However, it is unclear which delivery routes would yield both safety and maximal therapeutic benefits. We assessed whether a novel recombinant peptide (RCP sponge, that resembles human collagen, could act as a less invasive and beneficial scaffold in cell therapy for ischemic stroke. BMSCs from green fluorescent protein-transgenic rats were cultured and Sprague–Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAo. A BMSC-RCP sponge construct was transplanted onto the ipsilateral intact neocortex 7 days after MCAo. A BMSC suspension or vehicle was transplanted into the ipsilateral striatum. Rat motor function was serially evaluated and histological analysis was performed 5 weeks after transplantation. The results showed that BMSCs could proliferate well in the RCP sponge and the BMSC-RCP sponge significantly promoted functional recovery, compared with the vehicle group. Histological analysis revealed that the RCP sponge provoked few inflammatory reactions in the host brain. Moreover, some BMSCs migrated to the peri-infarct area and differentiated into neurons in the BMSC-RCP sponge group. These findings suggest that the RCP sponge may be a promising candidate for animal protein-free scaffolds in cell therapy for ischemic stroke in humans.

  15. Effects of human recombinant-interferon β in experimental autoimmune encephalomyelitis in guinea pigs.

    Science.gov (United States)

    Aritake, Kosuke; Koh, Chang-Sung; Inoue, Atsushi; Yabuuchi, Fumie; Kitagaki, Kunihiko; Ikoma, Yukihiro; Hayashi, Shigehiro

    2010-11-01

    Although clinical data for beneficial effects of Betaferon, human recombinant-interferon (r-IFN) β-1b, are accumulating, what is less evident is how and why it works. The present study was carried out to examine whether Betaferon suppresses progression of experimental autoimmune encephalomyelitis (EAE). The EAE model was employed in guinea pigs in vivo, and mononuclear cell proliferation and 2',5'-oligoadenylate synthetase activity were assessed in vitro. Betaferon was more reactive in two assays of guinea pigs, mitogen-induced proliferation of peripheral blood mononuclear cells and 2',5'-oligoadenylate synthetase activity of blood, than in rats and rabbits. Guinea pigs were immunized actively by antigen, porcine myelin basic protein. The neurological deficits were assessed by clinical signs scored daily. Guinea pig Betaferon, replaced with guinea pig albumin (GPA), at 1.2 and 12.0 MIU/kg/day or vehicle was administered subcutaneously daily for 20 days in the immunized guinea pigs. GPA-Betaferon suppressed the manifestation of ataxia or more progression of chronic neurological deficits significantly at 1.2 MIU/kg (p guinea pigs with evidence for higher susceptibility of animal cells/tissues to the human cytokine, in contrast with rodents and rabbits.

  16. Human xanthine oxidase recombinant in E. coli: A whole cell catalyst for preparative drug metabolite synthesis.

    Science.gov (United States)

    Ferreira Antunes, Márcia; Eggimann, Fabian Kurt; Kittelmann, Matthias; Lütz, Stephan; Hanlon, Steven P; Wirz, Beat; Bachler, Thorsten; Winkler, Margit

    2016-10-10

    Human xanthine oxidoreductase (XOR), which is responsible for the final steps of the purine metabolism pathway and involved in oxidative drug metabolism, was successfully expressed in Escherichia coli BL21(DE3) Gold. Recombinant human (rh) XOR yielded higher productivity with the gene sequence optimized for expression in E.coli than with the native gene sequence. Induction of XOR expression with lactose or IPTG resulted in complete loss of activity whereas shake flasks cultures using media rather poor in nutrients resulted in functional XOR expression in the stationary phase. LB medium was used for a 25L fermentation in fed-batch mode, which led to a 5 fold increase of the enzyme productivity when compared to cultivation in shake flasks. Quinazoline was used as a substrate on the semi-preparative scale using an optimized whole cell biotransformation protocol, yielding 73mg of the isolated product, 4-quinazolinone, from 104mg of starting material. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Structural consistency analysis of recombinant and wild-type human serum albumin

    Science.gov (United States)

    Cao, Hui-Ling; Sun, Li-Hua; Liu, Li; Li, Jian; Tang, Lin; Guo, Yun-Zhu; Mei, Qi-Bing; He, Jian-Hua; Yin, Da-Chuan

    2017-01-01

    Recombinant human serum albumin (rHSA) is potential alternatives for human serum albumin (HSA) which may ease severe shortage of HSA worldwide. In theory, rHSA and HSA are the same. Structure decides function. Therefore, the 3D structural consistency analysis of rHSA and HSA is outmost importance, which is the base of their function consistency. In this paper, the crystal structures of rHSA at resolution limit of 2.22 Å and HSA at 2.30 Å were determined by X-ray diffraction (XRD), which were deposited in the Protein Data Bank (PDB) with accession codes 4G03 (rHSA) and 4G04 (HSA). The differences between rHSA and HSA were systematically analyzed from the crystallization behavior, diffraction data and three-dimensional (3D) structure. The superimposed contrasted analysis indicated that rHSA and HSA achieved a structural similarity of 99% with an r.m.s. deviation of 0.397 Å for the corresponding overall Cα atoms. In addition, the number of α-helices in the rHSA or HSA molecule was verified to be 30. As a result, rHSA can potentially replace HSA. The study provides a theoretical and experimental basis for the clinical and additional applications of rHSA. Meanwhile, it is also a good example for applications of genetic engineering.

  18. The red queen model of recombination hotspots evolution in the light of archaic and modern human genomes.

    Science.gov (United States)

    Lesecque, Yann; Glémin, Sylvain; Lartillot, Nicolas; Mouchiroud, Dominique; Duret, Laurent

    2014-11-01

    Recombination is an essential process in eukaryotes, which increases diversity by disrupting genetic linkage between loci and ensures the proper segregation of chromosomes during meiosis. In the human genome, recombination events are clustered in hotspots, whose location is determined by the PRDM9 protein. There is evidence that the location of hotspots evolves rapidly, as a consequence of changes in PRDM9 DNA-binding domain. However, the reasons for these changes and the rate at which they occur are not known. In this study, we investigated the evolution of human hotspot loci and of PRDM9 target motifs, both in modern and archaic human lineages (Denisovan) to quantify the dynamic of hotspot turnover during the recent period of human evolution. We show that present-day human hotspots are young: they have been active only during the last 10% of the time since the divergence from chimpanzee, starting to be operating shortly before the split between Denisovans and modern humans. Surprisingly, however, our analyses indicate that Denisovan recombination hotspots did not overlap with modern human ones, despite sharing similar PRDM9 target motifs. We further show that high-affinity PRDM9 target motifs are subject to a strong self-destructive drive, known as biased gene conversion (BGC), which should lead to the loss of the majority of them in the next 3 MYR. This depletion of PRDM9 genomic targets is expected to decrease fitness, and thereby to favor new PRDM9 alleles binding different motifs. Our refined estimates of the age and life expectancy of human hotspots provide empirical evidence in support of the Red Queen hypothesis of recombination hotspots evolution.

  19. Population Pharmacokinetic Modelling of FE 999049, a Recombinant Human Follicle-Stimulating Hormone, in Healthy Women After Single Ascending Doses

    OpenAIRE

    Rose, Trine H?yer; R?shammar, Daniel; Erichsen, Lars; Grundemar, Lars; Ottesen, Johnny T.

    2016-01-01

    Objective The purpose of this analysis was to develop a population pharmacokinetic model for a novel recombinant human follicle-stimulating hormone (FSH) (FE 999049) expressed from a human cell line of foetal retinal origin (PER.C6?) developed for controlled ovarian stimulation prior to assisted reproductive technologies. Methods Serum FSH levels were measured following a single subcutaneous FE 999049 injection of 37.5, 75, 150, 225 or 450?IU in 27 pituitary-suppressed healthy female subjects...

  20. The red queen model of recombination hotspots evolution in the light of archaic and modern human genomes.

    Directory of Open Access Journals (Sweden)

    Yann Lesecque

    2014-11-01

    Full Text Available Recombination is an essential process in eukaryotes, which increases diversity by disrupting genetic linkage between loci and ensures the proper segregation of chromosomes during meiosis. In the human genome, recombination events are clustered in hotspots, whose location is determined by the PRDM9 protein. There is evidence that the location of hotspots evolves rapidly, as a consequence of changes in PRDM9 DNA-binding domain. However, the reasons for these changes and the rate at which they occur are not known. In this study, we investigated the evolution of human hotspot loci and of PRDM9 target motifs, both in modern and archaic human lineages (Denisovan to quantify the dynamic of hotspot turnover during the recent period of human evolution. We show that present-day human hotspots are young: they have been active only during the last 10% of the time since the divergence from chimpanzee, starting to be operating shortly before the split between Denisovans and modern humans. Surprisingly, however, our analyses indicate that Denisovan recombination hotspots did not overlap with modern human ones, despite sharing similar PRDM9 target motifs. We further show that high-affinity PRDM9 target motifs are subject to a strong self-destructive drive, known as biased gene conversion (BGC, which should lead to the loss of the majority of them in the next 3 MYR. This depletion of PRDM9 genomic targets is expected to decrease fitness, and thereby to favor new PRDM9 alleles binding different motifs. Our refined estimates of the age and life expectancy of human hotspots provide empirical evidence in support of the Red Queen hypothesis of recombination hotspots evolution.

  1. Recombinant human parathyroid hormone related protein 1-34 and 1-84 and their roles in osteoporosis treatment.

    Directory of Open Access Journals (Sweden)

    Hua Wang

    Full Text Available Osteoporosis is a common disorder characterized by compromised bone strength that predisposes patients to increased fracture risk. Parathyroid hormone related protein (PTHrP is one of the candidates for clinical osteoporosis treatment. In this study, GST Gene Fusion System was used to express recombinant human PTHrP (hPTHrP 1-34 and 1-84. To determine whether the recombinant hPTHrP1-34 and 1-84 can enhance renal calcium reabsorption and promote bone formation, we examined effects of recombinant hPTHrP1-34 and 1-84 on osteogenic lineage commitment in a primary bone marrow cell culture system and on osteoporosis treatment. Results revealed that both of recombinant hPTHrP1-34 and 1-84 increased colony formation and osteogenic cell differentiation and mineralization in vitro; however, the effect of recombinant hPTHrP1-84 is a little stronger than that of hPTHrP1-34. Next, ovariectomy was used to construct osteoporosis animal model (OVX to test activities of these two recombinants in vivo. HPTHrP1-84 administration elevated serum calcium by up-regulating the expression of renal calcium transporters, which resulted in stimulation of osteoblastic bone formation. These factors contributed to augmented bone mass in hPTHrP1-84 treated OVX mice but did not affect bone resorption. There was no obvious bone mass alteration in hPTHrP1-34 treated OVX mice, which may be, at least partly, associated with shorter half-life of hPTHrP1-34 compared to hPTHrP1-84 in vivo. This study implies that recombinant hPTHrP1-84 is more effective than hPTHrP1-34 to enhance renal calcium reabsorption and to stimulate bone formation in vivo.

  2. Hot topic: Holder pasteurization of human milk affects some bioactive proteins.

    Science.gov (United States)

    Guerra, André F; Mellinger-Silva, Caroline; Rosenthal, Amauri; Luchese, Rosa H

    2018-04-01

    The aim of this research was to investigate the effect of Holder pasteurization (HoP; 62.5°C, 30 min) on the protein profile and activities of glutathione peroxidase (GPx) and lysozyme (LZ) in human milk. Over 6 mo of lactation, human milk samples were analyzed before (raw) and after HoP for GPx and LZ activity and electrophoresis protein profile. Holder pasteurization reduced human milk lactoferrin, immunoglobulin fractions, and GPx activity. In addition, GPx activity, which is high in colostrum and transitional milk, was naturally reduced over the 6-mo lactation period. In contrast, HoP did not affect human milk LZ activity. Besides its critical cellular antioxidant role in protecting the organism from oxidative damage, GPx decreases the redox potential of milk, stimulating the growth of anaerobic microorganisms, such as the probiotic Bifidobacterium. Considering the role of lactoferrin in infant health, we conclude that an important part of its function has been inactivated by pasteurization. These compounds should be replaced by human milk banks after the HoP step to recover lost functionality. Otherwise, an alternative technology to HoP that better retains human milk properties should be used by milk banks to eliminate the risk of transmission of infectious agents. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Utility of P19 Gene-Silencing Suppressor for High Level Expression of Recombinant Human Therapeutic Proteins in Plant Cells

    Directory of Open Access Journals (Sweden)

    Maryam Zangi

    2016-07-01

    Full Text Available Background: The potential of plants, as a safe and eukaryotic system, is considered in the production of recombinant therapeutic human protein today; but the expression level of heterologous proteins is limited by the post-transcriptional gene silencing (PTGS response in this new technology. The use of viral suppressors of gene silencing can prevent PTGS and improve transient expression level of foreign proteins. In this study, we investigated the effect of p19 silencing suppressor on recombinant human nerve growth factor expression in Nicotiana benthamiana. Materials and Methods: The p19 coding region was inserted in the pCAMBIA using NcoI and BstEII recognition sites. Also, the cloned synthesized recombinant human NGF (rhNGF fragment was cloned directly into PVX vector by ClaI and SalI restriction enzymes. The co-agroinfiltration of rhNGF with p19 viral suppressor of gene silencing was evaluated by dot-blot and SDS-PAGE. The amount of expressed rhNGF protein was calculated by AlphaEaseFC software. Results: Co-agroinfiltration of hNGF with P19 suppressor showed about forty-fold increase (8% total soluble protein (TSP when compared to the absence of P19 suppressor (0.2%TSP. Conclusion: The results presented here confirmed that the use of P19 gene silencing suppressor derived from tomato bushy stunt virus (TBSV could efficiently increase the transient expression of recombinant proteins in Nicotiana benthamiana manifold.

  4. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

    Science.gov (United States)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

    2015-04-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    DEFF Research Database (Denmark)

    Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role...

  6. Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

    Directory of Open Access Journals (Sweden)

    Bruno Gottstein

    2014-06-01

    Full Text Available Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2 and cathepsin L-1 (recCL1, were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES from adult stage liver flukes was assessed by receiver operator characteristic (ROC analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20, patients with other parasitic infections (n=87 and patients with malignancies (n=121. The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy employing the threshold (cut-off to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

  7. Comparative Assessment of ELISAs Using Recombinant Saposin-Like Protein 2 and recombinant Cathepsin L-1 from Fasciola hepatica for the Serodiagnosis of Human Fasciolosis

    Science.gov (United States)

    Gottstein, Bruno; Schneeberger, Marianne; Boubaker, Ghalia; Merkle, Bernadette; Huber, Cristina; Spiliotis, Markus; Müller, Norbert; Garate, Teresa; Doherr, Marcus G.

    2014-01-01

    Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory–secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n = 20), patients with other parasitic infections (n = 87) and patients with malignancies (n = 121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls. PMID:24922050

  8. Protective effects of recombinant human brain natriuretic peptide in perioperative period during open heart surgery.

    Science.gov (United States)

    Xu, Yunbin; Li, Yong; Bao, Weiguo; Qiu, Shi

    2018-03-01

    The aim of the present study was to evaluate the protective effects and safety aspects of recombinant human brain natriuretic peptide (rhBNP) on cardiac functions of patients undergoing open-heart surgery during perioperative period. In total, 150 patients undergoing open heart surgery in the Second Hospital of Shandong Universty from August 2015 to July 2016 were randomly divided into control group and observation group each with 75 cases. Patients in control group were treated by routine rehabilitation while patients in the observation group were treated by both the routine rehabilitation and rhBNP. All the observations were made before operation, after operation and 7 days after operation. The changes of N-terminal pro-brain natriuretic peptide (NT-proBNP) of patients, the left ventricular ejection fraction (LVEF), cardiac function [Cardiac output (CO), pulmonary capillary wedge pressure (PAWP) and central venous pressure (CVP)] of patients were measured. Further, respirator support time, ICU stay time, incidence of complications and vital signs (BP, HR, SaO2) of patients in the two groups were also compared. NT-proBNP levels of all patients improved after operation but it decreased in both groups after 7 days of operation. The decrease of NT-proBNP levels in observation group was significantly higher than that of control group. Whereas, LVEF, CO, PAWP and CVP of patients in both the groups increased after operation but effects were significantly higher in the observation group after 7 days of medication. Respirator support time and ICU stay time of patients in observation group were significantly shorter than those in control group, and the incidence of postoperative complications of patients in the observation group were significantly lower than the control group. Moreover, BP, HR and SaO2 of patients in observation group were significantly elevated in comparison to control group (P<0.05). Recombinant human brain natriuretic peptide (rhBNP) could significantly

  9. Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Farooqahmed S Kittur

    Full Text Available Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P (20 U/ml provides 2-fold better cytoprotection (44% to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M (21%. The cytoprotective effect of the asialo-rhuEPO(P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2 and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

  10. Cytotoxic effects of Mangifera indica L. kernel extract on human breast cancer (MCF-7 and MDA-MB-231 cell lines) and bioactive constituents in the crude extract.

    Science.gov (United States)

    Abdullah, Al-Shwyeh Hussah; Mohammed, Abdulkarim Sabo; Abdullah, Rasedee; Mirghani, Mohamed Elwathig Saeed; Al-Qubaisi, Mothanna

    2014-06-25

    Waterlily Mango (Mangifera indica L.) is thought to be antioxidant-rich, conferred by its functional phytochemicals. The potential anticancer effects of the ethanolic kernel extract on breast cancer cells (MDA-MB-231 and MCF-7) using MTT, anti-proliferation, neutral red (NR) uptake and lactate dehydrogenase (LDH) release assays were evaluated. Cytological studies on the breast cancer cells were also conducted, and phytochemical analyses of the extract were carried out to determine the likely bioactive compounds responsible for such effects. Results showed the extract induced cytotoxicity in MDA-MB-231 cells and MCF-7 cells with IC50 values of 30 and 15 μg/mL, respectively. The extract showed significant toxicity towards both cell lines, with low toxicity to normal breast cells (MCF-10A). The cytotoxic effects on the cells were further confirmed by the NR uptake, antiproliferative and LDH release assays. Bioactive analyses revealed that many bioactives were present in the extract although butylated hydroxytoluene, a potent antioxidant, was the most abundant with 44.65%. M. indica extract appears to be more cytoxic to both estrogen positive and negative breast cancer cell lines than to normal breast cells. Synergistic effects of its antioxidant bioactives could have contributed to the cytotoxic effects of the extract. The extract of M. indica, therefore, has potential anticancer activity against breast cancer cells. This potential is worth studying further, and could have implications on future studies and eventually management of human breast cancers.

  11. Fruit quality and bioactive compounds relevant to human health of sweet cherry (Prunus avium L.) cultivars grown in Italy.

    Science.gov (United States)

    Ballistreri, Gabriele; Continella, Alberto; Gentile, Alessandra; Amenta, Margherita; Fabroni, Simona; Rapisarda, Paolo

    2013-10-15

    The fruit quality characteristics, phenolic compounds and antioxidant capacities of 24 sweet cherry (Prunus avium L.) cultivars grown on the mountainsides of the Etna volcano (Sicily, Italy) were evaluated. High-performance liquid chromatographic methods were used to identify and quantify sugars, organic acids and phenolics. A total of seven phenolic compounds were characterised as hydroxycinnamic acid derivatives (neochlorogenic acid, p-coumaroylquinic acid and chlorogenic acid) and anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside, pelargonidin 3-rutinoside and peonidin 3-rutinoside). The total anthocyanin content ranged from 6.21 to 94.20mg cyanidin 3-glucoside equivalents/100g fresh weight (FW), while the total phenol content ranged from 84.96 to 162.21mg gallic acid equivalents/100g FW. The oxygen radical absorbance capacity (ORAC) assay indicated that fruit of all genotypes possessed considerable antioxidant activity. The high level of phenolic compounds and antioxidant capacity of some sweet cherry fruits implied that they might be sources of bioactive compounds that are relevant to human health. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Encapsulating Bioactive Hydrogels Improve Rat Heart Function Post Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Andre Chow

    2017-11-01

    Full Text Available Tissue engineering offers an exciting possibility for cardiac repair post myocardial infarction. We assessed the effects of combined polyethylene glycol hydrogel (PEG, human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM, and erythropoietin (EPO therapy in a rat model of myocardial infarction. PEG with/out iPSC-CMs and EPO; iPSC-CMs in saline; or saline alone was injected into infarcted hearts shortly after infarction. Injection of almost any combination of the therapeutics limited acute elevations in chamber volumes. After 10 weeks, attenuation of ventricular remodeling was identified in all groups that received PEG injections, while ejection fractions were significantly increased in the gel-EPO, cell, and gel-cell-EPO groups. In all treatment groups, infarct thickness was increased and regions of muscle were identified within the scar. However, no grafted cells were detected. Hence, iPSC-CM-encapsulating bioactive hydrogel therapy can improve cardiac function post myocardial infarction and increase infarct thickness and muscle content despite a lack of sustained donor-cell engraftment.

  13. Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

    Directory of Open Access Journals (Sweden)

    Zanotto Carlo

    2011-11-01

    Full Text Available Abstract Background Human papilloma virus (HPV-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1 have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species. Methods A new fowlpox virus recombinant encoding HPV-L1 (FPL1 was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays. Results The FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector. Conclusion This FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

  14. Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2

    International Nuclear Information System (INIS)

    Vita, N.; Magazin, M.; Marchese, E.; Lupker, J.; Ferrara, P.

    1990-01-01

    We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2

  15. Twin arginine translocation system in secretory expression of recombinant human growth hormone.

    Science.gov (United States)

    Bagherinejad, Mohammad Reza; Sadeghi, Hamid Mir-Mohammad; Abedi, Daryoush; Chou, C Perry; Moazen, Fatemeh; Rabbani, Mohammad

    2016-12-01

    Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

  16. Pharmacokinetics of recombinant human erythropoietin applied subcutaneously to children with chronic renal failure.

    Science.gov (United States)

    Braun, A; Ding, R; Seidel, C; Fies, T; Kurtz, A; Schärer, K

    1993-02-01

    The single-dose pharmacokinetics of recombinant human erythropoietin (rHuEPO) given SC was investigated in 20 patients aged 7-20 years at different stages of chronic renal failure. In a pilot study we confirmed the lower bioavailability of the drug in 2 children when given SC compared with the IV route (24% and 43%, respectively). Following administration of 4,000 units/m2, rHuEPO SC effective serum erythropoietin concentrations increased from a mean baseline level (+/- SD) of 23 +/- 13 units/l to a mean peak concentration of 265 +/- 123 units/l, which was reached after 14.3 +/- 9.4 h, followed by a slow decline until baseline values were attained at 72 h. Mean residence time was 30 +/- 9 h and mean elimination half-time 14.3 +/- 7 h. The single-dose kinetics of SC rHuEPO in children with different degrees of renal failure are comparable to those in adult patients. Possibly, the higher efficacy of SC rHuEPO in patients with renal anaemia compared with IV rHuEPO is related to its prolonged action.

  17. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution

    DEFF Research Database (Denmark)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross......-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N......-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI...

  18. Inhibition of the recombinant human endostatin adenavirus on experimental choroidal neovascularization in rats

    Directory of Open Access Journals (Sweden)

    Li-Juan Chen

    2017-06-01

    Full Text Available AIM: To investigate the inhibition of the recombinant human endostatin adenavirus(Ad-Eson the experimental choroidal neovascularization(CNVmodels by intravitreous injection. METHODS: Experimental CNV models were induced by semiconductor laser in 30 male Brown Norway(BNrats and randomly divided into 3 groups with 10 rats in each group. At 21d after photocoagulation, the single administration group were given intravitreous injection with Ad-Es 0.01mL; the repeated administration group were given intravitreous injection with Ad-Es 0.01mL and a repeated injection 7d later; the saline control group were given intravitreous injection with saline 0.01mL. At 7d after final administration, the leakage of fundus fluorescein angiography(FFAwas observed. Various CNV areas were measured by using laser confocal microscopy of choroidal flatmount method. Pathology and ultrastructure were observed with light microscopy, the expressions of CD105 were measured by immunohistochemistry. RESULTS: The leakage of CNV of the administration group abviously decreased as compared with those in the saline group, the leakage of repeated administration group decreased compared with that of single administration group(PPCONCLUSION: Ad-Es can effectively inhibit semiconductor laser induced CNV in BN rats, and the inhibition effect of repeated administration group is better than that of single administration group. It may be a useful new method in the treatment of CNV.

  19. Induction of circulating phospholipase A2 by intravenous administration of recombinant human tumour necrosis factor

    Directory of Open Access Journals (Sweden)

    Waldemar Pruzanski

    1992-01-01

    Full Text Available We have examined the effects of intravenous infusion of recombinant human tumour necrosis factor (rh-TNF on serum activity of phospholipase A2 (PLA2 in patients with malignancies. Nine patients received a 24 h continuous intravenous infusion ranging from 1.0 × 105 U/m2 to 3.0 × 105 U/m2; 14 patients received a 5 day continuous intravenous infusion ranging from 0.5 × 105 U/m2/day to 3.0 105 U/m2/day. Twenty one of 23 patients responded with marked increases in serum PLA2 activity that were detectable 3 h after the beginning of the rh-TNF infusion and reached maximum levels at 18 h with a mean increase of 16.2-fold. In patients receiving a 5 day rh-TNF infusion, the highest levels of PLA2 were observed after the first day of infusion. Serum PLA2 activity declined continuously to 2.9-fold above baseline at the end of the infusion. A significant correlation was noted between the dose of infused rh-TNF and the maximum increase in PLA2 activity. To our knowledge, this is the first time that an association between intravenous TNF administration and induction of circulating PLA2 in man has been established.

  20. Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

    Directory of Open Access Journals (Sweden)

    Li-Xi Niu

    2015-04-01

    Full Text Available Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9 light chain (hEKL gene was designed and artificially synthesized with built-in codon blas towards Escherichia colicodon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3. Recombinant hEKL protein with a maltose binding protein (MBP tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95% by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate. Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen. From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104U/mg.

  1. Annulated heterocyclic bioisosteres of norarecoline. Synthesis and molecular pharmacology at five recombinant human muscarinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Ebert, B; Brann, M R

    1995-01-01

    A series of O-alkylated analogs of 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO) were synthesized and characterized as ligands for muscarinic acetylcholine receptors (mAChRs). O-Methyl-THAO (4a), O-ethyl-THAO (4b), O-isopropyl-THAO (4c), and O-propargyl-THAO (4d) were shown to be potent...... inhibitors of the binding of tritiated quinuclidinyl benzilate (QNB), pirenzepine (PZ), and oxotremorine-M (Oxo-M) to tissue membrane preparations. In the [3H]-Oxo-M binding assay, receptor affinities in the low nanomolar range were measured for 4a (IC50 = 0.010 microM), 4b (IC50 = 0.003 microM), 4c (IC50...... = 0.011 microM), and 4d (IC50 = 0.0008 microM). Pharmacological effects (EC50 or Ki values) and intrinsic activities (per cent of maximal carbachol responses) were determined using five recombinant human mAChRs (m1-m5) and the functional assay, receptor selection and amplification technology (R...

  2. Height Outcome of Recombinant Human Growth Hormone Treatment in Achondroplasia Children: A Meta-Analysis.

    Science.gov (United States)

    Miccoli, Mario; Bertelloni, Silvano; Massart, Francesco

    2016-01-01

    Although recombinant human growth hormone (rhGH) is not approved to treat short stature of achondroplasia (ACH), some studies suggested growth improvement during short-term rhGH treatment. A meta-analysis of rhGH therapy efficacy in ACH children was performed. From 12 English-language studies, 558 (54.0% males) rhGH-treated ACH children were enrolled. Administration of rhGH (median dosage 0.21 mg/kg/ week; range 0.16-0.42 mg/kg/week) improved height (Ht) from baseline [-5.069 standard deviation score (SDS; 95% CI -5.109 to -5.029); p < 0.0001] to 12 [-4.325 SDS (95% CI -4.363 to -4.287); p < 0.0001] and 24 months [-4.073 SDS (95% CI -4.128 to -4.019); p < 0.0001]. Then, Ht remained approximately constant up to 5 years [-3.941 SDS (95% CI -4.671 to -3.212); p < 0.0001]. In ACH children, rhGH treatment increased Ht from -5.0 to -4.0 SDS during 5 years, but insufficient data are available on both the adult Ht and the changes of body proportions. © 2016 S. Karger AG, Basel.

  3. FSHbeta gene mutation in a female with delayed puberty and hypogonadism: response to recombinant human FSH.

    Directory of Open Access Journals (Sweden)

    S Alain

    2010-01-01

    Full Text Available We report a woman with primary amenorrhoea and infertility associated with an isolated deficiency of pituitary FSH that does not respond to GnRH administration. Serum inhibin B was undetectable and antimullerian hormone (AMH was within the normal range. Ultra sound examination revealed a small uterus and small ovaries with few small follicles. We identified an homozygous 1-bp (G deletion at codon 79 in FSHbeta gene suggesting a complete loss of function. The patient underwent studies of ovarian responsiveness to recombinant human FSH according to the following protocol: 150UI/d for five days following by 75 UI/d for 10 days. Estradiol plasma level started to increase from day 5 associated to a sharp increase of inhibine B and a decrease of LH. During the same time, we observed an excessive development of multiple follicles resulting in an arrest of the treatment to avoid hyperstimulation. The present study confirm that follicles up to 5 mm in diameter had developed in the absence of FSH and that FSH is required for the growth of follicles beyond the two-layer granulose stage.

  4. Cortical bone growth and maturational changes in dwarf rats induced by recombinant human growth hormone

    Science.gov (United States)

    Martinez, D. A.; Orth, M. W.; Carr, K. E.; Vanderby, R. Jr; Vailas, A. C.

    1996-01-01

    The growth hormone (GH)-deficient dwarf rat was used to investigate recombinant human (rh) GH-induced bone formation and to determine whether rhGH facilitates simultaneous increases in bone formation and bone maturation during rapid growth. Twenty dwarf rats, 37 days of age, were randomly assigned to dwarf plus rhGH (GH; n = 10) and dwarf plus vehicle (n = 10) groups. The GH group received 1.25 mg rhGH/kg body wt two times daily for 14 days. Biochemical, morphological, and X-ray diffraction measurements were performed on the femur middiaphysis. rhGH stimulated new bone growth in the GH group, as demonstrated by significant increases (P < 0.05) in longitudinal bone length (6%), middiaphyseal cross-sectional area (20%), and the amount of newly accreted bone collagen (28%) in the total pool of middiaphyseal bone collagen. Cortical bone density, mean hydroxyapatite crystal size, and the calcium and collagen contents (microgram/mm3) were significantly smaller in the GH group (P < 0.05). Our findings suggest that the processes regulating new collagen accretion, bone collagen maturation, and mean hydroxyapatite crystal size may be independently regulated during rapid growth.

  5. Efficacy of protamine zinc recombinant human insulin for controlling hyperglycemia in dogs with diabetes mellitus.

    Science.gov (United States)

    Maggiore, A Della; Nelson, R W; Dennis, J; Johnson, E; Kass, P H

    2012-01-01

    Alternative insulin preparations are needed when NPH insulin is ineffective in diabetic dogs. This study evaluated the efficacy of recombinant human protamine zinc insulin (rhPZI) for treating diabetic dogs. rhPZI is effective for treating diabetic dogs. Six newly diagnosed and 11 insulin-treated diabetic dogs. Prospective clinical trial. Dogs were treated with rhPZI for 60 days. Control of glycemia was assessed on days 7, 14, 30, and 60 by evaluation of history, physical examination, body weight, serum fructosamine concentration, and blood glucose concentrations measured before and 2, 4, 6, 8, and 10 hours after rhPZI administration. Adjustments in dosage of rhPZI were made as needed to control glycemia. rhPZI administration resulted in a significant decrease in 10-hour mean blood glucose (MBG(10h) ; 299 ± 115 versus 457 ± 38 mg/dL, X ± SD, P = .0003) and serum fructosamine (478 ± 83 versus 557 ± 104 μmol/L, P = .006) concentration at day 60, compared with day 1, respectively. By day 60, polyuria and polydipsia had improved in 14, body weight was stable or increased in 16, MBG(10h) had decreased in 16, and serum fructosamine concentration had decreased in 11 of 17 dogs, compared with day 1. Hypoglycemia (diabetic dogs and can be considered as an alternative treatment in diabetic dogs that are poorly controlled using other insulin preparations. Copyright © 2011 by the American College of Veterinary Internal Medicine.

  6. Outcomes of Alveolar Ridge Preservation With Recombinant Human Bone Morphogenetic Protein-2: A Systematic Review.

    Science.gov (United States)

    Moslemi, Neda; Khoshkam, Vahid; Rafiee, Sahar; Bahrami, Naghmeh; Aslroosta, Hoori

    2018-02-01

    The main focused question of this systematic review was as follows: Does the application of recombinant human bone morphogenetic protein-2 (rhBMP-2) placed in extraction sockets reduce the alveolar ridge changes? A systematic literature search was performed up to February 2017. Clinical studies published in English were included. Outcome variables of interest were as follows: changes in alveolar ridge width and height, the quality of new bone, patient's safety, adverse events, and postoperative complications. Seven articles were included. Because of the vast heterogeneity and high risk of bias among the studies, performing a meta-analysis deemed not feasible. Application of rhBMP-2 in the extraction socket was more effective in the reduction of ridge width compared with that of ridge height. The superiority of 1.5 mg/mL rhBMP-2/absorbable collagen sponge over the carrier alone on alveolar ridge width/height remodeling was more significant when it was applied in the sockets with ≥50% buccal bone dehiscence. The limited available data showed that rhBMP-2 did not improve the quality of new bone. Antibodies against rhBMP-2 were detected in the serum in 1 trial. Within the limits of this review, 1.5 mg/mL rhBMP-2 might be beneficial for preserving the alveolar ridge width within extraction sockets given as to whether the cost-effectiveness is justifiable. Studies with lower risk of bias should be performed to confirm the above findings.

  7. Development of the method for microbiological purity testing of recombinant human interleukin-7-based product

    Directory of Open Access Journals (Sweden)

    O. Yu. Galkin

    2017-06-01

    Full Text Available The goal of the work was justification of standardization parameters of the product based on recombinant human IL-7 by “microbiological purity” parameter and development of its measurement method with consideration of the requirements of guideline documents regarding quality of medicinal pro­ducts. Suitabi­lity assessment of microbiological purity testing procedures was performed. It has been established that the studied­ product in standard testing methods possessed antimicrobial activity against test microorganisms Basillus subtilis ATCC 6633 (on soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic, Candida albicans АТСС 10231 (on soybean casein digest agar and Sabouraud dextrose agar containing an antibiotic, and possessed no antimicrobial activity against test microorganisms Staphylococcus aureus ATCC 6538 (on soybean casein digest agar and Pseudomonas aeruginosa ATCC 9027 (on soybean casein digest agar. The efficacy of lecithin and polysorbate as substances eliminating antibacterial effect in combination with 50-fold dilution of the test sample has been suggested and proven for the purpose of antimicrobial activity neutralization. The developed total aerobic microbial count measurement procedure for the test product conforms to acceptance criteria provided by SPU (2.6.12, and can be used for its microbiological purity control.

  8. Resistance to Recombinant Human Erythropoietin Therapy in a Rat Model of Chronic Kidney Disease Associated Anemia.

    Science.gov (United States)

    Garrido, Patrícia; Ribeiro, Sandra; Fernandes, João; Vala, Helena; Rocha-Pereira, Petronila; Bronze-da-Rocha, Elsa; Belo, Luís; Costa, Elísio; Santos-Silva, Alice; Reis, Flávio

    2015-12-25

    This study aimed to elucidate the mechanisms explaining the persistence of anemia and resistance to recombinant human erythropoietin (rHuEPO) therapy in a rat model of chronic kidney disease (CKD)-associated anemia with formation of anti-rHuEPO antibodies. The remnant kidney rat model of CKD induced by 5/6 nephrectomy was used to test a long-term (nine weeks) high dose of rHuEPO (200 UI/kg bw/week) treatment. Hematological and biochemical parameters were evaluated as well as serum and tissue (kidney, liver and/or duodenum) protein and/or gene expression of mediators of erythropoiesis, iron metabolism and tissue hypoxia, inflammation, and fibrosis. Long-term treatment with a high rHuEPO dose is associated with development of resistance to therapy as a result of antibodies formation. In this condition, serum EPO levels are not deficient and iron availability is recovered by increased duodenal absorption. However, erythropoiesis is not stimulated, and the resistance to endogenous EPO effect and to rHuEPO therapy results from the development of a hypoxic, inflammatory and fibrotic milieu in the kidney tissue. This study provides new insights that could be important to ameliorate the current therapeutic strategies used to treat patients with CKD-associated anemia, in particular those that become resistant to rHuEPO therapy.

  9. Allelic recombination between distinct genomic locations generates copy number diversity in human β-defensins

    Science.gov (United States)

    Bakar, Suhaili Abu; Hollox, Edward J.; Armour, John A. L.

    2009-01-01

    β-Defensins are small secreted antimicrobial and signaling peptides involved in the innate immune response of vertebrates. In humans, a cluster of at least 7 of these genes shows extensive copy number variation, with a diploid copy number commonly ranging between 2 and 7. Using a genetic mapping approach, we show that this cluster is at not 1 but 2 distinct genomic loci ≈5 Mb apart on chromosome band 8p23.1, contradicting the most recent genome assembly. We also demonstrate that the predominant mechanism of change in β-defensin copy number is simple allelic recombination occurring in the interval between the 2 distinct genomic loci for these genes. In 416 meiotic transmissions, we observe 3 events creating a haplotype copy number not found in the parent, equivalent to a germ-line rate of copy number change of ≈0.7% per gamete. This places it among the fastest-changing copy number variants currently known. PMID:19131514

  10. How bio-questionable are the different recombinant human erythropoietin copy products in Thailand?

    Science.gov (United States)

    Halim, Liem Andhyk; Brinks, Vera; Jiskoot, Wim; Romeijn, Stefan; Praditpornsilpa, Kearkiat; Assawamakin, Anunchai; Schellekens, Huub

    2014-05-01

    The high prevalence of pure red cell aplasia in Thailand has been associated with the sharp increase in number of recombinant human erythropoietin (rhEPO) copy products, based on a classical generic regulatory pathway, which have entered the market. This study aims to assess the quality of rhEPO copy products being used in Thailand. Twelve rhEPO copy products were purchased from pharmacies in Thailand, shipped under controlled cold chain conditions to the Netherlands and characterized using (1) high performance size-exclusion chromatography, (2) asymmetrical flow field-flow fractionation, (3) sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with (4) Western blotting and additionally tested for (5) host cell protein impurities as well as (6) endotoxin contamination. Some of the tested rhEPO copy products showed high aggregate levels and contained a substantial amount of protein fragments. Also, one of rhEPO copy products had a high endotoxin level, exceeding the FDA limit. Our observations show that some of the tested copy products on the Thai market differ significantly from the originator rhEPO product, Epogen®. This comparison study supports a link between the quality attributes of copy rhEPO products and their immunogenicity.

  11. A novel recombinant human thrombopoietin therapy for the management of immune thrombocytopenia in pregnancy.

    Science.gov (United States)

    Kong, Zhangyuan; Qin, Ping; Xiao, Shan; Zhou, Hai; Li, Hong; Yang, Renchi; Liu, Xiaofan; Luo, Jianmin; Li, Zhichun; Ji, Guochao; Cui, Zhongguang; Bai, Yusheng; Wu, Yuxia; Shao, Linlin; Peng, Jun; Ma, Jun; Hou, Ming

    2017-08-31

    The aim of this study was to determine the safety and efficacy of recombinant human thrombopoietin (rhTPO) for the management of immune thrombocytopenia (ITP) during pregnancy. Pregnant patients with ITP were enrolled in the study if they had a platelet count less than 30 × 10 9 /L, were experiencing bleeding manifestations, had failed to respond to corticosteroids and/or intravenous immunoglobulin (IVIG), and had developed refractoriness to platelet transfusion. Thirty-one patients received rhTPO at an initial dose of 300 U/kg once daily for 14 days. Twenty-three patients responded (74.2%), including 10 complete responders (>100 × 10 9 /L) and 13 responders (30-100 × 10 9 /L). It appears that rhTPO ameliorated the bleeding symptoms remarkably, even in the nonresponders. rhTPO was well tolerated. Dizziness, fatigue, and pain at an injection site were reported in 1 patient each. No congenital disease or developmental delays were observed in the infants in a median follow-up of 53 (range, 39-68) weeks. In conclusion, rhTPO is a potentially safe and effective treatment choice for patients with ITP during pregnancy. Our work has paved the way for further study on the clinical application of rhTPO and other thrombopoietic agents for the management of ITP during pregnancy. This study is registered at www.clinicaltrials.gov as NCT02391272. © 2017 by The American Society of Hematology.

  12. Kinetic investigation of recombinant human hyaluronidase PH20 on hyaluronic acid.

    Science.gov (United States)

    Fang, Shiping; Hays Putnam, Anna-Maria A; LaBarre, Michael J

    2015-07-01

    The kinetic investigation of hyaluronidases using physiologically relevant hyaluronic acid (HA or hyaluronan) substrate will provide useful and important clues to their catalytic behavior and function in vivo. We present here a simple and sensitive method for kinetic measurement of recombinant human hyaluronidase PH20 (rHuPH20) on HA substrates with sizes ranging from 90 to 752 kDa. The method is based on 2-aminobenzamide labeling of hydrolyzed HA products combined with separation by size exclusion-ultra performance liquid chromatography coupled with fluorescence detection. rHuPH20 was found to follow Michaelis-Menten kinetics during the initial reaction time. Optimal reaction rates were observed in the pH range of 4.5-5.5. The HA substrate size did not have significant effects on the initial rate of the reaction. By studying HA substrates of 215, 357, and 752 kDa, the kinetic parameters Km, Vmax, and kcat were determined to be 0.87-0.91 mg/ml, 1.66-1.74 NM s(-1), and 40.5-42.4 s(-1), respectively. This method allows for direct measurement of kinetics using physiologically relevant HA substrates and can be applied to other hyaluronidase kinetic measurements. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Potency Evaluation of Recombinant Human Erythropoietin in Brazil: Assessment of Reproducibility Using a Practical Approach

    Directory of Open Access Journals (Sweden)

    Michele Cardoso do Nascimento

    2015-08-01

    Full Text Available In this study, we compared the results of potency determination of recombinant human erythropoietin (rhEPO obtained between 2010 and 2012 by the National Institute of Quality Control in Health (INCQS/Fiocruz, i.e., the National Control Laboratory (NCL, and by a manufacturer of rhEPO. In total, 47 different batches of commercially prepared rhEPO (alpha isoform were analyzed. All results, including those of the control and warning limits, remained within the limits recommended by European Pharmacopoeia (Ph. Eur.. All relative error (RE values were less than ± 30%, wh ereas most were approximately ± 20%. Applying the Bland-Altman plot, only two of 47 values remained outside the limits of agreement (LA. In addition, agreement of potency determination between INCQS and the manufacturer coefficient of variation of reproducibility (% CVR was considered satisfactory. Taken together, our results demonstrate (i. the potency assay of rhEPO performed at INCQS, is standardized and controlled, (ii. the comparison of our results with those of the manufacturer, revealed an adequate inter-laboratory variation, and (iii. the critical appraisal proposed here appears to be a feasible tool to assess the reproducibility of biological activity, providing additional information regarding monitoring and production consistency to manufacturers and NCLs.

  14. Safety of recombinant human granulocyte-macrophage colony-stimulating factor in healing pediatric severe burns.

    Science.gov (United States)

    Chi, Y F; Chai, J K; Luo, H M; Zhang, Q X; Feng, R

    2015-03-31

    We explored the safety of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for healing burns in children. Subjects were randomly assigned to two groups: the experimental group received external rhGM-CSF gel, and the control group received rhGM-CSF gel matrix components, applied to the burn surface. Neither group was given any other drugs that promote wound healing. Each day we recorded the pulse, body temperature, and respiration status in the two groups. We detected the blood routine, urine routine, and hepatic and renal function before the patients received drug treatment and after 72 h. The wound scab and healing states in the two groups were recorded every 4 days to evaluate wound healing rate and time taken for complete healing. Adverse reactions and their rate of occurrence were also recorded. The median time of healing was 15 days in the experimental group and 19 days in the control group (log-rank χ(2) = 5.139, P 0.05). Compared with saline treatment of severe burns, rhGM-CSF can effectively shorten the healing time without significant adverse reactions, and is an effective and safe treatment for burns in children.

  15. Early postnatal treatment of hypogonadotropic hypogonadism with recombinant human FSH and LH

    DEFF Research Database (Denmark)

    Main, Katharina M; Schmidt, Ida M; Toppari, Jorma

    2002-01-01

    the normal range (0.05-0.17 IU/l and 79-112 pg/ml respectively). METHODS: From 7.9 to 13.7 months of age, the patient was treated with recombinant human LH and FSH in doses of 20 and 21.3 IU s.c. twice weekly respectively. RESULTS: During treatment concentrations of LH, FSH, inhibin B and estradiol increased...... postnatally, to mimic the physiological development, would improve testicular growth and fertility potential later in life. DESIGN: Our patient presented with micropenis. Serum hormone concentrations were measured monthly after delivery: LH and testosterone were undetectable, and FSH and inhibin B were below...... to values within normal limits (0.7-1.88 IU/l, 0.17-3.24 IU/l, 121-268 pg/ml and 40-55 pmol/l respectively), whereas serum testosterone remained undetectable. Penile length increased from 1.6 to 2.4 cm and testicular volume, assessed by ultrasound, increased by 170%. No significant adverse events were...

  16. Cortical bone growth and maturational changes in dwarf rats induced by recombinant human growth hormone

    Science.gov (United States)

    Martinez, D. A.; Orth, M. W.; Carr, K. E.; Vanderby, R. Jr; Vailas, A. C.

    1996-01-01

    The growth hormone (GH)-deficient dwarf rat was used to investigate recombinant human (rh) GH-induced bone formation and to determine whether rhGH facilitates simultaneous increases in bone formation and bone maturation during rapid growth. Twenty dwarf rats, 37 days of age, were randomly assigned to dwarf plus rhGH (GH; n = 10) and dwarf plus vehicle (n = 10) groups. The GH group received 1.25 mg rhGH/kg body wt two times daily for 14 days. Biochemical, morphological, and X-ray diffraction measurements were performed on the femur middiaphysis. rhGH stimulated new bone growth in the GH group, as demonstrated by significant increases (P bone length (6%), middiaphyseal cross-sectional area (20%), and the amount of newly accreted bone collagen (28%) in the total pool of middiaphyseal bone collagen. Cortical bone density, mean hydroxyapatite crystal size, and the calcium and collagen contents (microgram/mm3) were significantly smaller in the GH group (P bone collagen maturation, and mean hydroxyapatite crystal size may be independently regulated during rapid growth.

  17. Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli

    Science.gov (United States)

    Seo, Kyung Hye; Yang, Hyo-Jin; Kim, Sook-Kyung; Choi, Jun-Hyuk

    2013-01-01

    High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli. PMID:23409149

  18. Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Min-Ji Kim

    Full Text Available High-level expression of recombinant human growth hormone (hGH in Escherichia coli (E. coli leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC, size-exclusion chromatography (SEC, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF mass spectrometry, and circular dichroism (CD. These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.

  19. Comparative Study on the Antivirus Activity of Shuang?Huang?Lian Injectable Powder and Its Bioactive Compound Mixture against Human Adenovirus III In Vitro

    OpenAIRE

    Ma, Qinhai; Liang, Dedong; Song, Shuai; Yu, Qintian; Shi, Chunyu; Xing, Xuefeng; Luo, Jia-Bo

    2017-01-01

    Shuang?Huang?Lian injectable powder (SHL)?a classical purified herbal preparation extracted from Scutellaria baicalensis, Lonicera japonica, and Forsythia suspense?has been used against human adenovirus III (HAdV3) for many years. The combination herb and its major bioactive compounds, including chlorogenic acid, baicalin, and forsythia glycosides A, are effective inhibitors of the virus. However, no comprehensive studies are available on the antiviral effects of SHL against HAdV3. Moreover, ...

  20. "EFFECT OF HIGH VERSUS LOW DOSES OF HUMAN RECOMBINANT ERYTHROPOIETIN ON THE ANEMIA OF PREMATURITY"

    Directory of Open Access Journals (Sweden)

    A. Mohammadzadeh

    2005-05-01

    Full Text Available Recombinant human erythropoietin (rh-EPO is known to accelerate erythropoiesis in preterm infants. The purpose of this study was to compare the effectiveness of early treatment with two doses of rh-EPO (high vs. low dose in the management of anemia of prematurity. Twenty preterm infants with hematocrit (Hct < 30% when infant’s age was between 2 to 3 weeks after birth or Hct <25% when infant’s age was more than 3 weeks after birth, were divided randomly in two groups, each group including 10 babies. Infants in high dose group received 500 u/kg rh-EPO twice per week and the low dose group received 500 u/kg rh-EPO weekly. All infants were fed human milk supplemented with enteral iron. Hematocrit and reticulocyte counts were determined for each infant at the start of the study, 3 days after start of treatment and one week after the end of treatment. The means of gestational age in high dose and low dose groups were 31.4 ± 2.2 and 31.3±2.0 weeks, respectively. Means of birth weight in high dose and low dose groups were 1366 ± 243 and 1438±249 gr, respectively. The two groups were significantly different in reticulocyte count at 3 days after treatment (P = 0.047 and in hematocrit at the end of study (P < 0.0001. We concluded the early treatment of anemia of prematurity with high dose rh-EPO with supplemental iron significantly increases hematocrit and reticulocyte in preterm infants and reduce the need for blood transfusion in these high risk neonates.

  1. The effect of nicotine on osteoinduction by recombinant human bone morphogenetic protein 2.

    Science.gov (United States)

    Tamura, K; Togo, Y; Kaihara, S; Hussain, A; Takahashi, K; Bessho, K

    2014-08-01

    Nicotine, one of the constituents of tobacco, is known to have an adverse effect on human health. We sought to clarify the interaction between nicotine and recombinant human bone morphogenetic protein 2 (rhBMP-2) in terms of osteogenesis in vitro and osteoinduction in vivo. Nicotine did not inhibit or stimulate alkaline phosphatase (ALP) activity or the amount of osteocalcin in C2C12 cells in the presence of rhBMP-2 in vitro. Ectopic bone formation using a collagen sponge containing rhBMP-2 was evaluated with and without nicotine after 21 days using radiographic, histological, biochemical, and immunohistochemical analyses. ALP activity in the medium-dose group (2.2±0.9IU/mg protein; P=0.047) and the high-dose group (2.0±0.1IU/mg protein; P=0.03) was significantly lower than in the control group. The calcium content in the medium-dose group (35.4±12.9μg/mg tissue; P=0.0099) and high-dose group (34.8±10.5μg/mg tissue; P=0.006) was significantly lower than in the control group. The number of vascular endothelial growth factor-positive cells in the high-dose group (671.9±57.3cells/mm(2); P=0.03) was significantly lower than in the control group. Results showed that nicotine did not inhibit the stimulatory effect of rhBMP-2 in vitro, but a high dose of nicotine inhibited bone formation in vivo by adversely affecting vascularization. Copyright © 2014 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  2. Controlled release of recombinant human cementum protein 1 from electrospun multiphasic scaffold for cementum regeneration.

    Science.gov (United States)

    Chen, Xiaofeng; Liu, Yu; Miao, Leiying; Wang, Yangyang; Ren, Shuangshuang; Yang, Xuebin; Hu, Yong; Sun, Weibin

    2016-01-01

    Periodontitis is a major cause for tooth loss, which affects about 15% of the adult population. Cementum regeneration has been the crux of constructing the periodontal complex. Cementum protein 1 (CEMP1) is a cementum-specific protein that can induce cementogenic differentiation. In this study, poly(ethylene glycol) (PEG)-stabilized amorphous calcium phosphate (ACP) nanoparticles were prepared by wet-chemical method and then loaded with recombinant human CEMP1 (rhCEMP1) for controlled release. An electrospun multiphasic scaffold constituted of poly(ε-caprolactone) (PCL), type I collagen (COL), and rhCEMP1/ACP was fabricated. The effects of rhCEMP1/ACP/PCL/COL scaffold on the attachment proliferation, osteogenic, and cementogenic differentiations of human periodontal ligament cells, (PDLCs) were systematically investigated. A critical size defect rat model was introduced to evaluate the effect of tissue regeneration of the scaffolds in vivo. The results showed that PEG-stabilized ACP nanoparticles formed a core-shell structure with sustained release of rhCEMP1 for up to 4 weeks. rhCEMP1/ACP/PCL/COL scaffold could suppress PDLCs proliferation behavior and upregulate the expression of cementoblastic markers including CEMP1 and cementum attachment protein while downregulating osteoblastic markers including osteocalcin and osteopontin when it was cocultured with PDLCs in vitro for 7 days. Histology analysis of cementum after being implanted with the scaffold in rats for 8 weeks showed that there was cementum-like tissue formation but little bone formation. These results indicated the potential of using electrospun multiphasic scaffolds for controlled release of rhCEMP1 for promoting cementum regeneration in reconstruction of the periodontal complex.

  3. Human recombinant anti-thyroperoxidase autoantibodies: in vitro cytotoxic activity on papillary thyroid cancer expressing TPO.

    Science.gov (United States)

    Rebuffat, S A; Morin, M; Nguyen, B; Castex, F; Robert, B; Péraldi-Roux, S

    2010-03-02

    Thyroid cancers are difficult to treat due to their limited responsiveness to chemo- and radiotherapy. There is thus a great interest in and a need for alternative therapeutic approaches. We studied the cytotoxic activity of anti-thyroperoxidase autoantibodies (anti-TPO aAbs, expressed in baculovirus/insect cell (B4) and CHO cells (B4') or purified from patients' sera) against a papillary thyroid cancer (NPA) cell line. Anti-TPO aAbs from patients' sera led to a partial destruction of NPA cell line by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) and exhibited an anti-proliferative activity. Comparison of the cytotoxic activity of anti-TPO aAbs shows that B4' induced an anti-proliferative effect and a better ADCC than B4, but a lower one than anti-TPO aAbs from patients' sera. Antibody-dependent cell-mediated cytotoxicity was increased when human peripheral blood mononuclear cells were used as effector cells, suggesting that FcgammaRs, CD64, CD32 and CD16 are involved. Indeed, anti-TPO aAbs from patients' sera, but not B4 and B4', exhibited CDC activity. These data indicate that anti-TPO aAbs display moderate ADCC and anti-proliferative activities on NPA cells; IgG glycosylation appears to be important for cytotoxic activity and ADCC efficiency depends on FcgammaR-bearing cells. Finally, recombinant human anti-TPO aAbs cannot yet be considered as an optimal tool for the development of a novel therapeutic approach for thyroid cancer.

  4. Human Recombinant Antimannan Immunoglobulin G1 Antibody Confers Resistance to Hematogenously Disseminated Candidiasis in Mice

    Science.gov (United States)

    Zhang, Mason X.; Bohlman, M. Charlotte; Itatani, Carol; Burton, Dennis R.; Parren, Paul W. H. I.; St. Jeor, Stephen C.; Kozel, Thomas R.

    2006-01-01

    Mannan is a major cell wall component found in Candida species. Natural antimannan antibody is present in sera from most normal adults, but its role in host resistance to hematogenously disseminated candidiasis is unknown. The purpose of this study was to develop recombinant human antimannan antibody and to study its protective function. A phage Fab display combinatorial library containing Fab genes from bone marrow lymphocytes was screened with Candida albicans yeast cells and chemically purified mannan. One antimannan Fab, termed M1, was converted to a full-length immunoglobulin G1 antibody, M1g1, and M1g1 was produced in CHO cells. The M1g1 epitope was found in C. albicans serotypes A and B, Candida tropicalis, Candida guilliermondii, Candida glabrata, and Candida parapsilosis. Its expression was active at both 23°C and 37°C and uniform over the cell surface. BALB/c mice passively immunized with M1g1 were more resistant than control mice to a lethal hematogenous infection by C. albicans, as evidenced by extension of survival in an M1g1 dose-dependent manner (P, 0.08 to <0.001) and by reduction in number of infection foci and their size in the kidney. In vitro studies found that M1g1 promoted phagocytosis and phagocytic killing of C. albicans yeast cells by mouse peritoneal macrophages and was required for activation of the mouse complement cascade. Thus, human antimannan antibody may have a protective role in host resistance to systemic candidiasis. PMID:16368991

  5. Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

    Science.gov (United States)

    Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

    2018-03-25

    Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

  6. One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.

    Science.gov (United States)

    Boerma, Jan Simon; Vermeulen, Nico P E; Commandeur, Jan N M

    2014-01-25

    Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed: p-benzoquinone imines, o-imine methide and arene-oxide. Recently, detection of 2'-(glutathion-S-yl)-deschloro-diclofenac (DDF-SG), resulting from chlorine substitution, suggested the existence of a fourth type of P450-dependent reactive intermediate whose inactivation by GSH is completely dependent on presence of glutathione S-transferase. In this study, fourteen recombinant cytochrome P450s and three flavin-containing monooxygenases were tested for their ability to produce oxidative DF metabolites and their corresponding GSH conjugates. Concerning the hydroxymetabolites and their GSH conjugates, results were consistent with previous studies. Unexpectedly, all tested recombinant P450s were able to form DDF-SG to almost similar extent. DDF-SG formation was found to be partially independent of NADPH and even occurred by heat-inactivated P450. However, product formation was fully dependent on both GSH and glutathione-S-transferase P1-1. DDF-SG formation was also observed in reactions with horseradish peroxidase in absence of hydrogen peroxide. Because DDF-SG was not formed by free iron, it appears that DF can be bioactivated by iron in hemeproteins. This was confirmed by DDF-SG formation by other hemeproteins such as hemoglobin. As a mechanism, we propose that DF is subject to heme-dependent one-electron oxidation. The resulting nitrogen radical cation, which might activate the chlorines of DF, then undergoes a GST-catalyzed nucleophilic aromatic substitution reaction in which the chlorine atom of the DF moiety is replaced by GSH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Protection against Cutaneous Leishmaniasis Induced by Recombinant Antigens in Murine and Nonhuman Primate Models of the Human Disease

    Science.gov (United States)

    Campos-Neto, Antonio; Porrozzi, Renato; Greeson, Kay; Coler, Rhea N.; Webb, John R.; Seiky, Yasir A. W.; Reed, Steven G.; Grimaldi, Gabriel

    2001-01-01

    Leishmaniasis affects approximately 2 million people each year throughout the world. This high incidence is due in part to the lack of an efficacious vaccine. We present evidence that the recombinant leishmanial antigens LmSTI1 and TSA, which we identified and characterized previously, induce excellent protection in both murine and nonhuman primate (rhesus monkey) models of human cutaneous leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an animal model that is evolutionarily close to humans qualifies this antigen combination as a promising candidate subunit vaccine against human leishmaniasis. PMID:11349082

  8. Recombinant human erythropoietin alpha improves the efficacy of radiotherapy of a human tumor xenograft, affecting tumor cells and microvessels

    Energy Technology Data Exchange (ETDEWEB)

    Loevey, J. [Dept. of Radiotherapy, National Inst. of Oncology, Budapest (Hungary); Bereczky, B.; Gilly, R.; Kenessey, I.; Raso, E.; Simon, E.; Timar, J. [Dept. of Tumor Progression, National Inst. of Oncology, Budapest (Hungary); Dobos, J. [Dept. of Tumor Progression, National Inst. of Oncology, Budapest (Hungary); National Koranyi Inst. of TBC and Pulmonology, Budapest (Hungary); Vago, A. [Central Lab., National Inst. of Oncology, Budapest (Hungary); Kasler, M. [Head and Neck Surgery, National Inst. of Oncology, Budapest (Hungary); Doeme, B. [National Koranyi Inst. of TBC and Pulmonology, Budapest (Hungary); Tovari, J. [National Koranyi Inst. of TBC and Pulmonology, Budapest (Hungary); 1. Inst. of Pathology and Experimental Cancer Research, Semmelweis Univ., Budapest (Hungary)

    2008-01-15

    Background and purpose: tumor-induced anemia often occurs in cancer patients, and is corrected by recombinant human erythropoietins (rHuEPOs). Recent studies indicated that, besides erythroid progenitor cells, tumor and endothelial cells express erythropoietin receptor (EPOR) as well; therefore, rHuEPO may affect their functions. Here, the effect of rHuEPO{alpha} on irradiation in EPOR-positive human squamous cell carcinoma xenograft was tested. Material and methods: A431 tumor-bearing SCID mice were treated from the tumor implantation with rHuEPO{alpha} at human-equivalent dose. Xenografts were irradiated (5 Gy) on day 14, and the final tumor mass was measured on day 22. The systemic effects of rHuEPO{alpha} on the hemoglobin level, on tumor-associated blood vessels and on hypoxia-inducible factor-(HIF-)1{alpha} expression of the tumor xenografts were monitored. The proliferation, apoptosis and clonogenic capacity of A431 cancer cells treated with rHuEPO{alpha} and irradiation were also tested in vitro. Results: in vitro, rHuEPO{alpha} treatment alone did not modify the proliferation of EPOR-positive A431 tumor cells but enhanced the effect of irradiation on proliferation, apoptosis and clonogenic capacity. In vivo, rHuEPO{alpha} administration compensated the tumor-induced anemia in SCID mice and decreased tumoral HIF-1{alpha} expression but had no effect on tumor growth. At the same time rHuEPO{alpha} treatment significantly increased the efficacy of radiotherapy in vivo (tumor weight of 23.9 {+-} 4.7 mg and 34.9 {+-} 4.6 mg, respectively), mediated by increased tumoral blood vessel destruction. Conclusion: rHuEPO{alpha} treatment may modulate the efficacy of cancer radiotherapy not only by reducing systemic hypoxia and tumoral HIF-1{alpha} expression, but also by destroying tumoral vessels. (orig.)

  9. Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response.

    Science.gov (United States)

    Wakasa, Yuhya; Takaiwa, Fumio

    2016-01-01

    Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail.

  10. In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola compared with its reference medicinal product (GONAL-f.

    Directory of Open Access Journals (Sweden)

    Renato Mastrangeli

    Full Text Available Recombinant human follicle-stimulating hormone (r-hFSH is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency, as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value, which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048, although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%. A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]. These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability

  11. Isolation of Human Intestinal Bacteria Capable of Producing the Bioactive Metabolite Isourolithin A from Ellagic Acid.

    Science.gov (United States)

    Selma, María V; Beltrán, David; Luna, María C; Romo-Vaquero, María; García-Villalba, Rocío; Mira, Alex; Espín, Juan C; Tomás-Barberán, Francisco A

    2017-01-01

    Urolithins are intestinal microbial metabolites produced from ellagitannin- and ellagic acid-containing foods such as walnuts, strawberries, and pomegranates. These metabolites, better absorbed than their precursors, can contribute significantly to the beneficial properties attributed to the polyphenols ellagitannins and ellagic acid (EA). However, both the ability of producing the final metabolites in this catabolism (urolithins A, B and isourolithin A) and the health benefits associated with ellagitannin consumption differ considerably among individuals depending on their gut microbiota composition. Three human urolithin metabotypes have been previously described, i.e., metabotype 0 (urolithin non-producers), metabotype A (production of urolithin A as unique final urolithin) and metabotype B (urolithin B and/or isourolithin A are produced besides urolithin A). Although production of some intermediary urolithins has been recently attributed to intestinal species from Eggerthellaceae family named Gordonibacter urolithinfaciens and Gordonibacter pamelaeae , the identification of the microorganisms responsible for the complete transformation of EA into the final urolithins, especially those related to metabotype B, are still unknown. In the present research we illustrate the isolation of urolithin-producing strains from human feces of a healthy adult and their ability to transform EA into different urolithin metabolites, including isourolithin A. The isolates belong to a new genus from Eggerthellaceae family. EA transformation and urolithin production arisen during the stationary phase of the growth of the bacteria under anaerobic conditions. The HPLC-DAD-MS analyses demonstrated the sequential appearance of 3,8,9,10-tetrahydroxy-urolithin (urolithin M6), 3,8,9-trihydroxy-urolithin (urolithin C) and 3,9-dihydroxy-urolithin (isourolithin A) while 3,8-dihydroxy-urolithin (urolithin A) and 3-hydroxy-urolithin (urolithin B) were not detected. For the first time

  12. Isolation of Human Intestinal Bacteria Capable of Producing the Bioactive Metabolite Isourolithin A from Ellagic Acid

    Directory of Open Access Journals (Sweden)

    María V. Selma

    2017-08-01

    Full Text Available Urolithins are intestinal microbial metabolites produced from ellagitannin- and ellagic acid-containing foods such as walnuts, strawberries, and pomegranates. These metabolites, better absorbed than their precursors, can contribute significantly to the beneficial properties attributed to the polyphenols ellagitannins and ellagic acid (EA. However, both the ability of producing the final metabolites in this catabolism (urolithins A, B and isourolithin A and the health benefits associated with ellagitannin consumption differ considerably among individuals depending on their gut microbiota composition. Three human urolithin metabotypes have been previously described, i.e., metabotype 0 (urolithin non-producers, metabotype A (production of urolithin A as unique final urolithin and metabotype B (urolithin B and/or isourolithin A are produced besides urolithin A. Although production of some intermediary urolithins has been recently attributed to intestinal species from Eggerthellaceae family named Gordonibacter urolithinfaciens and Gordonibacter pamelaeae, the identification of the microorganisms responsible for the complete transformation of EA into the final urolithins, especially those related to metabotype B, are still unknown. In the present research we illustrate the isolation of urolithin-producing strains from human feces of a healthy adult and their ability to transform EA into different urolithin metabolites, including isourolithin A. The isolates belong to a new genus from Eggerthellaceae family. EA transformation and urolithin production arisen during the stationary phase of the growth of the bacteria under anaerobic conditions. The HPLC-DAD-MS analyses demonstrated the sequential appearance of 3,8,9,10-tetrahydroxy-urolithin (urolithin M6, 3,8,9-trihydroxy-urolithin (urolithin C and 3,9-dihydroxy-urolithin (isourolithin A while 3,8-dihydroxy-urolithin (urolithin A and 3-hydroxy-urolithin (urolithin B were not detected. For the first time

  13. Immune bioactivity in shellfish toward serum-free cultured human cell lines.

    Science.gov (United States)

    Kong, Z L; Chiang, L C; Fang, F; Shinohara, K; Pan, P

    1997-01-01

    The biologically functional effect of eight kinds of hot-water extracts of shellfish on cultured human cell lines was examined in a serum-free medium model. Meretrix lusoria and Sinonovacula constricta extracts enhanced IgM secretion of both hybridoma HB4C5 and SI102 cells when cultured with the respective extracts. The purified principle exhibited remarked activity in the adsorbed fraction in hydroxyapatite and Concanavalin A columns. The extracts of Corbicula fluminea, Crassostreas gigas, Meretrix lusoria, Anadara granosa, and Sinonovacula constricta enhanced in nitroblue tetrazolium (NBT)-reducing ability of macrophage U-M cells. Meretrix lusoria, Anadara granosa, and Sinonovacula constricta were specifically cytotoxic to both cultures of MCF-7 breast cancer cells and HuH-6KK hepatoblastoma. These findings imply that the extracts of shellfish that were examined exhibited a differential effect on immune cells and tumor cells.

  14. Human TMEM30a promotes uptake of antitumor and bioactive choline phospholipids into mammalian cells.

    Science.gov (United States)

    Chen, Rui; Brady, Erin; McIntyre, Thomas M

    2011-03-01

    Antitumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. 1-O-Alkyl-2-carboxymethyl-sn-glycero-3-phosphocholine (Edelfosine) like other short-chained phospholipids--inflammatory platelet-activating factor (PAF) and apoptotic oxidatively truncated phospholipids--are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in a process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show that import of choline phospholipids into S. cerevisiae ΔLem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a-GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. Short hairpin RNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [(3)H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show that extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short chain choline phospholipids such as Edelfosine, PAF, and proapoptotic phospholipids.

  15. Growth Hormone Safety Workshop Position Paper: a critical appraisal of recombinant human growth hormone therapy in children and adults

    DEFF Research Database (Denmark)

    Allen, David B; Backeljauw, Philippe; Bidlingmaier, Martin

    2015-01-01

    Recombinant human GH (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, the statem......Recombinant human GH (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however......, the statement highlighted a number of areas for on-going surveillance of long-term safety, including cancer risk, impact on glucose homeostasis, and use of high dose pharmacological rhGH treatment. Over the intervening years, there have been a number of publications addressing the safety of rhGH with regard...

  16. Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Hård, K.; Mekking, A.; Damm, J.B.L.; Kamerling, J.P.; Boer, W. de; Wijnands, R.A.

    1990-01-01

    Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides were separated from

  17. Aberrant recombination and repair during immunoglobulin class switching in BRCA1-deficient human B cells

    DEFF Research Database (Denmark)

    Björkman, Andrea; Qvist, Per; Du, Likun

    2015-01-01

    cancer type 2 susceptibility protein (BRCA2)-deficient cells. Thus, BRCA1, together with its interaction partners, seems to play an important role in repairing DSBs generated during class switch recombination by promoting the classical NHEJ pathway. This may not only provide a general mechanism......Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement...... in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ...

  18. Genome-Wide Patterns of Recombination in the Opportunistic Human Pathogen Pseudomonas aeruginosa

    Science.gov (United States)

    Dettman, Jeremy R.; Rodrigue, Nicolas; Kassen, Rees

    2015-01-01

    The bacterium Pseudomonas aeruginosa is a significant cause of acute nosocomial infections as well as chronic respiratory infections in patients with cystic fibrosis (CF). Recent reports of the intercontinental spread of a CF-specific epidemic strain, combined with high intrinsic levels of antibiotic resistance, have made this opportunistic pathogen an important public health concern. Strain-specific differences correlate with variation in clinical outcomes of infected CF patients, increasing the urgency to understand the evolutionary origin of genetic factors conferring important phenotypes that enable infection, virulence, or resistance. Here, we describe the genome-wide patterns of homologous and nonhomologous recombination in P. aeruginosa, and the extent to which the genomes are affected by these diversity-generating processes. Based on whole-genome sequence data from 32 clinical isolates of P. aeruginosa, we examined the rate and distribution of recombination along the genome, and its effect on the reconstruction of phylogenetic relationships. Multiple lines of evidence suggested that recombination was common and usually involves short stretches of DNA (200–300 bp). Although mutation was the main source of nucleotide diversity, the import of polymorphisms by homologous recombination contributed nearly as much. We also identified the genomic regions with frequent recombination, and the specific sequences of recombinant origin within epidemic strains. The functional characteristics of the genes contained therein were examined for potential associations with a pathogenic lifestyle or adaptation to the CF lung environment. A common link between many of the high-recombination genes was their functional affiliation with the cell wall, suggesting that the products of recombination may be maintained by selection for variation in cell-surface molecules that allows for evasion of the host immune system. PMID:25480685

  19. Determination of Coreceptor Usage of Human Immunodeficiency Virus Type 1 from Patient Plasma Samples by Using a Recombinant Phenotypic Assay

    OpenAIRE

    Trouplin, Virginie; Salvatori, Francesca; Cappello, Fanny; Obry, Veronique; Brelot, Anne; Heveker, Nikolaus; Alizon, Marc; Scarlatti, Gabriella; Clavel, François; Mammano, Fabrizio

    2001-01-01

    We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populati...

  20. Molecular interaction and energy transfer between human serum albumin and bioactive component Aloe dihydrocoumarin

    Science.gov (United States)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Li, Lin; Tang, Ya-Lin

    2008-10-01

    Aloe dihydrocoumarin is an antioxidant and a candidate of immunomodulatory drug on the immune system and can balance physiological reactive oxygen species (ROS) levels which may be useful to maintain homeostasis. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydrocoumarin with human serum albumin (HSA) has been investigated by fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydrocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. A Förster type fluorescence resonance energy transfer mechanism is involved in this quenching of Trp fluorescence by Aloe dihydrocoumarin. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydrocoumarin with HSA causes a conformational change of the protein, with the loss of α-helix stability and the gain of β-sheet and β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FT-IR experiments along with the docking studies suggest that Aloe dihydrocoumarin binds to residues located in subdomain IIA of HSA.

  1. Binding of the bioactive component Aloe dihydroisocoumarin with human serum albumin

    Science.gov (United States)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Tang, Ya-Lin

    2008-11-01

    Aloe dihydroisocoumarin, one of new components isolated from Aloe vera, can scavenge reactive oxygen species. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydroisocoumarin with human serum albumin (HSA) has been investigated by using fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydroisocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. An isoemissive point at 414 nm is seen, indicating that the quenching of HSA fluorescence depends on the formation of Aloe dihydroisocoumarin-HSA complex, which is further confirmed by fluorescence dynamic result. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydroisocoumarin with HSA causes a conformational change of the protein, with the gain of α-helix, β-sheet and random coil stability and the loss of β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FTIR experiments along with the docking studies suggest that Aloe dihydroisocoumarin binds to residues located in subdomain IIA of HSA.

  2. [Adenovirus mediated expression of recombinant human single chain interleukin-27(rhscIL-27) fusion gene in hepatoma cells].

    Science.gov (United States)

    Cheng, Xiao-gang; Zhang, Ya-qing; Ye, Wan; Zou, Qiang; Chen, Wei; Jin, Hong; Xu, Yan; Zhang, Shao-lan

    2010-06-01

    To explore the adenovirus mediated expression of recombinant human single chain interleukin-27(rhscIL-27) fusion gene in hepatoma cells. The rhscIL-27 fusion gene was subcloned into the shuttle plasmid pAdTrack-CMV and then clone the homologous recombinant adenovirus genomic plasmid pAdEasy in bacteria. The identified recombinant plasmid AdIL-27 was tranfected into 293 cells, and then the adenovirus did the package and amplification. The HepG2 cells were infected with AdIL-27 and the target gene expression was determined by RT-PCR and ELISA. The biological activity of rhscIL-27 was detected by IFN-gamma inducing assay. Restriction endonuclease and gene sequencing confirmed that the recombinant adenovirus vector of rhscIL-27 fusion gene was successfully constructed. The expression of rhscIL-27 fusion gene was observed at 48 h after the transfection of the HepG2 cells with AdIL-27. The IFN-gamma inducing assay showed that the rhscIL-27 protein has the ability inducing IFN-gamma secretion. By using adenovirus expression system, rhscIL-27 fusion gene with biological activity is expressed successfully in hepatoma cells. This experiment laid a foundation for gene therapy of hepatoma with IL-27.

  3. Distribution of 131I-labeled recombinant human erythropoietin in maternal and fetal organs following intravenous administration in pregnant rats

    International Nuclear Information System (INIS)

    Yilmaz, O.; Lambrecht, F.Y.; Durkan, K.; Gokmen, N.; Erbayraktar, S.

    2007-01-01

    The aim of the present study was to demonstrate the possible transplacental transmission of 131 I labeled recombinant human erythropoietin ( 131 I-rh-EPO) in pregnant rats and its distribution through maternal and fetal organs. Six Wistar Albino Rats in their pregnancy of 18 days were used 131 I labeled recombinant human erythropoietin (specific activity = 2.4 μCi/IU) was injected into the tail vein of rats. After 30 minutes labeled erythropoietin infusion maternal stomach, kidney, lung, liver, brain and heart as well as fetus were removed. Then, the same organs were removed from each fetus. Measuring weight of maternal and fetal organs as well as placenta were followed by radioactivity count via Cd(Te) detector. 131 I labeled recombinant human erythropoietin was found to be able to pass rat placenta and its distribution order in fetal organs was similar to those of maternal organs. Besides, as measurements were performed closer to cornu uteri, uptakes were decreasing in every fetus and its corresponding placenta. (author)

  4. Increased preoperative collection of autologous blood with recombinant human erythropoietin therapy in tertiary care hospitals of Jammu

    Directory of Open Access Journals (Sweden)

    Kumkum Sharma

    2013-01-01

    Full Text Available Introduction: To study whether the administration of recombinant human erythropoietin increases the amount of autologous blood that can be collected before orthopaedic surgery. Materials and Methods: We conducted a randomized controlled trial of recombinant human erythropoietin in 68 adults scheduled for elective orthopedic procedures. The patients received either erythropoietin 600 units/kg of body weight or placebo intravenously every 5 th day prior to each phlebotomy for 21 days during which time up to 5 units of blood was collected. Patients were excluded from donation when their hematocrit values were less than 33%. All patients received iron sulphate 325mg orally 3 times daily. The mean number of units collected per patient was 4.33 ± 0.4 for erythropoietin group and 3.05± 0.71 for the placebo group. Results: The mean packed red cell volume donated by patients who received erythropoietin was 32% greater than that donated by patients who received placebo (196.3 vs. 169.4 ml, p<0.05. 68% in the placebo group and 9% of patients treated with erythropoietin were unable to donate ≥4 units. No adverse effects were attributed to erythropoietin. While participating in the study, complications developed in 2 patients one in each group necessitating their removal from the study. Conclusion: We conclude that recombinant human erythropoietin increases the ability of the patients about to undergo elective surgery to donate autologous blood units.

  5. Brucella melitensis VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis.

    Science.gov (United States)

    Mirkalantari, Shiva; Zarnani, Amir-Hassan; Nazari, Mahboobeh; Irajian, Gholam Reza; Amirmozafari, Nour

    2017-03-03

    The numerous drawbacks of current serological tests for diagnosis of brucellosis which mainly results from cross reactivity with LPS from other gram-negative bacteria have generated an increasing interest to find more specific non-LPS antigens. Previous studies had indicated that Brucella VirB12 protein, a cell surface protein and component of type IV secretion system, induces antibody response during animal infection. However, this protein has not yet been tested as a serological diagnostic marker in human brucellosis. Recombinant VirB12 protein was prepared and evaluated the efficacy of it in an indirect enzyme-linked immunosorbent assay (ELISA) for brucellosis with sera collected from different region of Iran and the results were compared with a commercial ELISA kit. Sera from human brucellosis patients strongly reacted to the purified recombinant VirB12. The sensitivity, specificity, accuracy, negative predictive value and positive predictive value of recombinant VirB12-based ELISA related to the commercial-ELISA method were 87.8, 94, 90, 80 and 96.6% respectively. We concluded that antigenic VirB12 have a property value that can be considered as a candidate for using in serodiagnostic tests for human brucellosis.

  6. Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis.

    Science.gov (United States)

    Pathak, Prachi; Kumar, Ashu; Thavaselvam, Duraipandian

    2017-07-11

    Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay). Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23). The rOmp2a was found to be

  7. The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2.

    Science.gov (United States)

    Duggirala, S S; Rodgers, J B; DeLuca, P P

    1996-07-01

    Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.

  8. Effects of recombinant human erythropoietin injections on physical self in endurance athletes.

    Science.gov (United States)

    Ninot, Grégory; Connes, Philippe; Caillaud, Corrine

    2006-04-01

    This study examined the time course of mean self-esteem and physical self scores in three groups: male endurance athletes treated with recombinant human erythropoietin (rHuEPO group, n = 6), a placebo group (n = 5) injected with a sodium chloride solution and a control group who did not receive any injection (n = 6). Each participant completed the Physical Self Inventory twice a day (between 07.00 and 09.00 h and between 19.00 and 21.00 h). Using a 10 cm visual analog scale, the participants assessed global self-esteem, physical self-worth and the sub-domains of physical condition, sport competence, attractive body and physical strength (Fox & Corbin, 1989). This was conducted over three consecutive periods: in the 2 weeks before the course of injections, during the 6 weeks of injections and for 4 weeks after the injections. Aerobic capacity was assessed before and after 4 weeks of treatment. The results showed a significant increase in aerobic physical fitness in the rHuEPO group and a significant increase in perceived physical condition and physical strength scores at the end of treatment. The main psychological result was that endurance athletes were highly sensitive to the effects of rHuEPO on physical fitness. The perception of increased physical condition may lead to a stronger commitment to training. The rHuEPO injections presented a dangerous hedonic effect linked to endurance training. These results confirm the need to tackle rHuEPO abuse at any time during the training season.

  9. Effects of Recombinant Human Bone Morphogenetic Protein-2 on Vertical Bone Augmentation in a Canine Model.

    Science.gov (United States)

    Hsu, Yung-Ting; Al-Hezaimi, Khalid; Galindo-Moreno, Pablo; O'Valle, Francisco; Al-Rasheed, Abdulaziz; Wang, Hom-Lay

    2017-09-01

    Vertical bone augmentation (VBA) remains unpredictable and challenging for most clinicians. This study aims to compare hard tissue outcomes of VBA, with and without recombinant human bone morphogenetic protein (rhBMP)-2, under space-making titanium mesh in a canine model. Eleven male beagle dogs were used in the study. Experimental ridge defects were created to form atrophic ridges. VBA was performed via guided bone regeneration using titanium mesh and allografts. In experimental hemimandibles, rhBMP-2/absorbable collagen sponge was well mixed with allografts prior to procedures, whereas a control buffer was applied within controls. Dogs were euthanized after a 4-month healing period. Clinical and radiographic examinations were performed to assess ridge dimensional changes. In addition, specimens were used for microcomputed tomography (micro-CT) assessment and histologic analysis. Membrane exposure was found on five of 11 (45.5%) rhBMP-2-treated sites, whereas it was found on nine of 11 (81.8%) non-rhBMP-2-treated sites. Within 4 months of healing, rhBMP-2-treated sites showed better radiographic bone density, greater defect fill, and significantly more bone gain in ridge height (P 0.05). Under light microscope, predominant lamellar patterns were found in the specimen obtained from rhBMP-2 sites. With inherent limitations of the canine model and the concern of such a demanding surgical technique, current findings suggest that the presence of rhBMP-2 in a composite graft allows an increase of vertical gain, with formation of ectopic bone over the titanium mesh in comparison with non-rhBMP-2 sites.

  10. Recombinant human erythropoietin stimulates angiogenesis and wound healing in the genetically diabetic mouse.

    Science.gov (United States)

    Galeano, Mariarosaria; Altavilla, Domenica; Cucinotta, Domenico; Russo, Giuseppina T; Calò, Margherita; Bitto, Alessandra; Marini, Herbert; Marini, Rolando; Adamo, Elena B; Seminara, Paolo; Minutoli, Letteria; Torre, Valerio; Squadrito, Francesco

    2004-09-01

    The effects of recombinant human erythropoietin (rHuEPO) in diabetes-related healing defects were investigated by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ-m(+/+)Lept(db) mice (db(+)/db(+)) and their normoglycemic littermates (db(+/+)m). Animals were treated with rHuEPO (400 units/kg in 100 microl s.c.) or its vehicle alone (100 microl). Mice were killed on different days (3, 6, and 12 days after skin injury) for measurement of vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis, for monitoring angiogenesis by CD31 expression, and for evaluating histological changes. Furthermore, we evaluated wound-breaking strength at day 12. At day 6, rHuEPO injection in diabetic mice resulted in an increase in VEGF mRNA expression (vehicle = 0.33 +/- 0.1 relative amount of mRNA; rHuEPO = 0.9 +/- 0.09 relative amount of mRNA; P < 0.05) and protein wound content (vehicle = 23 +/- 5 pg/wound; rHuEPO = 92 +/- 12 pg/wound; P < 0.05) and caused a marked increase in CD31 gene expression (vehicle = 0.18 +/- 0.05 relative amount of mRNA; rHuEPO = 0.98 +/- 0.21 relative amount of mRNA; P < 0.05) and protein synthesis. Furthermore, rHuEPO injection improved the impaired wound healing and, at day 12, increased the wound-breaking strength in diabetic mice (vehicle = 12 +/- 2 g/mm; rHuEPO 21 +/- 5 g/mm; P < 0.05). Erythropoietin may have a potential application in diabetes-related wound disorders.

  11. Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds.

    Science.gov (United States)

    Galeano, Mariarosaria; Altavilla, Domenica; Bitto, Alessandra; Minutoli, Letteria; Calò, Margherita; Lo Cascio, Patrizia; Polito, Francesca; Giugliano, Giovanni; Squadrito, Giovanni; Mioni, Chiara; Giuliani, Daniela; Venuti, Francesco S; Squadrito, Francesco

    2006-04-01

    Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental burn wounds. Randomized experiment. Research laboratory. C57BL/6 male mice weighing 25-30 g. Mice were immersed in 80 degrees C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 microL subcutaneously) or its vehicle alone (100 microl/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

  12. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Directory of Open Access Journals (Sweden)

    Niels Jacob Aachmann-Andersen

    Full Text Available The membrane-assisted isoform immunoassay (MAIIA quantitates erythropoietin (EPO isoforms as percentages of migrated isoforms (PMI. We evaluated the effect of recombinant human EPO (rhEPO on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13; high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13; or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3 % (mean (SD. High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2% (p<0.00001 and 45.2 (7.3% (p<0.00001. Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8% (p<0.00001 and 46.1 (10.4% (p<0.00001. In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4% (p=0.029; low-dose Epoetin beta: 73.1 (17.8% (p=0.039. In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

  13. Immunosafety of recombinant human C1-inhibitor in hereditary angioedema: evaluation of ige antibodies.

    Science.gov (United States)

    Hack, C Erik; Relan, Anurag; Baboeram, Aartie; Oortwijn, Beatrijs; Versteeg, Serge; van Ree, Ronald; Pijpstra, Rienk

    2013-04-01

    Recombinant human C1-inhibitor (rhC1INH) purified from milk of transgenic rabbits is used for the treatment of acute attacks in patients with hereditary angioedema (HAE) due to C1-inhibitor (C1INH) deficiency. The objective was to investigate the risk of rhC1INH inducing IgE antibodies or eliciting anaphylactic reactions. In subjects treated with rhC1INH, we retrospectively analysed the frequency and clinical relevance of pre-exposure and potentially newly induced IgE antibodies against rabbit and other animal allergens including cow's milk by the ImmunoCAP(®) Specific IgE blood test system. 130 HAE patients and 14 healthy subjects received 300 administrations of rhC1INH, 65 subjects (47.4 %) on one occasion; 72 (52.6 %) on at least two occasions (range 2-12; median 2). Five subjects had pre-existing anti-rabbit epithelium IgE; the subject with the highest levels and a previously undisclosed rabbit allergy developed an anaphylactic reaction upon first exposure to rhC1INH, whereas the other four subjects with lower pre-existing IgE levels (Class 1-3), did not. No other anaphylactic reactions were identified in any of the subjects exposed to rhC1INH. Analysis of post-exposure samples revealed that the risk of inducing new or boosting existing IgE responses to rabbit or cow's milk allergens was negligible. The propensity of rhC1INH to induce IgE antibodies following repeated administration of rhC1INH is low. Subjects with substantially elevated anti-rabbit epithelium IgE antibodies and/or clinical allergy to rabbits may have an increased risk for an allergic reaction. No other risk factors for allergic reactions to rhC1INH have been identified.

  14. Population pharmacokinetics of recombinant human C1 inhibitor in patients with hereditary angioedema.

    Science.gov (United States)

    Farrell, Colm; Hayes, Siobhan; Relan, Anurag; van Amersfoort, Edwin S; Pijpstra, Rienk; Hack, C Erik

    2013-12-01

    To characterize the pharmacokinetics (PK) of recombinant human C1 inhibitor (rhC1INH) in healthy volunteers and hereditary angioedema (HAE) patients. Plasma levels of C1INH following 294 administrations of rhC1INH in 133 subjects were fitted using nonlinear mixed-effects modelling. The model was used to simulate maximal C1INH levels for the proposed dosing scheme. A one-compartment model with Michaelis-Menten elimination kinetics described the data. Baseline C1INH levels were 0.901 [95% confidence interval (CI): 0.839-0.968] and 0.176 U ml(-1) (95% CI: 0.154-0.200) in healthy volunteers and HAE patients, respectively. The volume of distribution of rhC1INH was 2.86 l (95% CI: 2.68-3.03). The maximal rate of elimination and the concentration corresponding to half this maximal rate were 1.63 U ml(-1) h(-1) (95% CI: 1.41-1.88) and 1.60 U ml(-1) (95% CI: 1.14-2.24), respectively, for healthy volunteers and symptomatic HAE patients. The maximal elimination rate was 36% lower in asymptomatic HAE patients. Peak C1INH levels did not change upon repeated administration of rhC1INH. Bodyweight was found to be an important predictor of the volume of distribution. Simulations of the proposed dosing scheme predicted peak C1INH concentrations above the lower level of the normal range (0.7 U ml(-1)) for at least 94% of all patients. The population PK model for C1INH supports a dosing scheme on a 50 U kg(-1) basis up to 84 kg, with a fixed dose of 4200 U above 84 kg. The PK of rhC1INH following repeat administration are consistent with the PK following the first administration. © 2013 The British Pharmacological Society.

  15. Nonclinical comparability studies of recombinant human arylsulfatase A addressing manufacturing process changes.

    Science.gov (United States)

    Wright, Teresa; Li, Aiqun; Lotterhand, Jason; Graham, Anne-Renee; Huang, Yan; Avila, Nancy; Pan, Jing

    2018-01-01

    Recombinant human arylsulfatase A (rhASA) is in clinical development for the treatment of patients with metachromatic leukodystrophy (MLD). Manufacturing process changes were introduced to improve robustness and efficiency, resulting in higher levels of mannose-6-phosphate and sialic acid in post-change (process B) compared with pre-change (process A) rhASA. A nonclinical comparability program was conducted to compare process A and process B rhASA. All doses were administered intrathecally. Pharmacodynamic comparability was evaluated in immunotolerant MLD mice, using immunohistochemical staining of lysosomal-associated membrane protein-1 (LAMP-1). Pharmacokinetic comparability was assessed in juvenile cynomolgus monkeys dosed once with 6.0 mg (equivalent to 100 mg/kg of brain weight) process A or process B rhASA. Biodistribution was compared by quantitative whole-body autoradiography in rats. Potential toxicity of process B rhASA was evaluated by repeated rhASA administration at doses of 18.6 mg in juvenile cynomolgus monkeys. The specific activities for process A and process B rhASA were 89 U/mg and 106 U/mg, respectively, which were both well within the target range for the assay. Pharmacodynamic assessments showed no statistically significant differences in LAMP-1 immunohistochemical staining in the spinal cord and in most of the brain areas assessed between process A and B rhASA-dosed mice. LAMP-1 staining was reduced with both process A and B rhASA compared with vehicle, supporting its activity. Concentration-time curves in cerebrospinal fluid and serum of cynomolgus monkeys were similar with process A and B rhASA. Process A and B rhASA were similar in terms of their pharmacokinetic parameters and biodistribution data. No process B rhASA-related toxicity was detected. In conclusion, manufacturing process changes did not affect the pharmacodynamic, pharmacokinetic or safety profiles of process B rhASA relative to process A rhASA.

  16. Combination of recombinant human growth hormone and propranolol decreases hypermetabolism and inflammation in severely burned children.

    Science.gov (United States)

    Jeschke, Marc G; Finnerty, Celeste C; Kulp, Gabriela A; Przkora, Rene; Mlcak, Ronald P; Herndon, David N

    2008-03-01

    Recombinant human growth hormone (rhGH) is a salutary modulator of posttraumatic metabolic responses. However, rhGH administration is associated with deleterious side effects, such as hyperglycemia, increased free fatty acids, and triglycerides, which limit its use. Administration of beta-blocker attenuates cardiac work and resting energy expenditure after severe thermal injury and improves fat metabolism and insulin sensitivity. Therefore, the combination of rhGH plus propranolol appears ideal. The aim of the present study was to determine whether rhGH plus propranolol improves hypermetabolism and the inflammatory and acute phase response after severe burn without causing adverse side effects. Prospective randomized control trial. Shriners Hospitals for Children. Fifteen pediatric patients with burns > 40% total body surface area, 0.1-16 yrs of age, admitted within 7 days after burn. Fifteen children were matched for burn size, age, gender, inhalation injury, and infection and served as controls. Patients in the experimental group received rhGH (0.2 mg/kg/day) and propranolol (to decrease heart rate by 15%) for > or = 15 days. Outcome measurements included resting energy expenditure, body composition, acute phase proteins, and cytokines. Both cohorts were similar in age, burn size, gender, and accompanying injuries. Percent predicted resting energy expenditure significantly decreased in patients receiving rhGH/propranolol (Delta -5% +/- 8%) compared with controls (Delta +35% +/- 20%) (p < .05). rhGH/propranolol administration significantly decreased serum C-reactive protein, cortisone, aspartate aminotransferase, alanine aminotransferase, free fatty acids, interleukin-6, interleukin-8, and macrophage inflammatory protein-1beta when compared with controls, while growth hormone/propranolol increased serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, growth hormone, prealbumin, and interleukin-7 when compared with placebo (p < .05). rh

  17. [Recombinant human growth hormone treatment in short children with renal disease--our first experience].

    Science.gov (United States)

    Spasojević-Dimitrijeva, Brankica; Kostić, Mirjana; Peco-Antić, Amira; Kruscić, Divna; Cvetković, Mirjana; Milosevski-Lomić, Gordana; Paripović, Dusan

    2010-01-01

    Growth retardation is a hallmark of chronic illnesses such as chronic kidney disease in children, and it is associated with increased morbidity and mortality. The growth hormone (GH) resistance observed in uraemia can be overcome by supraphysiological doses of exogenous GH. We would like to present our first results of recombinant human growth hormone (rhGH) treatment, mainly in children on haemodialysis. Sixteen children, aged 4.5-17.1 years (mean age 11.25 +/- 3.57) with height below -2.0 standard deviation score (SDS) for age or height velocity below -2.0 SDS for age, were selected to receive rhGH therapy at our Nephrology and Haemodialysis Department. Most of them were on haemodialysis (14 children) with mean spent time 2.88 +/- 2.68 years (0-9 years) before the initiation of rhGH therapy. One half of patients were prepubertal (8 children) and the second half were in early puberty (testicular volume between 4 and 8 ml for boys and breast development B2 or B3 in girls). All patients received 28-30 IU/m2 rhGH per week by daily subcutaneous injection. The year before rhGH therapy served as a control period. During the first year of treatment, mean height velocity in haemodialysis patients increased from 2.25 cm/year to 6.59 cm/year (p children did not improve significantly during the first year of rhGH treatment (from -3.01 SDS to -2.77 SDS, p = 0.063). Neither weight nor the body mass index varied compared with the pretreatment period. Two patients developed worsened secondary hyperparathyroidism and were excluded from the study, but the relationship with rhGH remains uncertain. Mean height velocity significantly improved during rhGH therapy in haemodialysis patients. No significant side-effects were observed in children during three-year treatment with GH.

  18. Effects of low-dose recombinant human erythropoietin treatment on cognitive performance.

    Science.gov (United States)

    Viuff, Søren Lundgaard; Plenge, Ulla; Belhage, Bo; Boushel, Robert; Koester, Thomas

    2017-09-01

    High-dose recombinant human erythropoietin (rhEpo) has been shown to improve cognitive performance in both healthy volunteers and in patients suffering from diseases affecting the brain. The aim of this study was to examine whether administration of low-dose and even micro-dose rhEpo improves cognitive performance in healthy volunteers. We enrolled 25 healthy volunteers in a double-blind, randomised, placebo-controlled study to receive either low-dose rhEpo (n = 8, 60 IU/kg/week), micro-dose rhEpo (n = 9, 20 IU/kg/week), or saline (n = 8) for four weeks. Two cognitive performance-tests, the Raven Standard Progressive Matrices (Raven) and the Number Finder (NUFI), were performed during the first and last day of the study period. Semi-structured interviews were conducted weekly and were coded according to a scale. Subjects receiving micro-dose rhEpo improved significantly measured by the Raven score (p = 0.04), and subjects receiving low-dose rhEpo treatment improved significantly measured by the NUFI score (p = 0.047), whereas no improvement was found in experienced cognitive performance in any of the groups. We found no significant difference in either Raven, NUFI or self-reported results between the groups. In this small study, we found no significant effect of low-dose or micro-dose rhEpo on visual attention, cognitive performance in complex cognitive tasks or self-experienced cognitive performance compared with placebo. The Aase and Ejnar Danielsen's Foundation. Danish Ministry of Science, Innovation and Higher Education. ClinicalTrials.gov identifier: NCT03093506. Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

  19. Heterotopic ossification related to the use of recombinant human BMP-2 in osteonecrosis of femoral head.

    Science.gov (United States)

    Shi, Lijun; Sun, Wei; Gao, Fuqiang; Cheng, Liming; Li, Zirong

    2017-07-01

    Despite the wide use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in bone defect, its application in treating osteonecrosis of femoral head (ONFH) is yet to be elucidated. The heterotopic ossification (HO) after rhBMP-2 usage in some orthopedic surgeries has been reported previously; however, only a few studies describe this complication in the treatment of ONFH.The present study investigated whether the rhBMP-2 application would increase the risk of HO formation in selected ONFH patients with nonvascularized bone grafting surgery and enhance the surgical results of nonvascularized bone grafting as compared to patients who did not receive intraoperative rhBMP-2.A retrospective analysis was performed on 94 patients (141 hips) who, with Association Research Circulation Osseous (ARCO) stages IIb, IIc, and IIIa ONFH, underwent nonvascularized bone grafting surgery. The first 46 patients (66 hips) received intraoperative rhBMP-2. The postoperative radiographic results (X-ray and CT scan) and Harris hip score (HHS) were reviewed in each patient to record the incidence of HO formation and evaluate the clinical efficacy of rhBMP-2, respectively.HO formation frequently occurred in patients receiving intraoperative rhBMP-2 (8/66 hips) than those not receiving the protein (1/75 hips) (P = .02). HHS improved from preoperatively at the final follow-up (P < .01) in the BMP-positive group, with a survival rate of 83.3%. In the BMP-negative group, the HHS improved from preoperatively at the end of the follow-up (P < .01), and the survival rate was 72.0%.rhBMP-2 has osteoinductive property and might serve as an adjuvant therapy in the surgical treatment of ONFH. However, the incidence of HO formation might increase when used in high doses.

  20. Experimental research on recombinant human endostatin-induced cardiomyocyte apoptosis in rats

    Directory of Open Access Journals (Sweden)

    Jing QIN

    2014-03-01

    Full Text Available Objective To explore the recombinant human endostatin (rh-ES-induced cardiotoxicity in rats and its mechanism. Methods Twenty four female Wistar rats were randomly divided into four groups (6 each. Rats in low, moderate and high dose group received rh-ES with a dosage of 3, 6 and 12mg/(kg·d, respectively, by intraperitoneal injection, and rats in control group received the same amount of normal saline alone. Half of rats in each group were sacrificed by spinal dislocation after 4 weeks and 8 weeks of the treatment. Pathomorphologic and ultrastructural changes in rat's myocardial tissue were evaluated by light microscopy and transmission electron microscopy. Cardiomyocyte apoptosis was detected with TdT-mediated dUTP nick end labeling (TUNEL assay. Microvessel density (MVD in myocardial tissue was measured by immunohistochemically marking endothelial cell with CD34. Results No pathomorphologic and ultrastrucural changes were found under light microscope and transmission electron microscope in the low dose and moderate dose groups, but cardiomyocyte damage were found in the high dose group. TUNEL assay revealed more apoptotic cells in high and moderate (only 8 weeks dose groups than in control group (P=0.033, P=0.000, and the apoptosis index was highest in the high dose group at 8 weeks. In addition, compared with the control group, MVD significantly increased in high dose groups at 4 weeks and 8 weeks (P<0.05. Conclusions rh-ES induces the cardiotoxicity in rats, and cardiomyocyte apoptosis is involved in the pathological course of cardiac toxicity. DOI: 10.11855/j.issn.0577-7402.2014.01.02

  1. Recombinant human TSH in differentiated thyroid cancer: a nuclear medicine perspective

    Energy Technology Data Exchange (ETDEWEB)

    Zanotti-Fregonara, P. [CEA, DSV, I2BM, SHFJ, LMNRB, Orsay (France); Rubello, D. [Osped S Maria Misericordia, IRCCS, IOV, Dept Nucl Med, PET Ctr, I-45100 Rovigo (Italy); Hindie, E. [Hop St Louis, Dept Nucl Med, Paris (France)

    2008-07-01

    The use of recombinant human thyroid-stimulating hormone (rhTSH) in differentiated thyroid cancer (DTC) is widely discussed in the literature with regard to the diagnostic and therapeutic aspects of the management of DTC patients. However, some controversy about the appropriate indications, advantages and potential disadvantages of the use of rhTSH may still exist within the community of nuclear medicine physicians. In our opinion, the clinical benefits of rhTSH in avoiding hypothyroidism outweigh its somewhat lesser diagnostic accuracy. However, we disagree on designating rhTSH as the 'golden standard' to obtain TSH stimulation, as suggested by some authors. Thus, the first follow-up examination after ablation, which is determinant for patients' prognostic classification, can be either done under rhTSH stimulation or after hormone withdrawal. In our practice, and for higher risk patients, we still favour performing the initial follow-up after thyroid hormone withdrawal. rhTSH also shows the ability to enhance radioiodine concentration into thyroid cells. This characteristic is obviously of great interest among the nuclear medicine community. In clinical practice, it seems preferable to perform {sup 131}I treatment for metastatic disease during hypothyroidism. rhTSH may find its utility for the treatment of specific populations of patients, i.e. those in whom hormone withdrawal is medically contraindicated or in whom adequate endogenous TSH levels cannot be obtained due to reduced pituitary reserve or continued thyroxine production by metastatic tissue. In conclusion, rhTSH has demonstrated to be a reliable alternative to hypothyroidism for the stimulation of Tg in the follow-up of thyroid cancer patients. However, its use must be more carefully chosen in the therapeutic setting. Our feeling is that rhTSH should no tbe used for remnant ablation in high-risk patients and for the treatment of metastatic disease, except for specific populations of

  2. Effect of hemodilution on coagulation and recombinant factor VIIa efficacy in human blood in vitro.

    Science.gov (United States)

    Darlington, Daniel N; Delgado, Angel V; Kheirabadi, Bijan S; Fedyk, Chriselda G; Scherer, Michael R; Pusateri, Anthony E; Wade, Charles E; Cap, Andrew P; Holcomb, John B; Dubick, Michael A

    2011-11-01

    This study evaluates the effect of hemodilution by various common resuscitation fluids, and the efficacy of activated recombinant factor VII (rFVIIa) on coagulation parameters in human blood in vitro. Samples from normal healthy volunteers (n = 9) were hemodiluted from 0% to 90% with normal saline, or 0%, 40%, 60%, and 80% with 5% albumin, Hespan, Hextend, normal saline, or lactated Ringer's, and incubated at 37°C ± 1°C for 30 minutes with and without rFVIIa (1.26 μg/mL). There was a strong correlation between the dilution of hemoglobin (Hb), platelets, or fibrinogen and coagulation parameters. Hemodilution 0% to 90% changed coagulation parameters (prothrombin time [PT], activated partial thromboplastin time [aPTT], and thromboelastography) in an exponential fashion; the greatest changes occurred after hemodilution lowered Hb time (K). Hemodilution with Hextend and Hespan decreased maximum amplitude and α angle >5% albumin, lactated Ringer's, or normal saline. rFVIIa significantly improved PT at 60% and 80% dilutions, and aPTT at 80% dilution. There was a significant effect of dilution, but not fluid type, on the efficacy of rFVIIa to change PT and aPTT, and the onset of clotting (R). We have strong in vitro evidence that Hb 5% albumin or the crystalloids. rFVIIa significantly decreased PT at all dilutions and aPTT at the highest dilution. The effectiveness of rFVIIa on PT and aPTT was significantly affected by the degree of dilution, but not by the type of fluid.

  3. Improvement of in vivo efficacy of recombinant human erythropoietin by encapsulation in PEG–PLA micelle

    Directory of Open Access Journals (Sweden)

    Shi YN

    2012-12-01

    Full Text Available Yanan Shi,1,2,* Wan Huang,1,* Rongcai Liang,1–3 Kaoxiang Sun,2,3 Fangxi Zhang,2,3 Wanhui Liu,2,3 Youxin Li1–31College of Life Science, Jilin University, Changchun, China; 2State Key Laboratory of Long-acting and Targeting Drug Delivery System, Luye Pharmaceutical Co, Ltd, Yantai, China; 3School of Pharmacy, Yantai University, Yantai, China*These authors contributed equally to this workAbstract: To improve the pharmacokinetics and stability of recombinant human erythropoietin (rhEPO, rhEPO was successfully formulated into poly(ethylene glycol–poly(d,l-lactide (PEG–PLA di-block copolymeric micelles at diameters ranging from 60 to 200 nm with narrow polydispersity indices (PDIs; PDI < 0.3 and trace amount of protein aggregation. The zeta potential of the spherical micelles was in the range of −3.78 to 4.65 mV and the highest encapsulation efficiency of rhEPO in the PEG–PLA micelles was about 80%. In vitro release profiles indicated that the stability of rhEPO in the micelles was improved significantly and only a trace amount of aggregate was found. Pharmacokinetic studies in rats showed highly enhanced plasma retention time of the rhEPO-loaded PEG-PLA micelles in comparison with the native rhEPO group. Increased hemoglobin concentrations were also found in the rat study. Native polyacrylamide gel electrophoresis results demonstrated that rhEPO was successfully encapsulated into the micelles, which was stable in phosphate buffered saline with different pHs and concentrations of NaCl. Therefore, PEG–PLA micelles can be a potential protein drug delivery system.Keywords: rhEPO, PEG–PLA micelle, in vitro, pharmacokinetics, pharmacodynamics

  4. Pharmacokinetics and tissue distribution of recombinant human tumor necrosis factor-alpha in mice

    International Nuclear Information System (INIS)

    Ferraiolo, B.L.; Moore, J.A.; Crase, D.; Gribling, P.; Wilking, H.; Baughman, R.A.

    1988-01-01

    The serum pharmacokinetics and the major organs of accumulation of recombinant human tumor necrosis factor-alpha (rHuTNF) were determined in BDF1 mice after intravenous and intramuscular administration. Serum concentrations of immunoreactive protein were determined by enzyme-linked immunosorbent assay, and radioactivity was quantitated by beta and gamma scintigraphy. The serum pharmacokinetics of labeled and unlabeled rHuTNF were identical when administered by the intravenous route. After intravenous doses of 165 to 320 micrograms/kg, the clearance was 2.9-3.6 ml/hr, the initial volume of distribution was 1.4-1.6 ml (70-80 ml/kg), and the half-life was 18.5-19.2 min. Intramuscular administration of 320 micrograms/kg resulted in a peak serum concentration of 112 ng/ml. The time of the peak concentration was 1 hr, and the bioavailability of the intramuscular dose was 12%. The data suggest that the disposition of this protein may be biexponential. If this is the case, the terminal phase would appear to account for less than 1% of the total AUC. Since serum concentrations in the terminal phase are at the sensitivity limit of the assay, a single half-life is reported. 125I-Labeled and metabolically labeled 3H-rHuTNF were used to examine tissue distribution. After intravenous 125I-rHuTNF administration, the rank order of accumulation of the 125I-radiolabel in the major organs (per cent dose per organ over 1440 min) was: liver greater than kidney greater than lung greater than heart greater than spleen. This rank order of accumulation was confirmed by intravenous 3H-rHuTNF administration

  5. Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults

    Directory of Open Access Journals (Sweden)

    Piotr Szpakowski

    2015-01-01

    Full Text Available Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG, the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18 and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4+ T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

  6. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

    Directory of Open Access Journals (Sweden)

    Pacífico Lucila G

    2007-01-01

    Full Text Available Abstract Background Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL in the presence of Polymyxin B (10 μg/mL. Results The levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves. Conclusion This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.

  7. Recombinant human lactoferrin as a biomaterial for bone tissue engineering: mechanism of antiapoptotic and osteogenic activity.

    Science.gov (United States)

    Amini, Ashley A; Nair, Lakshmi S

    2014-06-01

    Lactoferrin is a bioactive globular protein with unique properties towards musculo-skeletal cells and anabolic to bone in vivo. Even though the potent anti-apoptotic and osteogenic activity of lactoferrin has been reported, the mechanism of action has not been fully elucidated. The study demonstrates that the anti-apoptotic effect of rhLF towards MC3T3 pre-osteoblast cells is mediated by Wnt5a/PKA pathway and the stabilization of β-catenin by rhLF is dependent on PKA/LRP6 signaling pathway. The study also investigates the feasibility of developing rhLF as a biomaterial for cell delivery. The injectable rhLF cell delivery vehicles are prepared by enzymatic crosslinking of tyramine-modified rhLF in the presence of hydrogen peroxide and horseradish peroxidase. The modified rhLF shows bioactivity similar to unmodified rhLF. The rhLF gels support encapsulated MC3T3 cell viability, proliferation, and differentiation, as well as phosphorylation of signaling proteins. In conclusion, the study demonstrates the involvement of Wnt5a, LRP6, and PKA signaling in rhLF-mediated bioactivity towards MC3T3 cells and the feasibility of developing an injectable cell delivery vehicle from rhLF. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. UV-dependent production of 25-hydroxyvitamin D2 in the recombinant yeast cells expressing human CYP2R1

    International Nuclear Information System (INIS)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2013-01-01

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D 3 or vitamin D 2 was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D 2 was produced without additional vitamin D 2 . Endogenous ergosterol was likely converted into vitamin D 2 by UV irradiation and thermal isomerization, and then the resulting vitamin D 2 was converted to 25-hydroxyvitamin D 2 by CYP2R1. This novel method for producing 25-hydroxyvitamin D 2 without a substrate could be useful for practical purposes

  9. Construction and characterization of recombinant fowlpox viruses expressing human papilloma virus E6 and E7 oncoproteins.

    Science.gov (United States)

    Pozzi, Eleana; Basavecchia, Valeria; Zanotto, Carlo; Pacchioni, Sole; Morghen, Carlo De Giuli; Radaelli, Antonia

    2009-06-01

    Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype and the expression of the E6 and E7 proteins, which can bind to the p53 and p105Rb host cell-cycle regulatory proteins, is related to its tumorigenicity. Virus-like-particle (VLP)-based immunogens developed recently are successful as prophylactic HPV vaccines. However, given the high number of individuals infected already with HPV and the absence of expression of the L1 structural protein in HPV-infected or HPV-transformed cells, an efficient therapeutic vaccine targeting the non-structural E6 and E7 oncoproteins is required. In this study, two new fowlpox virus (FPV) recombinants encoding the HPV-16 E6 and E7 proteins were engineered and evaluated for their correct expression in vitro, with the final aim of developing a therapeutic vaccine against HPV-related cervical tumors. Although vaccinia viruses expressing the HPV-16 and HPV-18 E6 and E7 oncoproteins have already been studied, due to their natural host-range restriction to avian species and their ability to elicit a complete immune response, FPV recombinants may represent efficient and safer vectors also for immunocompromised hosts. The results indicate that FPV recombinants can express correctly the E6 and E7 oncoproteins, and they should represent appropriate vectors for the expression of these oncoproteins in human cells.

  10. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Raheem Ullah

    Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  11. A Randomized Case-Controlled Study of Recombinant Human Granulocyte Colony Stimulating Factor for the Treatment of Sepsis in Preterm Neutropenic Infants

    OpenAIRE

    Aktaş, Doğukan; Demirel, Bilge; Gürsoy, Tuğba; Ovalı, Fahri

    2015-01-01

    To investigate the efficacy and safety of recombinant human granulocyte colony-stimulating factor, recombinant human granulocyte-macrophage colony-stimulating factor (rhG-CSF) to treat sepsis in neutropenic preterm infants. Methods: Fifty-six neutropenic preterm infants with suspected or culture-proven sepsis hospitalized in Zeynep Kamil Maternity and Children's Educational and Training Hospital, Kozyatağı/Istanbul, Turkey between January 2008 and January 2010 were enrolled. Patients were ...

  12. Evidence for repair of ultraviolet light-damaged herpes virus in human fibroblasts by a recombination mechanism

    International Nuclear Information System (INIS)

    Hall, J.D.; Featherston, J.D.; Almy, R.E.

    1980-01-01

    Human cells were either singly or multiply infected with herpes simplex virus (HSV-1) damaged by ultraviolet (uv) light, and the fraction of cells able to produce infectious virus was measured. The fraction of virus-producing cells was considerably greater for multiply infected cells than for singly infected cells at each uv dose examined. These high survival levels of uv-irradiated virus in multiply infected cells demonstrated that multiplicity-dependent repair, possibly due to genetic exchanges between damaged HSV-1 genomes, was occurring in these cells. To test whether uv light is recombinogenic for HSV-1, the effect of uv irradiation on the yield of temperature-resistant viral recombinants in cells infected with pairs of temperature-sensitive mutants was also investigated. The results of these experiments showed that the defective functions in these mutant host cells are not required for multiplicity-dependent repair or uv-stimulated viral recombination in herpes-infected cells

  13. Genetic analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene.

    Directory of Open Access Journals (Sweden)

    Elizabeth B Liu

    Full Text Available In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487 was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event.

  14. A fine-scale recombination map of the human-chimpanzee ancestor reveals faster change in humans than in chimpanzees and a strong impact of GC-biased gene conversion.

    Science.gov (United States)

    Munch, Kasper; Mailund, Thomas; Dutheil, Julien Y; Schierup, Mikkel Heide

    2014-03-01

    Recombination is a major determinant of adaptive and nonadaptive evolution. Understanding how the recombination landscape has evolved in humans is thus key to the interpretation of human genomic evolution. Comparison of fine-scale recombination maps of human and chimpanzee has revealed large changes at fine genomic scales and conservation over large scales. Here we demonstrate how a fine-scale recombination map can be derived for the ancestor of human and chimpanzee, allowing us to study the changes that have occurred in human and chimpanzee since these species diverged. The map is produced from more than one million accurately determined recombination events. We find that this new recombination map is intermediate to the maps of human and chimpanzee but that the recombination landscape has evolved more rapidly in the human lineage than in the chimpanzee lineage. We use the map to show that recombination rate, through the effect of GC-biased gene conversion, is an even stronger determinant of base composition evolution than previously reported.

  15. BG-4, a novel bioactive peptide from Momordica charantia, inhibits lipopolysaccharide-induced inflammation in THP-1 human macrophages

    Science.gov (United States)

    Background: Bitter melon (Momordica charantia) is a commonly used food crop for management of a variety of diseases most notably for control of diabetes, a disease associated with aberrant inflammation. Purpose: To evaluate the anti-inflammatory property of BG-4, a novel bioactive peptide isolated f...

  16. Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: Safety, pharmacokinetics, and distribution

    Energy Technology Data Exchange (ETDEWEB)

    Vuillemenot, Brian R., E-mail: bvuillemenot@bmrn.com [BioMarin Pharmaceutical Inc., Novato, CA (United States); Kennedy, Derek [BioMarin Pharmaceutical Inc., Novato, CA (United States); Reed, Randall P.; Boyd, Robert B. [Northern Biomedical Research, Inc., Muskegon, MI (United States); Butt, Mark T. [Tox Path Specialists, LLC, Hagerstown, MD (United States); Musson, Donald G.; Keve, Steve; Cahayag, Rhea; Tsuruda, Laurie S.; O' Neill, Charles A. [BioMarin Pharmaceutical Inc., Novato, CA (United States)

    2014-05-15

    CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20 mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3–14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS. - Highlights: • TPP1 enzyme replacement therapy to the CNS is in development for CLN2 disease. • Toxicology, pharmacokinetics, and CNS distribution were assessed in monkeys. • TPP1 infusion directly to the brain did not result in any safety concerns. • A positive pharmacokinetic and distribution profile resulted from TPP1 infusion. • This study demonstrates the feasibility of ICV administered

  17. Treatment effects of recombinant human soluble thrombomodulin in patients with severe sepsis: a historical control study.

    Science.gov (United States)

    Yamakawa, Kazuma; Fujimi, Satoshi; Mohri, Tomoyoshi; Matsuda, Hiroki; Nakamori, Yasushi; Hirose, Tomoya; Tasaki, Osamu; Ogura, Hiroshi; Kuwagata, Yasuyuki; Hamasaki, Toshimitsu; Shimazu, Takeshi

    2011-01-01

    Cross-talk between the coagulation system and inflammatory reactions during sepsis causes organ damage followed by multiple organ dysfunction syndrome or even death. Therefore, anticoagulant therapies have been expected to be beneficial in the treatment of severe sepsis. Recombinant human soluble thrombomodulin (rhTM) binds to thrombin to inactivate coagulation, and the thrombin-rhTM complex activates protein C to produce activated protein C. The purpose of this study was to examine the efficacy of rhTM for treating patients with sepsis-induced disseminated intravascular coagulation (DIC). This study comprised 65 patients with sepsis-induced DIC who required ventilatory management. All patients fulfilled the criteria of severe sepsis and the International Society on Thrombosis and Haemostasis criteria for overt DIC. The initial 45 patients were treated without rhTM (control group), and the following 20 consecutive patients were treated with rhTM (0.06 mg/kg/day) for six days (rhTM group). The primary outcome measure was 28-day mortality. Stepwise multivariate Cox regression analysis was used to assess which independent variables were associated with mortality. Comparisons of Sequential Organ Failure Assessment (SOFA) score on sequential days between the two groups were analyzed by repeated measures analysis of variance. Cox regression analysis showed 28-day mortality to be significantly lower in the rhTM group than in the control group (adjusted hazard ratio, 0.303; 95% confidence interval, 0.106 to 0.871; P = 0.027). SOFA score in the rhTM group decreased significantly in comparison with that in the control group (P = 0.028). In the post hoc test, SOFA score decreased rapidly in the rhTM group compared with that in the control group on day 1 (P < 0.05). We found that rhTM administration may improve organ dysfunction in patients with sepsis-induced DIC. Further clinical investigations are necessary to evaluate the effect of rhTM on the pathophysiology of sepsis

  18. The value of recombinant human TSH-aided 131I treatment in differentiated thyroid carcinoma patients

    International Nuclear Information System (INIS)

    Ding Yong; Long Yahong; Tian Jiahe; Xu Baixuan; Xing Jialiu; Fang Yi; Wei Lijing; Zong Zhaoyi

    2013-01-01

    Objective: To evaluate the efficacy and safety of recombinant human TSH(rhTSH)-aided 131 I treatment for DTC. Methods: A total of 144 patients with DTC who underwent total or near total thyroidectomy were retrospectively analyzed. The rhTSH-aided 131 I treatment of 3.7 GBq was performed in 72 cases (Group Ⅰ: euthyroid). Another 72 cases received radioiodine ablation treatment of 3.7 GBq after 4 to 6 weeks of thyroxine withdrawal (Group Ⅱ: hypothyroidism). Serum endogenous TSH, FT 3 , FT 4 and Tg were measured. The life qualities of both groups were observed, such as intolerance to cold, weight gain, constipation, motor retardation, skin dryness, periorbital edema and bone pain. Absence of visible uptake or uptake rate less than 1% was taken as complete ablation. The efficacy of 131 I treatment was evaluated. The life quality of both groups was evaluated by χ 2 test, and the effect of 131 I treatment was analyzed by t test. Results: Serum TSH was effectively improved in both groups before 131 I treatment. In group Ⅰ, TSH was higher than that of group Ⅱ ((141.26 ± 27.30) mU/L vs (70.57 ± 51.13) mU/L; t=2.435, P<0.05), and FT 3 , FT 4 were not significantly different before or after the injection of rhTSH. Tg was well stimulated in both groups with no statistical difference. Group Ⅱ exhibited more side effects, which included intolerance to cold 80.56% (58/72), weight gain 86.11% (62/72), constipation 15.28% (11/72), motor retardation 22.22% (16/72), skin dryness 56.94% (41/72), bone pain 2.78% (2/72), and no periorbital edema was found. Group Ⅰ had a higher quality of life than group Ⅱ, only few side effects were observed including dizziness and nausea 2.78% (2/72), bone pain 2.78% (2/72), and transient tachycardia 1.39% (1/72). The effect of 131 I treatment was evaluated by whole body scans with a diagnostic dose of 131 I. The complete ablation rate was 70.83% (51/72) in group Ⅰ and 66.67% (48/72) in group Ⅱ (χ 2 =0.58, P>0.05). Conclusion: The

  19. Recombinant human growth hormone treatment in short children with renal disease: Our first experience

    Directory of Open Access Journals (Sweden)

    Spasojević-Dimitrijeva Brankica

    2010-01-01

    Full Text Available Introduction. Growth retardation is a hallmark of chronic illnesses such as chronic kidney disease in children, and it is associated with increased morbidity and mortality. The growth hormone (GH resistance observed in uraemia can be overcome by supraphysiological doses of exogenous GH. Objective. We would like to present our first results of recombinant human growth hormone (rhGH treatment, mainly in children on haemodialysis. Methods. Sixteen children, aged 4.5-17.1 years (mean age 11.25±3.57 with height below -2.0 standard deviation score (SDS for age or height velocity below -2.0 SDS for age, were selected to receive rhGH therapy at our Nephrology and Haemodialysis Department. Most of them were on haemodialysis (14 children with mean spent time 2.88±2.68 years (0-9 years before the initiation of rhGH therapy. One half of patients were prepubertal (8 children and the second half were in early puberty (testicular volume between 4 and 8 ml for boys and breast development B2 or B3 in girls. All patients received 28-30IU/m² rhGH per week by daily subcutaneous injection. The year before rhGH therapy served as a control period. Results. During the first year of treatment, mean height velocity in haemodialysis patients increased from 2.25 cm/year to 6.59 cm/year (p<0.0001 and in the second year it was 5.25 cm/ year (p=0.004. The mean height SDS in haemodialysis children did not improve significantly during the first year of rhGH treatment (from -3.01 SDS to -2.77 SDS, p=0.063. Neither weight nor the body mass index varied compared with the pretreatment period. Two patients developed worsened secondary hyperparathyroidism and were excluded from the study, but the relationship with rhGH remains uncertain. Conclusion. Mean height velocity significantly improved during rhGH therapy in haemodialysis patients. No significant side-effects were observed in children during three-year treatment with GH.

  20. Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: Safety, pharmacokinetics, and distribution

    International Nuclear Information System (INIS)

    Vuillemenot, Brian R.; Kennedy, Derek; Reed, Randall P.; Boyd, Robert B.; Butt, Mark T.; Musson, Donald G.; Keve, Steve; Cahayag, Rhea; Tsuruda, Laurie S.; O'Neill, Charles A.

    2014-01-01

    CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20 mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3–14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS. - Highlights: • TPP1 enzyme replacement therapy to the CNS is in development for CLN2 disease. • Toxicology, pharmacokinetics, and CNS distribution were assessed in monkeys. • TPP1 infusion directly to the brain did not result in any safety concerns. • A positive pharmacokinetic and distribution profile resulted from TPP1 infusion. • This study demonstrates the feasibility of ICV administered

  1. Strontium- and cobalt-substituted bioactive glasses seeded with human umbilical cord perivascular cells to promote bone regeneration via enhanced osteogenic and angiogenic activities.

    Science.gov (United States)

    Kargozar, Saeid; Lotfibakhshaiesh, Nasrin; Ai, Jafar; Mozafari, Masoud; Brouki Milan, Peiman; Hamzehlou, Sepideh; Barati, Mahmood; Baino, Francesco; Hill, Robert G; Joghataei, Mohammad Taghi

    2017-08-01

    Designing and developing new biomaterials to accelerate bone healing are currently under progress. In this study, we attempted to promote osteogenesis using strontium- and cobalt-substituted bioactive glasses (BGs) seeded with human umbilical cord perivascular cells (HUCPVCs) in a critical size defect in the distal femur of rabbit animal model. The BG particles were successfully synthesized in the form of granules using the melt-derived route. After being isolated, HUCPVCs were expanded and then characterized to use during in vitro and in vivo procedures. The in vitro effects of the synthesized glasses on the isolated HUCPVCs as well as on cell lines SaOS-2 (selected for screening the osteogenetic potential) and HUVEC (selected for screening the angiogenic potential) were assessed by analyzing cytotoxicity, cell attachment, bone-like nodule formation, and real time PCR. The results of in vitro tests indicated cytocompatibility of the synthesized BG particles. For in vivo study, the HUCPVCs-seeded BGs were implanted into the animal's body. Radiographic imaging, histology and immunohistology staining were performed on the harvested specimens at 4 and 12weeks post-surgery. The in vivo evaluation of the samples showed that all the cell/glass constructs accelerated bone healing process in comparison with blank controls. The best in vitro and in vivo results were associated to the BGs containing both strontium and cobalt ions. This group of bioactive glasses is able to promote both osteogenesis and angiogenesis and can therefore be highly suitable for the development of advanced functional bone substitutes. Bone regeneration is considered as an unmet clinical need. The most recent researches focused on incorporation of strontium (Sr 2+ ) and cobalt (Co 2+ ) ions into bioactive glasses structure. Strontium is an alkaline earth metal which is currently used in the treatment of osteoporosis. Also, cobalt is considered as another promising element in the bone regeneration

  2. Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Maheswara Reddy Mallu

    2016-07-01

    Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.

  3. Human histologic evaluation of anorganic bovine bone mineral combined with recombinant human platelet-derived growth factor BB in maxillary sinus augmentation: case series study.

    Science.gov (United States)

    Nevins, Myron; Garber, David; Hanratty, James J; McAllister, Bradley S; Nevins, Marc L; Salama, Maurice; Schupbach, Peter; Wallace, Steven; Bernstein, Simon M; Kim, David M

    2009-12-01

    The objective of this proof-of-principle study was to examine the potential for improved bone regenerative outcomes in maxillary sinus augmentation procedures when recombinant human platelet-derived growth factor BB (0.3 mg/mL) is combined with particulate anorganic bovine bone mineral. The surgical outcomes in all treated sites were uneventful at 6 to 8 months, with sufficient regenerated bone present to allow successful placement of maxillary posterior implants. Large areas of dense, well-formed lamellar bone were seen throughout the intact core specimens in more than half of the grafted sites. Abundant numbers of osteoblasts were noted in concert with significant osteoid in all sites, indicating ongoing osteogenesis. A number of cores demonstrated efficient replacement of the normally slowly resorbing anorganic bovine bone mineral matrix particles with newly formed bone when the matrix was saturated with recombinant human platelet-derived growth factor BB.

  4. [Construction of a recombinant adenovirus vector harboring human transforming growth factor-beta type II receptor-IgG1Fc fusion gene].

    Science.gov (United States)

    Jia, Li; Xue, Jian-xin; Lu, You

    2008-12-01

    To construct a recombinant adenoviral vector harboring human transforming growth factor-beta type II receptor-IgG1Fc (TbetaRII-IgG1Fc) fusion gene. The cDNA fragments of human TbetaRII and IgG1Fc genes were amplified by RT-PCR and fused with overlap PCR to obtain the fusion gene TbetaRKK-IgG1Fc. The TbetaRII-IgG1Fc gene was cloned into the shuttle plasmid pAdTrack-CMV, which was linearized and transfected into E.coli BJ 5183 strain containing the adenoviral backbone vector. The recombinant adenovirus vector was constructed by homologous recombination. The recombinant adenoviral plasmid was linearized and transfected into 293 cells, followed by amplification and purification of the virus and detection of TbetaRII-IgG1Fc mRNA expression by RT-PCR. The functional activity of the recombinant adenoviral plasmid was assessed using enzyme-linked immunosorbent assay (ELISA). The results of restriction endonuclease digestion and DNA sequencing indicated correct sequence of the target TbetaRII-IgG1Fc fusion gene. The recombinant adenoviral plasmid expressed hTbetaRII-IgG1Fc and neutralized TGF-beta1 in vitro after infection of the human lung fibroblasts (HLF), as confirmed by RT-PCR and ELISA. The recombinant adenoviral plasmid capable of neutralizing TGF-beta1 in vitro is constructed successfully.

  5. A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats

    Directory of Open Access Journals (Sweden)

    van Remmerden Yvonne

    2010-06-01

    Full Text Available Abstract Background Respiratory syncytial virus (RSV is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (rRSV and an rRSV lacking the G gene (ΔG were constructed based on a clinical RSV isolate (strain 98-25147-X. Results Growth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for ΔG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days. Conclusion Collectively, the data indicate that a single dose immunization with the highly attenuated ΔG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since ΔG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.

  6. Recombinant human growth differentiation factor-9 improves oocyte reprogramming competence and subsequent development of bovine cloned embryos.

    Science.gov (United States)

    Su, Jianmin; Hu, Guangdong; Wang, Yongsheng; Liang, Dong; Gao, Mingqing; Sun, Hongzheng; Zhang, Yong

    2014-08-01

    Previously, we found that oocyte-secreted factors (OSFs) secreted by denuded oocytes during in vitro maturation (IVM) enhance subsequent development of bovine somatic cell nuclear transfer (SCNT) embryos. This treatment requires many oocytes during IVM. Hence, the aim of this study was to investigate whether supplementing with recombinant growth differentiation factor-9 (GDF9), one of crucial OFSs, in oocyte maturation medium could improve developmental competence of bovine oocytes and subsequent development of cloned embryos. Cumulus-oocyte complexes (COCs) from antral follicles of bovine ovaries collected from an abattoir were cultured with (SCNT+GDF9 group) or without (SCNT group) 200 ng/mL recombinant human GDF9 in oocyte maturation medium. After 22 h, metaphase II (MII) oocytes were used for SCNT. The presence of 200 ng/mL GDF9 significantly increased oocyte maturation rates, the cleavage rate, and blastocyst formation rates of bovine cloned embryos. The blastocyst total, inner cell mass (ICM) cell numbers, and ratio of ICM:TE were higher, whereas the rate of apoptosis in bovine cloned blastocysts was lower in the SCNT+GDF9 group than in the SCNT group. The histone modifications at various sites were also different between each group. These results suggest that COCs cultured with recombinant GDF9 in oocyte maturation medium improve oocyte developmental competence and subsequent developmental competence of cloned embryo in cattle.

  7. HUMAN PAPILLOMA VIRUS IMMUNOGEN CREATION ON THE BASE OF CHIMERIC RECOMBINANT PROTEIN L2E7

    Directory of Open Access Journals (Sweden)

    I. S. Malakhov

    2016-01-01

    Full Text Available The cervical cancer is one of the most common diseases in world. This malignancy is the seventh highest prevalence oncological disease worldwide and the second highest prevalence oncological disease of women in the world. Meanwhile women need to be infected by human papilloma virus (HPV is absolutely necessary for it further evolution, HPV DNA was found in 99.97% cases of disease. Except cervical cancer, HPV cause 85% of rectal cancer, 50% of the vulva, vagina and penis cancers, 20% of oropharyngeal cancer and 10% of larynx and esophagus cancers. In 2009, 14 000 women were diagnosed with cervical cancer in Russia. The growth in morbidity was 19% (in comparison with 1999. The most effective recognised measure for almost each infection prophylaxis is a vaccination. Two human papilloma virus vaccines are available in Russia nowadays — Gardasil and Cervarix, produced in Belgium and the Netherlands respectively. Cervarix is a bivalent vaccine based on virus-like particles (VLP of two types. Recombinant major capsid proteins L1 HPV 16 and HPV 18 express in baculovirus expression system and self-assembled into virus-like particles (about 70 percent of cervical cancers are caused by HPV 16 and HPV 18. VLP of each strain produced in different baculovirus vectors and then combined in single drug. Gardasil is like Cervarix with few exceptions. Producing organisms are fungi S. cerevisiae in this case, and this vaccine contains low-risk HPV 6 and HPV 11 VLP. Thus, Gardasil is quadrivalent HPV-6/11/16/18 vaccine. These vaccines are very effective in averting infection of disease and don’t have significant side-effects, however they have some disadvantages. Firstly, they have a high price because of necessity of their expression in eukaryotic cells. Secondly, they are strain-specific, so vaccines are completely effective only for virus’s strains which are represented in the vaccine. Thirdly, it`s the absence of therapeutic (treatment of established

  8. Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli.

    Science.gov (United States)

    Babaeipour, Valiollah; Khanchezar, Sirwan; Mofid, Mohammad Reza; Pesaran Hagi Abbas, Mahdi

    2015-01-01

    The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h⁻¹ at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l⁻¹ and 32.1 g.l⁻¹. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g⁻¹ DCW and 1.83 g.l⁻¹.h⁻¹, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen and PD-Grastim showed that all proteins have a similar secondary structure. Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity.

  9. A Validated RP-HPLC Method for the Determination of Recombinant Human Insulin in Bulk and Pharmaceutical Dosage Form

    Directory of Open Access Journals (Sweden)

    B. A. Moussa

    2010-01-01

    Full Text Available A modified RP-HPLC method was developed for the quantitative determination of recombinant human insulin in bulk and pharmaceutical dosage form with reduced retention time. Study of the effects of the column temperature, pH of the mobile phase and presence of vial additives (phenol and m-cresol, or impurities (A-21 Disamido on the accuracy of the assay were assessed. Separation was achieved using a Hypersil BDS C-18 column and the mobile phase was composed of solution A (aqueous solution of 28.3 anhydrous Na2SO4g/L, pH 2.3 and solution B (28.5 g anhydrous Na2SO4 g/L in 50:50 mixture of water and acetonitrile, pH 2.3 in a ratio 48:52 (v/v at 45–50 °C. The column temperature was 40 °C, the flow rate was 1 mL/min and detection was performed at 216 nm. The procedures were validated according to international conference on harmonization (ICH guidelines. Recovery study was done applying standard addition technique for further validation of the procedure. The retention time of recombinant human insulin was 19.7 min as compared to 29 min obtained by the reference method. Analytical conditions fluctuations or presence of vial additives or impurities did not show any significant effect on the accuracy of the method. The prepared standard insulin solution in 0.01 N HCl was found to be stable for 5 days. Statistical comparison showed no significant difference between the described method and reference method regarding the accuracy and precision. The modified method can be applied for routine quality control applications for determination of recombinant human insulin.

  10. Effect of relative hypoparathyroidism on the responsiveness to recombinant human erythropoietin in chronic hemodialysis patients: A single Saudi center experience

    Directory of Open Access Journals (Sweden)

    Khalid Al Saran

    2013-01-01

    Full Text Available Anemia is a common concomitant disorder in dialysis patients. The responsiveness to recombinant human erythropoietin in hemodialysis (HD patients with relative hypoparathyroidism [4 ≤ intact parathyroid hormone (iPTH ≤16.5 pmol/L] remains undetermined. We retrospectively studied 70 chronic hemodialysis patients who were divided into two groups: Group A (32 patients had 16.5 ≤ iPTH levels <33.5 pmol/L and Group B (38 patients had 4 ≥ iPTH≤16.5 pmol/L during the preceding six months without 1- (OH Vitamin D3 administration. The percentage of female gender was significantly higher in Group B compared with Group A (P = 0.018. In Groups A and B, the mean weekly recombinant human erythropoietin dose (U/kg/ week was 227.96 ± 95.24 vs. 154.1 ± 84.9 (P = 0.001 and the mean hemoglobin level was 11.15 ± 0.63 g/dL versus 11.62 ± 0.63 g/dL (P = 0.008. There was no significant statistical difference regarding the other biochemical markers (serum ferritin, iron saturation, serum Ca, serum alkaline phosphatase, C-reactive protein, serum B12, serum folate levels, residual renal function and Kt/v between the groups. If other factors related to anemia are excluded in chronic HD patients, the lower the iPTH level (relative hypoparathyroidism the better the responsiveness to recombinant human erythropoietin.

  11. Development of an immunodiagnosis method using recombinant PsCP for detection of Paragonimus skrjabini infection in human.

    Science.gov (United States)

    Yu, Shasha; Zhang, Xilin; Chen, Wenbi; Zheng, Hong; Ai, Guoping; Ye, Nan; Wang, Ying

    2017-01-01

    Paragonimiasis skrjabini is a kind of zoonosis and prevalent in 16 provinces in China, such as Chongqing, Fujian, Sichuan, and Yunnan. However, sensitive and efficient diagnostic methods for the infection are limited. In order to provide a more convenient and simple method for serologic diagnosis, the recombinant P. skrjabini cysteine protease (PsCP) was expressed, purified, and then used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting anti-PsCP antibodies in human. Given the positive/negative cutoff value as 0.606, the maximum dilution of human sera in which anti-PsCP antibodies could be detected was 1:12,800. In addition, the coefficients of variation (CVs) of inter-assay and intra-assay experiments were both below 10 %. Furthermore, the sensitivity of the PsCP-based ELISA was 95.5 %, and the indirect ELISA displays no cross-reactivity with human antisera against Echinococcus granulosus, Taenia solium, Schistosoma japonicum, and Trichinella spiralis, either. In conclusion, recombinant PsCP was readily produced and used to establish a simple PsCP-based ELISA that provided a highly specific and sensitive method for analysis of clinical samples. Besides, the method can also probably be used to diagnose P. skrjabini infection in animals.

  12. Both human pituitary growth hormone and recombinant DNA-derived human growth hormone cause insulin resistance at a postreceptor site.

    Science.gov (United States)

    Rosenfeld, R G; Wilson, D M; Dollar, L A; Bennett, A; Hintz, R L

    1982-05-01

    We have investigated the effects on carbohydrate metabolism of human GH produced by recombinant DNA technology (methionyl-hGH) compared with pituitary hGH. Twelve normal adult male subjects received four daily im injections of either methionyl-hGH or pituitary hGH in a double blind, crossover study. Oral glucose tolerance tests and assays of insulin binding to peripheral monocytes were performed before th initial administration and 12 h after the fourth injection of both hGH preparations. Both methionyl-hGH and pituitary hGH resulted in significant carbohydrate intolerance, with a rise in fasting plasma glucose from 96.6 +/- 2.9 to 105.9 +/- 3.0 mg/ml (mean +/- SEM) after pituitary hGH and from 96.2 +/- 1.5 to 107.5 +/- 3.3 mg/dl after methionyl-hGH (P less than 0.01). The area under the glucose tolerance curve increased by 34% after pituitary hGH and by 37% after methionyl-hGH. With both hGH preparations, carbohydrate intolerance was associated with marked hyperinsulinemia, with a rise in fasting plasma insulin levels from 9.4 +/- 1.2 to 33.2 +/- 7.8 microU/ml after pituitary hGH and from 7.4 +/- 1.1 to 45.8 +/- 11.1 microU/ml after methionyl-hGH (P less than 0.01). The integrated plasma insulin levels during the oral glucose tolerance test tripled after both hGH preparations. The pronounced insulin resistance could not be attributed to an alteration in insulin receptor concentrations. Both hGH preparations were associated with small reductions in insulin binding to monocytes at tracer concentrations, but the decline in binding was not statistically significant. The calculated binding sites per cell and Ke were not significantly altered by hGH administration. We conclude that methionyl-hGH and pituitary hGH are indistinguishable in their ability to induce insulin-resistant carbohydrate intolerance. This decrease in insulin sensitivity cannot be attributed to an alteration in insulin binding, and presumably represents a postreceptor defect in insulin action.

  13. The role of p53 in radiation activated recombination in human teratocarcinoma cells

    International Nuclear Information System (INIS)

    Ming Zeng; Hahn, Laura; Cerniglia, George; Lee, Jerry; EI-Deiry, Wafik; Stevens, Craig W.

    1997-01-01

    Purpose/Objective: We have previously demonstrated that ionizing radiation can activate a DNA recombination pathway in mammalian cells. In this project, we investigated the role of p53 in radiation activated recombination in ovarian tumor cell lines, and also the effect of p53 status on radiation sensitivity in this cell system. Materials and Methods: PA-1 teratocarcinoma cells, which express wild type p53, were transfected with an HPV16 E6 expression vector (PA-1/E6) which promotes p53 degradation, or transfected with a similar vector coding only for the neomycin phosphotransferase gene (PA-1/Neo). Approximately 3 weeks after this transfection, surviving cells were pooled and expanded. Nuclear extracts were made from each cell line three hours after cells were irradiated with doses ranging from 0 Gy to 10 Gy. Briefly, cells were lysed in sucrose buffer, and the nuclei and cytoplasm separated by centrifugation. Nuclei were lysed in low salt buffer followed by high salt buffer and centrifugation (as described by Johnson et al., Biotechniques 19:193-5, 1995). The ability of these nuclear extracts to rejoin or recombine EcoRI linearized pSV2neo was then determined. The effect of irradiation and P53 on stable transfection determined by assessing transfection of a Hygromycin marker vector (pSV2HPH). Radiation sensitivity was also determined. Results: Nuclear extracts from unirradiated cells had demonstrated end joining activity. PA-1/Neo had little end joining activity as measured by dimerization of linearized pSV2neo. Recutting of these dimers with EcoRI almost completely removed the dimer. PA-1/E6 demonstrated significantly more dimer formation (∼10 fold more) than PA-1/Neo. These dimers could only be reduced to ∼50% of PA-1/E6 control by redigestion with EcoRI. Nuclear extracts generated 3 hours after irradiation also had end joining activity. After 10Gy, PA-1/Neo demonstrated markedly elevated end joining activity to the level seen in unirradiated PA-1/E6. This

  14. [Interaction of human apolipoprotein AI and HIV-1 envelope proteins with the native and recombinant CD4 receptors].

    Science.gov (United States)

    Panin, L E; Kostina, N E

    2003-01-01

    The method of enzyme-linked immunosorbent assay (ELISA) was used to show an interaction of soluble recombinant CD4-receptor (rsCD4) with human apolipoprotein A-1. Competitive interactions between envelope proteins VIH-1 (gp120 and gp41), on the one hand, and human apolipoprotein A-1 with CD4 receptor, present in the cellular membranes of line MT4 human lymphocytes, were demonstrated by the method of flow cytofluorimetry. It was suggested that the competitive interactions between the above proteins could manifest in respect to the apolipoprotein A-1 receptor, which affects the involvement of the latter in the regulation of protein biosynthesis and which leads to a decrease in the body weight of HIV-infected patients.

  15. Preclinical evaluation of a recombinant adeno-associated virus vector expressing human alpha-1 antitrypsin made using a recombinant herpes simplex virus production method.

    Science.gov (United States)

    Chulay, Jeffrey D; Ye, Guo-Jie; Thomas, Darby L; Knop, David R; Benson, Janet M; Hutt, Julie A; Wang, Gensheng; Humphries, Margaret; Flotte, Terence R

    2011-02-01

    Recombinant adeno-associated virus (rAAV) vectors offer promise for gene therapy of alpha-1 antitrypsin (AAT) deficiency. A toxicology study in mice evaluated intramuscular injection of an rAAV vector expressing human AAT (rAAV-CB-hAAT) produced using a herpes simplex virus (HSV) complementation system or a plasmid transfection (TFX) method at doses of 3 × 10(11) vg (1.2 × 10(13) vg/kg) for both vectors and 2 × 10(12) vg (8 × 10(13) vg/kg) for the HSV-produced vector. The HSV-produced vector had favorable in vitro characteristics in terms of purity, efficiency of transduction, and hAAT expression. There were no significant differences in clinical findings or hematology and clinical chemistry values between test article and control groups and no gross pathology findings. Histopathological examination demonstrated minimal to mild changes in skeletal muscle at the injection site, consisting of focal chronic interstitial inflammation and muscle degeneration, regeneration, and vacuolization, in vector-injected animals. At the 3 × 10(11) vg dose, serum hAAT levels were higher with the HSV-produced vector than with the TFX-produced vector. With the higher dose of HSV-produced vector, the increase in serum hAAT levels was dose-proportional in females and greater than dose-proportional in males. Vector copy numbers in blood were highest 24 hr after dosing and declined thereafter, with no detectable copies present 90 days after dosing. Antibodies to hAAT were detected in almost all vector-treated animals, and antibodies to HSV were detected in most animals that received the highest vector dose. These results support continued development of rAAV-CB-hAAT for treatment of AAT deficiency.

  16. Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C)

    DEFF Research Database (Denmark)

    Lukovic, Dunja; Plasencia, Inés; Taberner, Francisco J

    2006-01-01

    A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two...... porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations....

  17. Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

    Science.gov (United States)

    Varghese, Anju; Raina, Opinder K; Chandra, Dinesh; Mirdha, Bijay R; Kelawala, Naresh H; Solanki, Jayesh B; Kumar, Niranjan; Ravindran, Reghu; Arun, Anandanarayanan; Rialch, Ajayta; Lalrinkima, Hniang; Kelawala, Rohan N; Samanta, Subhamoy

    2017-12-20

    Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

  18. HMC-1 human mast cells synthesize neurotensin (NT) precursor, secrete bioactive NT-like peptide(s) and express NT receptor NTS1.

    Science.gov (United States)

    Cochrane, David E; Carraway, Robert E; Harrington, Kimberly; Laudano, Melissa; Rawlings, Stephen; Feldberg, Ross S

    2011-12-01

    To determine if mast cells synthesize the inflammatory peptide, neurotensin (NT), secrete immunoreactive and bioactive NT, and express the NT receptor NTS1. HMC-1 cells, pleural mast cells from Sprague-Dawley rats, LAD2 mast cells, and human cord blood mast cells were used. HMC-1 cells were stimulated with NT, C48/80, mastoparan, or PGE(2). For changes in cutaneous vascular permeability, anesthetized rats were injected intravenously with Evans Blue dye and intradermally with saline, NT, histamine, diphenhydramine, and C48/80. RT-PCR was used to identify RNA transcripts. Histamine was measured by fluorometric assay. In vivo cutaneous vascular permeability assays, radio-immunoassays for NT, Western blotting for the NT precursor protein and NTS1 protein from HMC-1 cells and tissues from rats were used. Immunohistochemistry was used to identify NT precursor-like proteins in HMC-1 mast cells. HMC-1 cells express mRNAs for NT precursor, PC5A processing enzyme and NTS1 receptor. Human cord blood mast cells and LAD2 mast cells express mRNA transcripts for NT precursor and NTS1. Western blotting showed NT precursor and NTS1 receptor in HMC1. Rat tissues with high numbers of mast cells contained NT precursor proteins. NT-like peptides from HMC-1 displayed NT-like bioactivity. HMC-1 mast cells synthesize and secrete immunoreactive and bioactive NT-like peptide(s) and express the NT receptor, suggesting that NT from mast cells might serve autocrine and paracrine roles.

  19. METHODS FOR RECOMBINANT EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF HUMAN CANNABINOID RECEPTOR CB2

    Directory of Open Access Journals (Sweden)

    Alexei A. Yeliseev

    2013-03-01

    Full Text Available Cannabinoid receptor CB2 is a seven transmembrane-domain integral membrane protein that belongs to a large superfamily of G protein-coupled receptors (GPCR. CB2 is a part of the endocannabinoid system that plays vital role in regulation of immune response, inflammation, pain sensitivity, obesity and other physiological responses. Information about the structure and mechanisms of functioning of this receptor in cell membranes is essential for the rational development of specific pharmaceuticals. Here we review the methodology for recombinant expression, purification, stabilization and biochemical characterization of CB2 suitable for preparation of multi-milligram quantities of functionally active receptor. The biotechnological protocols include expression of the recombinant CB2 in E. coli cells as a fusion with the maltose binding protein, stabilization with a high affinity ligand and a derivative of cholesterol in detergent micelles, efficient purification by tandem affinity chromatography, and reconstitution of the receptor into lipid bilayers. The purified recombinant CB2 receptor is amenable to functional and structural studies including nuclear magnetic resonance spectroscopy and a wide range of biochemical and biophysical techniques.

  20. Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

    Science.gov (United States)

    Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

    1996-07-15

    Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

  1. Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival.

    OpenAIRE

    Lopez, A F; Williamson, D J; Gamble, J R; Begley, C G; Harlan, J M; Klebanoff, S J; Waltersdorph, A; Wong, G; Clark, S C; Vadas, M A

    1986-01-01

    A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethion...

  2. Construction of a recombinant eukaryotic human ZHX1 gene expression plasmid and the role of ZHX1 in hepatocellular carcinoma.

    Science.gov (United States)

    Wang, Jianping; Liu, Dejie; Liang, Xiaohong; Gao, Lifen; Yue, Xuetian; Yang, Yang; Ma, Chunhong; Liu, Jun

    2013-11-01

    The zinc-fingers and homeoboxes protein 1 (ZHX1) consists of 873 amino acid residues, is localized in the cell nucleus and appears to act as a transcriptional repressor. Previous studies have shown that ZHX1 interacts with nuclear factor Y subunit α (NF-YA), DNA methyltransferases (DNMT) 3B and ZHX2, all of which are involved in tumorigenesis. However, the exact role of ZHX1 in tumorigenesis remains unknown. The aim of the current study was to construct a recombinant eukaryotic expression plasmid containing the human ZHX1 (hZHX1) gene and to investigate the biological activities of ZHX1 in hepatocellular carcinoma (HCC). Reverse transcription-polymerase chain reaction (RT‑PCR) was used to amplify the N- and C-terminal fragments (ZHX1‑N and ZHX1‑C, respectively) of the hZHX1 gene. The two PCR fragments were cloned into the pEASY-T1 vector and subcloned into the pcDNA3 plasmid to generate a recombinant pcDNA3‑ZHX1 plasmid. Following identification by enzyme digestion and DNA sequencing, the recombinant pcDNA3‑ZHX1 plasmid was transfected into SMMC-7721 cells. The level of ZHX1 expression was detected by RT-PCR and western blot analysis. Cell growth curve assays were used to evaluate the effect of ZHX1 on cell proliferation. Moreover, the differential expression of ZHX1 between cancer and adjacent cirrhotic liver tissue was investigated by quantitative PCR (qPCR). Enzyme digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid, pcDNA3‑ZHX1. qPCR and western blot analysis demonstrated that ZHX1 was efficiently expressed in SMMC-7721 cells and overexpression of ZHX1 may inhibit the proliferation of SMMC-7721 cells. In addition, reduced ZHX1 expression is widespread among cancer tissues from HCC patients. In conclusion, a recombinant eukaryotic expression plasmid, pcDNA3‑ZHX1, was successfully constructed. In addition, the current results indicate that a low expression of ZHX1 may be responsible for hepatocarcinogenesis.

  3. Poly(norepinephrine)-coated open tubular column for the separation of proteins and recombination human erythropoietin by capillary electrochromatography.

    Science.gov (United States)

    Xiao, Xue; Zhang, Yamin; Wu, Jia; Jia, Li

    2017-12-01

    Recombinant human erythropoietin is an important therapeutic protein with high economic interest due to the benefits provided by its clinical use for the treatment of anemias associated with chronic renal failure and chemotherapy. In this work, a poly(norepinephrine)-coated open tubular column was successfully prepared based on the self-polymerization of norepinephrine under mild alkaline condition, the favorable film forming and easy adhesive properties of poly(norepinephrine). The poly(norepinephrine) coating was characterized by scanning electron microscopy and measurement of the electro-osmotic flow. The thickness of the coating was about 431 nm. The electrochromatographic performance of the poly(norepinephrine)-coated open tubular column was evaluated by separation of proteins. Some basic and acidic proteins including two variants of bovine serum albumin and two variants of β-lactoglobulin achieved separation in the poly(norepinephrine)-coated open tubular column. More importantly, the column demonstrated separation ability for the glycoforms of recombinant human erythropoietin. In addition, the column demonstrated good repeatability with the run-to-run, day-to-day, and column-to-column relative standard deviations of migration times of proteins less than 3.40%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. [Production of recombinant human interleukin-38 and its inhibitory effect on the expression of proinflammatory cytokines in THP-1 cells].

    Science.gov (United States)

    Yuan, X L; Li, Y; Pan, X H; Zhou, M; Gao, Q Y; Li, M C

    2016-01-01

    Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-β-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/μg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1β, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical applications.

  5. Recombinant human antithrombin expressed in the milk of non-transgenic goats exhibits high efficiency on rat DIC model.

    Science.gov (United States)

    Yang, Hai; Li, Qing-Wang; Han, Zeng-Sheng; Hu, Jian-Hong; Li, Wen-Ye; Liu, Zhi-Bin

    2009-11-01

    Plasma-derived antithrombin (pAT) is often used for the treatments of disseminated intravascular coagulation (DIC) patients. In this paper, the recombinant adenovirus vector encoding human antithrombin (AT) cDNA was constructed and directly infused into the mammary gland of two goats. The recombinant human antithrombin (rhAT) was purified by heparin affinity chromatography from the goat milk, and then used in the treatment of thirty lipopolysaccharide (LPS) induced DIC rats. A high expression level of rhAT up to 2.8 g/l was obtained in the milk of goats. After purification, the recovery rate and the purity of the rhAT were up to 54.7 +/- 3.2% and 96.2 +/- 2.7%, respectively. In blood of the DIC rat model treated with rhAT, the levels of antithrombin and thrombin-antithrombin (TAT) were augmented significantly; meanwhile the consumption of fibrinogen and platelet was reduced significantly, and the increase of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentration was restrained modest and non-significant. For the above DIC indexes, there were no differences between pAT and rhAT (P > 0.05). Our results demonstrated that the way we established is a pragmatic tool for large-scale production of rhAT, and the rhAT produced with this method has potential as a substitute for pAT in the therapy of DIC patients.

  6. Bioactive glasses materials, properties and applications

    CERN Document Server

    Ylänen, Heimo

    2011-01-01

    Due to their biocompatibility and bioactivity, bioactive glasses are used as highly effective implant materials throughout the human body to replace or repair damaged tissue. As a result, they have been in continuous use since shortly after their invention in the late 1960s and are the subject of extensive research worldwide.Bioactive glasses provides readers with a detailed review of the current status of this unique material, its properties, technologies and applications. Chapters in part one deal with the materials and mechanical properties of bioactive glass, examining topics such

  7. Population Pharmacokinetic Modelling of FE 999049, a Recombinant Human Follicle-Stimulating Hormone, in Healthy Women After Single Ascending Doses

    DEFF Research Database (Denmark)

    Rose, Trine Høyer; Röshammar, Daniel; Erichsen, Lars

    2016-01-01

    Objective: The purpose of this analysis was to develop a population pharmacokinetic model for a novel recombinant human follicle-stimulating hormone (FSH) (FE 999049) expressed from a human cell line of foetal retinal origin (PER.C6) developed for controlled ovarian stimulation prior to assisted...... effects population pharmacokinetic modelling in NONMEM 7.2.0. Results: A one-compartment model with first-order absorption and elimination rates was found to best describe the data. A transit model was introduced to describe a delay in the absorption process. The apparent clearance (CL/F) and apparent...... volume of distribution (V/F) estimates were found to increase with body weight. Body weight was included as an allometrically scaled covariate with a power exponent of 0.75 for CL/F and 1 for V/F. Conclusions: The single-dose pharmacokinetics of FE 999049 were adequately described by a population...

  8. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    Science.gov (United States)

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-06-10

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

  9. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  10. Radioimmunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands.

    Science.gov (United States)

    Falorni, A; Ortqvist, E; Persson, B; Lernmark, A

    1995-10-12

    Autoantibodies are an important marker of human autoimmune diseases and the development of simple, precise and reproducible immunoassays to detect autoantibodies is important to our understanding of human autoimmunity. GAD65 autoantibodies occur frequently in insulin-dependent diabetic patients and is a useful marker for IDDM. A RIA to detect immunoreactive GAD65 has not been described. In the present study we describe a semi-automated fluid-phase immunoassay for the rapid detection of GAD65 autoantibodies in human serum. We also developed a sensitive RIA to determine immunoreactive human GAD65 in biological fluids and in vitro cell systems. Using in vitro translated recombinant human GAD65 in a multiwell-adapted procedure, our GAD65Ab RIA combines high specificity and sensitivity with a high capacity to analyze a large number of samples. In this report the three critical steps in the GAD65Ab RIA, DNA preparation, in vitro translation and immunoprecipitation, have been optimized. In our RIA, GAD65Ab were detected in 116/155 (75%) new onset Swedish IDDM children and in 1/85 (1.2%) healthy controls. In an immunoassay to detect autoantibodies against the proinsulin converting enzyme 2 (PC-2) no such antibodies were detected in IDDM patients. In the GAD65 RIA the lower detection limit was 2 ng/ml (31 fmol/ml). Our data demonstrate that autoantigen radioligands produced by in vitro translation are useful in RIA for autoantibodies and autoantigens in studies of human autoimmunity.

  11. Replication-defective recombinant Semliki Forest virus encoding GM-CSF as a vector system for rapid and facile generation of autologous human tumor cell vaccines

    NARCIS (Netherlands)

    Withoff, S; Glazenburg, KL; van Veen, ML; Kraak, MMJ; Hospers, GAP; Storkel, S; de Vries, EGE; Wischut, J; Daemen, T

    2001-01-01

    This paper describes the production of recombinant Semliki Forest virus encoding murine or human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the capacity of these vectors to transduce murine and human tumor cells ex vivo. High-titer stocks (up to 3 x 10(9) particles/ml) of

  12. Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes. The UNAIDS Network for HIV Isolation and Characterization

    NARCIS (Netherlands)

    Cornelissen, M.; Kampinga, G.; Zorgdrager, F.; Goudsmit, J.

    1996-01-01

    Genetic subtypes of human immunodeficiency virus type 1 can be distinguished on the basis of phylogenetic analysis of their envelope (env) gene. A significant proportion of human immunodeficiency virus type 1 strains was retrospectively shown to result from recombination events between viruses

  13. Bioactivity of a Calcium Silicate-based Endodontic Cement (BioRoot RCS): Interactions with Human Periodontal Ligament Cells In Vitro.

    Science.gov (United States)

    Camps, Jean; Jeanneau, Charlotte; El Ayachi, Ikbale; Laurent, Patrick; About, Imad

    2015-09-01

    Tricalcium silicate-based materials are recognized as bioactive materials through their capacity to induce hard tissue formation both in the dental pulp and bone. Sealing the apex implies that the root canal filling materials interact with the periapical tissues. This work was designed to study the interactions of newly developed tricalcium silicate cement (BioRoot RCS; Septodont, Saint Maur Des Fosses, France) with apical tissue compared with a standard zinc oxide-eugenol sealer (Pulp Canal Sealer [PCS]; SybronEndo, Orange, CA). Cell viability was assessed by direct contact between human periodontal ligament (PDL) cells and BioRoot RCS or PCS. In addition, an in vitro tooth model was used to study the interactions between these materials and PDL cells. For this purpose, human extracted incisors were sectioned at the enamel-cementum junction; root canals were prepared, sterilized, and filled with lateral condensation with both materials. The root apices were dipped in the culture medium for 24 hours. These conditioned media were used to investigate their effects on human PDL cells. Cell proliferation was investigated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the secretion of angiogenic and osteogenic growth factors was quantified using an enzyme-linked immunosorbent assay. BioRoot RCS has less toxic effects on PDL cells than PCS and induced a higher secretion of angiogenic and osteogenic growth factors than PCS. Taken together, these preclinical results suggest that the calcium silicate cement (BioRoot RCS) has a higher bioactivity than the zinc oxide-eugenol sealer (PCS) on human PDL cells. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  14. I-124 labeled recombinant human annexin V produced by E. coli for apoptosis image using small animal PET

    Energy Technology Data Exchange (ETDEWEB)

    Jung, J. H.; Lee, I. S.; Woo, S. K.; Woo, G. S.; Chung, W. S.; Kang, J. H.; Cheon, G. J.; Choi, C. W.; Urn, S. M. [Korea Institute of Radiological and Medical Sciences, Daejeon (Korea, Republic of)

    2007-07-01

    Annexin V labeled with radioisotope and optical probe has been used to detect apoptosis. To evaluate annexin V as a multimodal apoptosis imaging agent, large-scale preparation of Annexin V (AV) is preliminary. The aim of this study is to produce and purify recombinant human Annexin V (rh-AV) in E. coli system and radiolabeled rh-AV evaluate in vitro and in vivo apoptosis model system. Annexin V cDNA was obtained from human placenta and rh-AV cloning vector used fusion E. coli vector. Expression vector was based on the E. coli pET system. Induction of rh-AV was used Isopropyl--D-thiogalactoside (IPTG) and purification was used TALON metal affinity resin and T7 - Taq. Purification yield confirmed through SDS-PAGE. In camptothecin (0, 50, 100 uM) induced Jurkat T cell apoptosis model, AV-PI flow cytometry analysis and in vitro binding assay of I-124 labeled rh - AV were performed and compared. Small animal PET images of I-124 labeled rh-AV were obtained in Fas-mediated hepatic apoptosis model. Optimum expression condition was at 37, 250 rpm, 8 hr in 2X YT media including 1mM IPTG, Through two step purification process, rh-AV confirmed about 35 Kd single band by SDS-PAGE. As camptothecin concentration increasing, annexin V-FITC positive % increased in flow cytometry analysis and uptake of I-124 labeled rh-AV also increased. Annexin V-FITC positive % was correlated with and uptake of I-124 labeled rh-AV (R{sup 2}=0.99). In Fas-mediated hepatic apoptosis model, I-124 labeled rh-AV was selectively localized in liver region in PET image. Recombinant Human annexin V was produced by E. coli system and purified using two step affinity chromatography. Radiolabeled rh-AV was useful for the evaluation of apoptosis in vitro and in vivo model. Recombinant human annexin V could be used as apoptosis imaging agent with various radiolabel and optical probe.

  15. Pro-inflammatory Effects of Bacterial Recombinant Human C-Reactive Protein are Caused by Contamination with Bacterial Products not by C-Reactive Protein Itself

    Science.gov (United States)

    Pepys, Mark B.; Hawkins, Philip N.; Kahan, Melvyn C.; Tennent, Glenys A.; Gallimore, J. Ruth; Graham, David; Sabin, Caroline A.; Zychlinsky, Arturo; de Diego, Juana

    2006-01-01

    Intravenous administration to human volunteers of a commercial preparation of recombinant human C-reactive protein (CRP) produced in E. coli was recently reported in this journal to induce an acute phase response of serum amyloid A protein (SAA) and of CRP itself, and to activate the coagulation system. The authors concluded that CRP is probably a mediator of atherothrombotic disease. Here we confirm that this recombinant CRP preparation was pro-inflammatory both for mouse macrophages in vitro and for mice in vivo, but show that pure natural human CRP had no such activity. Furthermore mice transgenic for human CRP, and expressing it throughout their lives, maintained normal concentrations of their most sensitive endogenous acute phase reactants, SAA and serum amyloid P component (SAP). The patterns of in vitro cytokine induction and of in vivo acute phase stimulation by the recombinant CRP preparation were consistent with contamination by bacterial products, and there was 46.6 EU of apparent endotoxin activity per mg of CRP in the bacterial product, compared to 0.9 EU per mg of our isolated natural human CRP preparation. The absence of any pro-inflammatory activity in natural CRP for macrophages or healthy mice strongly suggests that the in vivo effects of the recombinant preparation observed in humans were due to pro-inflammatory bacterial products and not human CRP. PMID:16254214

  16. Bioactive Constituents of Zanthoxylum rhetsa Bark and Its Cytotoxic Potential against B16-F10 Melanoma Cancer and Normal Human Dermal Fibroblast (HDF) Cell Lines.

    Science.gov (United States)

    Santhanam, Ramesh Kumar; Ahmad, Syahida; Abas, Faridah; Safinar Ismail, Intan; Rukayadi, Yaya; Tayyab Akhtar, Muhammad; Shaari, Khozirah

    2016-05-24

    Zanthoxylum rhetsa is an aromatic tree, known vernacularly as "Indian Prickly Ash". It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin), a berberine alkaloid (columbamine) and a triterpenoid (lupeol) from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF) and mouse melanoma (B16-F10) cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells.

  17. Bioactive Constituents of Zanthoxylum rhetsa Bark and Its Cytotoxic Potential against B16-F10 Melanoma Cancer and Normal Human Dermal Fibroblast (HDF Cell Lines

    Directory of Open Access Journals (Sweden)

    Ramesh Kumar Santhanam

    2016-05-01

    Full Text Available Zanthoxylum rhetsa is an aromatic tree, known vernacularly as “Indian Prickly Ash”. It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin, a berberine alkaloid (columbamine and a triterpenoid (lupeol from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF and mouse melanoma (B16-F10 cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells.

  18. HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles

    International Nuclear Information System (INIS)

    Haffar, O.K.; Smithgall, M.D.; Moran, P.A.; Travis, B.M.; Zarling, J.M.; Hu, S.L.

    1991-01-01

    Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultraviolet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, the authors results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS

  19. Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)–chitosan scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jing; Zhou, Aimei; Deng, Aipeng [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Yang [Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Gao, Lihu; Zhong, Zhaocai [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Shulin, E-mail: yshulin@njust.edu.cn [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2015-04-01

    Pore architecture of 3D scaffolds used in tissue engineering plays a critical role in the maintenance of cell survival, proliferation and further promotion of tissue regeneration. We investigated the pore size and structure, porosity, swelling as well as cell viability of a series of recombinant human collagen-peptide–chitosan (RHCC) scaffolds fabricated by lyophilization. In this paper, freezing regime containing a final temperature of freezing (T{sub f}) and cooling rates was applied to obtain scaffolds with pore size ranging from 100 μm to 120 μm. Other protocols of RHC/chitosan suspension concentration and ratio modification were studied to produce more homogenous and appropriate structural scaffolds. The mean pore size decreased along with the decline of T{sub f} at a slow cooling rate of 0.7 °C/min; a more rapid cooling rate under 5 °C/min resulted to a smaller pore size and more homogenous microstructure. High concentration could reduce pore size and lead to thick well of scaffold, while improved the ratio of RHC, lamellar and fiber structure coexisted with cellular pores. Human umbilical vein endothelial cells (HUVECs) were seeded on these manufactured scaffolds, the cell viability represented a negative correlation to the pore size. This study provides an alternative method to fabricate 3D RHC–chitosan scaffolds with appropriate pores for potential tissue engineering. - Highlights: • Fabrication of recombinant human collagen-chitosan scaffolds by freezing drying • Influence of freeze drying protocols on lyophilized scaffolds • Pore size, microstructure, porosity, swelling and cell viability were compared. • The optimized porous scaffold is suitable for cell (HUVEC) seeding.

  20. Comparison of Immobilized Metal Affinity Chromatography Ni-NTA and Co-TALON for the Purification of Recombinant Human Erythropoietin

    Directory of Open Access Journals (Sweden)

    Yana Rubiyana

    2015-12-01

    Full Text Available The purification of recombinant proteins is an important stage in biopharmaceutical research. A commonly used technique is immobilized metal affinity chromatography (IMAC. One of the main advantages of this type of chromatography is that the column can easily be regenerated for subsequent purification work. The mechanism of IMAC is based on bonding between metal ions immobilized on a matrix with a specific amino acid. Because of the strong interactions of the electron donor group on the imidazole ring, histidine is often used in the IMAC purification system. Two types of commercial IMAC resin use a nitrilotriacetic acid (NTA matrix: a nickel-based (Ni-NTA and cobalt-based (Co-NTA, better known as TALON. This study was aim to investigate the effect of the metal ions Ni2+ and Co2+ to purify recombinant human erythropoietin (rhEPO expressed in yeast system Pichia pastoris. The results indicated that both Ni-NTA and Co-TALON gave almost the same level of protein purity; however, Ni-NTA has a higher binding affinity than Co-TALON might be due to the higher stability complex of Ni+. The average amount of protein bound by Ni-NTA and Co-TALON was 183.5 and 38.7 µg/mL, respectively.

  1. Soft Tissue Swelling Associated with the Use of Recombinant Human Bone Morphogenetic Protein-2 in Long Bone Non-unions.

    Science.gov (United States)

    Young, Andrew; Mirarchi, Adam

    2015-01-01

    This report describes two cases of long bone non-union associated with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) and is the first of its kind. The first case describes a 25-year-old male who sustained a left diaphyseal femoral shaft fracture initially treated with operative fixation using an intramedullary nail, which subsequently loosened distally and was treated with exchange nailing and rhBMP-2 application. This patient developed acute local soft tissue inflammation post-operatively. The second case describes a 61-year-old female who sustained a right diaphyseal humeral shaft fracture that was initially treated with intramedullary nail fixation with subsequent distal interlock screw loosening. She underwent nail removal, and compression plating with rhBMP-2 placement, and postoperatively developed severe acute local tissue swelling centered over the rhBMP-2 sponge. Surgeons should be aware that rhBMP-2 may cause local acute tissue swelling and recombinant bone morphogenic proteins such as rhBMP-2 may have a role in the management for atrophic fracture non-unions. The authors recommend careful consideration prior to rhBMP-2 use in long bone non-unions.

  2. Evolution of R5 and X4 human immunodeficiency virus type 1 gag sequences in vivo: evidence for recombination

    International Nuclear Information System (INIS)

    Rij, Ronald P. van; Worobey, Michael; Visser, Janny A.; Schuitemaker, Hanneke

    2003-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection is in general established by CCR5-utilizing (R5) virus variants, which persist throughout the course of infection. R5 HIV-1 variants evolve into CXCR4-utilizing (X4) HIV-1 variants in approximately half of the infected individuals. We have previously observed an ongoing genetic evolution with a continuous divergence of envelope gp120 sequences of coexisting R5 and X4 virus variants over time. Here, we studied evolution of gag p17 sequences in two patients who developed X4 variants in the course of infection. In contrast to the envelope gp120 sequences, gag p17 sequences of R5 and X4 virus populations intermingled in phylogenetic trees and did not diverge from each other over time. Statistical evaluation using the Shimodaira-Hasegawa test indicated that the different genomic regions evolved along different topologies, supporting the hypothesis of recombination. Therefore, our data imply that recombination between R5 and X4 HIV-1 variants occurs in vivo

  3. Analysis of antigenic cross-reactivity between subgroup C avian pneumovirus and human metapneumovirus by using recombinant fusion proteins.

    Science.gov (United States)

    Luo, L; Sabara, M I; Li, Y

    2009-10-01

    Avian pneumovirus subgroup C (APV/C) has recently been reported to be more closely related to human metapneumovirus (hMPV) as determined by sequence analysis. To examine the antigenic relationship between APV/C and hMPV, the APV/C fusion (F) gene was cloned and expressed as an uncleaved glycoprotein in a baculovirus system. The reactivity of the APV/C F protein with antibodies against APV subgroups A, B, C, and hMPV was examined by Western blot analysis. The results showed that the expressed APV/C F protein was not only recognized by APV/C-specific antibodies but also by antibodies raised against hMPV. Previously expressed recombinant hMPV F protein also reacted with APV/C-specific antibodies, suggesting that there was significant antigenic cross-reactivity and a potential evolutionary relationship between hMPV and APV/C. Interestingly, the recombinant F proteins from APV/C and hMPV were not recognized by polyclonal antibodies specific to APV subgroups A and B.

  4. Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells.

    Science.gov (United States)

    Almeida, Aline G; Pinto, Rodrigo C V; Smales, C Mark; Castilho, Leda R

    2017-08-01

    To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4-8 °C. NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance. Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, L-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

  5. Study and characterisation of human HEK293 cell line as a platform for recombinant protein production

    OpenAIRE

    Liste Calleja, Leticia

    2015-01-01

    El present treball es centra en l'estudi de la producció de proteïnes recombinants en línies cel·∙lulars de mamífer. Concretament, s'ha realitzat l'estudi de tres estratègies de bioprocés, totes elles basades en el cultiu de cèl·∙lules HEK293. Com a proteïna model per a l'expressió de proteïnes heteròlogues s'ha triat la proteïna CapPCV2, la qual conforma la càpsida viral del Circovirus porcí serotip 2 (PCV2). Aquest virus és l'agent causal de PCVDS (porcine circovirus diseases o malaties der...

  6. Disruption of the p53-mediated G{sub 1}/S cell cycle checkpoint results in elevated rates of spontaneous genetic recombination in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Strasfeld, L.; Brainerd, E.; Meyn, M.S. [Yale Univ. School of Medicine, New Haven, CT (United States)

    1994-09-01

    A key feature of the cancer-prone inherited disease ataxia-telangiectasia (A-T) is genetic instability. We recently demonstrated that one aspect of genetic instability in A-T is a marked elevation in the spontaneous rates of intrachromosomal mitotic recombination. We have proposed a model for A-T that attributes these high recombination rates to a lack of DNA damage-sensitive cell cycle checkpoints. One prediction of this model is that disrupting p53 function in normal cells should increase their spontaneous rates of recombination by interfering with their p53-dependent G{sub 1}/S cell cycle checkpoint. To test this prediction, we transfected control and A-T fibroblast lines that each harbor a single integrated copy of lacZ-based recombination vector (pLrec) with derivatives of a eukaryotic expression vector (pRep5) that contain either a dominant-negative p53 mutant (143{sup val{yields}ala}) or a human papilloma virus E6 gene (HPV18 E6). Expression of either of these genes results in loss of p53 function and abolition of the G{sub 1}/S cell cycle checkpoint. Four independent p53{sup 143ala} transformants of the control line showed 25-80 fold elevations in spontaneous recombination rates when compared to their parent cell line. Elevations in spontaneous recombination rates were also detected following transfection with the HPV18 E6 gene. In contrast, four independent p53{sup 143ala} transformants of the A-T cell line showed no significant changes in their already high spontaneous recombination rates. We are now extending these observations to additional normal human fibroblast lines and carrying out molecular analyses of the products of these recombinational events. Our results support our hypothesis that the lack of a p53-dependent G{sub 1}/S cell cycle checkpoint contributes to the hyperrecombination seen in A-T.

  7. Human Rad51 filaments on double- and single-stranded DNA : Correlating regular and irregular forms with recombination function

    NARCIS (Netherlands)

    Ristic, D.; Modesti, M.; Van der Heijden, T.; Van Noort, J.; Dekker, C.; Kanaar, R.; Wyman, C.

    Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of

  8. Effects of recombinant human erythropoietin on the haemopoietic bone marrow monitored by magnetic resonance spectroscopy in patients with end-stage renal disease

    DEFF Research Database (Denmark)

    Jensen, K E; Stenver, D; Jensen, M

    1990-01-01

    Volume selective magnetic resonance (MR) proton spectroscopy was used to investigate changes in the haemopoietic bone marrow in patients with end-stage renal disease undergoing treatment with recombinant human erythropoietin (rHuEPO). Significant changes could be detected in the spectra 14 days...... change in the spectrum four days after treatment began, indicating that MR spectroscopy may detect early changes in the cellular composition of the bone marrow. This noninvasive method may be useful in evaluating treatment effects of recombinant human haemopoietic growth factors in the bone marrow...

  9. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

    Full Text Available Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

  10. Recombinant human thyrotropin-stimulated radioiodine therapy of large nodular goiters facilitates tracheal decompression and improves inspiration

    DEFF Research Database (Denmark)

    Bonnema, Steen Joop; Nielsen, Viveque E; Boel-Jørgensen, Henrik

    2008-01-01

    to placebo (n = 15) or 0.3 mg rhTSH (n = 14) 24 h before (131)I therapy. The smallest cross-sectional area of the trachea (SCAT; assessed by magnetic resonance imaging) and the pulmonary function were determined before, 1 wk, and 12 months after therapy. RESULTS: Data on goiter reduction have been reported......INTRODUCTION: The impact on tracheal anatomy and respiratory function of recombinant human (rh)TSH-stimulated (131)I therapy in patients with goiter is not clarified. METHODS: In a double-blinded design, patients (age 37-87 yr) with a large multinodular goiter (range, 99-440 ml) were randomized...... previously. In the placebo group, no significant changes in the lung function or SCAT were found throughout the study. In the rhTSH group, a slight decrease was observed in the forced vital capacity 1 wk after therapy, whereas the mean individual change in SCAT was significantly increased by 10.5% (95...

  11. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit the chemotaxis and enhance the oxidative burst response of human neutrophils in vitro. The present study describes the effect of recombinant GM-CSF on the neutrophil and monocyte function in patients with lymphoma...... undergoing GM-CSF treatment. Patients with either Hodgkin's or non-Hodgkin's lymphoma were treated with various dosages (2-16 micrograms kg-1 body weight per day for 5 days) of rhGM-CSF by intravenous or subcutaneous route. Prior to and on day 5 of rhGM-CSF treatment, neutrophil and monocyte chemotaxis...... by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence...

  12. Recombinant human granulocyte colony-stimulating factor increases circulating CD34-postive cells in patients with AIDS

    DEFF Research Database (Denmark)

    Nielsen, S D; Dam-Larsen, S; Nielsen, C

    1997-01-01

    In a gene therapy-based treatment of AIDS, it would be desirable to have as many transduced target cells as possible. A limiting factor is the number of target cells. In this study, we investigated whether it was possible to increase the absolute number of one possible target cell, i.......e., the circulating hematopoietic progenitor cells (CD34 cells) in patients with AIDS, using the recombinant human granulocyte colony-stimulating factor (G-CSF). Eight patients with AIDS were treated with G-CSF for neutropenia (... cells. The median increase in CD34 cells was from 0.8 to 2.2 x 10(6)/l. Finally, using a highly sensitive HIV-1 RNA PCR, we found that treatment of AIDS patients with G-CSF did not lead to enhanced HIV replication. These observations indicate that G-CSF may be used to mobilize CD34 cells in patients...

  13. Seaweed Bioactivity

    DEFF Research Database (Denmark)

    Zaharudin, Nazikussabah Binti

    α-amylase and α-glucosidase were identified. The edible seaweeds that were showing high potential for inhibiting the enzymes were selected to investigate their effect on the postprandial blood glucose and insulin levels following a starch load in a human meal study. In vitro studies and a human...... postprandial blood glucose and insulin concentrations following a starch load in a human meal study. There was no significant effect in plasma glucose but both brown seaweeds lowered postprandial insulin response following consumption of Laminaria digitata or Undaria pinnatifida compared to the control meal....... Maintenance of normal plasma glucose concentration is essential for the human health. Diet and exercise play important role to control blood sugar level. Limiting intake of high Glycaemic Index (GI) foods as part of a balanced diet is known to be important. In addition, having the right food intake such ad...

  14. Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris.

    Science.gov (United States)

    Dai, Mengmeng; Yu, Changming; Fang, Ting; Fu, Ling; Wang, Jing; Zhang, Jun; Ren, Jun; Xu, Junjie; Zhang, Xiaopeng; Chen, Wei

    2015-01-01

    Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.

  15. Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage.

    Science.gov (United States)

    Hsu, Chuan-Chih; Chen, Sheng-Hsien; Lin, Cheng-Hsien; Yung, Ming-Chi

    2014-10-01

    Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65-270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage

  16. Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways

    Directory of Open Access Journals (Sweden)

    Lim HC

    2016-06-01

    Full Text Available Hyun-Chang Lim,1,* Ok Hyung Nam,2,* Mi-joo Kim,3 Ahmed El-Fiqi,4,5 Hyung-Mun Yun,3 Yoo-Mi Lee,3 Guang-Zhen Jin,4,5 Hae-Hyoung Lee,5,6 Hae-Won Kim,4–6 Eun-Cheol Kim3 1Department of Periodontology, 2Department of Pediatric Dentistry, 3Department of Oral and Maxillofacial Pathology, Research Center for Tooth and Periodontal Regeneration (MRC, School of Dentistry, Kyung Hee University, Seoul, 4Department of Nanobiomedical Science, BK21 PLUS NBM Global Research Center for Regenerative Medicine, 5Institute of Tissue Regeneration Engineering, 6Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan, Republic of Korea *These authors contributed equally to this work as first authors Abstract: Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs and deliver the odontogenic drug dexamethasone (DEX to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs. DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and β3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK, Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN

  17. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    NARCIS (Netherlands)

    C. Buecker (Christa); H.H. Chen; J.M. Polo (Jose); L. Daheron (Laurence); L. Bu (Lei); T.S. Barakat (Tahsin Stefan); P. Okwieka (Patricia); A. Porter (Andrew); J.H. Gribnau (Joost); K. Hochedlinger (Konrad); N. Geijsen (Niels)

    2010-01-01

    textabstractMurine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human

  18. Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression

    Science.gov (United States)

    Mueller, Christian; Chulay, Jeffrey D.; Trapnell, Bruce C.; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A.; McElvaney, Noel G.; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N.; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R.; Ye, Guo-jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P.; Vonderheide, Robert H.; Hulme, Maigan A.; Brusko, Todd M.; Wilson, James M.; Flotte, Terence R.

    2013-01-01

    Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy. PMID:24231351

  19. Human Treg responses allow sustained recombinant adeno-associated virus-mediated transgene expression.

    Science.gov (United States)

    Mueller, Christian; Chulay, Jeffrey D; Trapnell, Bruce C; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A; McElvaney, Noel G; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R; Ye, Guo-Jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P; Vonderheide, Robert H; Hulme, Maigan A; Brusko, Todd M; Wilson, James M; Flotte, Terence R

    2013-12-01

    Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1-AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.

  20. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    Science.gov (United States)

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  1. MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

    Directory of Open Access Journals (Sweden)

    Cunha-Neto E.

    1999-01-01

    Full Text Available The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

  2. Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows.

    Directory of Open Access Journals (Sweden)

    Yuhang Wang

    Full Text Available Bile salt-stimulated lipase (BSSL is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL.

  3. Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows

    Science.gov (United States)

    Ding, Fangrong; Wang, Tao; Liu, Wenjie; Lindquist, Susanne; Hernell, Olle; Wang, Jianwu; Li, Jing; Li, Ling; Zhao, Yaofeng; Dai, Yunping; Li, Ning

    2017-01-01

    Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL. PMID:28475629

  4. Enhancement of radiosensitivity of recombinant Ad-p53 gene on human lung adenocarcinoma cell with different p53 status

    International Nuclear Information System (INIS)

    Pang Dequan; Wang Peiguo; Wang Ping; Zhang Weiming

    2008-01-01

    Objective: To investigate the enhancement of radiosensitivity of recombinant Ad-p53 gene on human lung adenocarcinoma cell lines(A549 and GLC-82) with different p53 status in vitro. Methods: Two human lung adenocarcinoma cell lines of A549 and GLC-82 were examined on their difference in p53 status with immunohistochemistry stain and PCR-SSCP technique. Expand Ad-wtp53 was transfected into tumor cells. Clonogenic assays were performed to evaluate the inhibition effect on cell growth and the degree of sensitization to irradiation. Apoptosis and cell cycle changes were determined using the flow cytometry assay. Results: The A549 cell line presented positive P53 expression while GLC-82 negative. GLC-82 bore mutant p53 on the exon 7. The wtp53 gene could be efficiently expressed in the two cell lines and greatly inhibit the cell growth. Its efficiency didn't depend on the intrinsic p53 genetic status. After irradiation, its function of inducing G 1 arrest and apoptosis on GLC-82 cell line was much stronger than the A549 cell line. In both the A549 and GLC-82 cell lines, the combination of Ad-p53 plus radiation resulted in more apoptosis than the others. There was no significant difference between two groups. Conclusions: Ad-p53 can depress the tumor growth and enhance the radiosensitivity of human lung adenocarcinoma cells. And this effect is independent of endogenous p53 status. (authors)

  5. Functions of Human Rad51 and Other Recombination Factors in DNA Double-Strand Break Repair

    National Research Council Canada - National Science Library

    Sigurdsson, Stefan

    2004-01-01

    ... of. DNA double strand breaks. Genetic and biochemical studies have suggested that the function of genes of the RAD52 group is highly conserved from yeast to humans and interestingly the efficiency of DNA double strand break...

  6. Establishment of human sperm-specific voltage-dependent anion channel 3 recombinant vector for the production of a male contraceptive vaccine

    Directory of Open Access Journals (Sweden)

    Asmarinah Asmarinah

    2012-05-01

    Full Text Available Background: The aim of this study was to construct a recombinant vector of human sperm specific VDAC3 gene for production of VDAC3 antibody, which is potential as male contraception vaccine.Methods: Target fragment sequence of VDAC3 gene was obtained through amplification of human sperm VDAC3 cDNA with primers covering exon 5 to exon 8. Its PCR product in size of 435 bp was cloned to the pET101/D-TOPO expression vector (5753 bp. E. coli bacteria were transformed with this vector. Cloning of VDAC3 fragment gene to the vector was confirmed by the using of XbaI restriction enzyme and PCR colony method with primers covering exons 5-8 of the human VDAC3 gene.Results: Alignment analysis of amplified fragment covering exon 5 to exon 8 of VDAC3 gene showed 94% homology to human VDAC3 gene from databank. After cloning to the expression vector and transformation to E. coli competent cells, twelve colonies could grow in culture media. Gel electrophoresis of sliced VDAC3 recombinant vector showed a single band in the size of 6181 bp in 8 colonies. After application of PCR colony and amplicon sequencing, the result showed a single band in the size of 435 bp and fragment sequence with 94% identity to human VDAC3 gene.Conclusion: The construction of human sperm specific VDAC3 gene recombinant vector was established in this study. In the future, this recombinant vector will be used to produce VDAC3 antibody for the development of a male contraception vaccine. (Med J Indones. 2012;21:61-5Keywords: Contraception, recombinant vector, sperm, VDAC3

  7. Stability and compatibility of recombinant adeno-associated virus under conditions commonly encountered in human gene therapy trials.

    Science.gov (United States)

    Gruntman, Alisha M; Su, Lin; Su, Qin; Gao, Guangping; Mueller, Christian; Flotte, Terence R

    2015-04-01

    Recombinant adeno-associated virus (rAAV) vectors are rapidly becoming the first choice for human gene therapy studies, as clinical efficacy has been demonstrated in several human trials and proof-of-concept data have been demonstrated for correction of many others. When moving into human use under the auspices of an FDA Investigational New Drug (IND) application, it is necessary to demonstrate the stability of vector material under various conditions of storage, dilution, and administration when used in humans. Limited data are currently available in the literature regarding vector compatibility and stability, leading most IND sponsors to repeat all necessary studies. The current study addresses this issue with an rAAV vector (rAAV1-CB-chAATmyc) containing AAV2-inverted terminal repeat sequences packaged into an AAV1 capsid. Aliquots of vector were exposed to a variety of temperatures, diluents, container constituents, and other environmental conditions, and its functional biological activity (after these various treatments) was assessed by measuring transgene expression after intramuscular injection in mice. rAAV was found to be remarkably stable at temperatures ranging from 4°C to 55°C (with only partial loss of potency after 20 min at 70°C), at pH ranging from 5.5 to 8.5, after contact with mouse or human serum (with or without complement depletion) or with gadolinium and after contact with glass, polystyrene, polyethylene, polypropylene, and stainless steel. The only exposure resulting in near-total loss of vector activity (10,000-fold loss) was UV exposure for 10 min. The stability of rAAV1 preparations bodes well for future dissemination of this therapeutic modality.

  8. Species association of hepatitis B virus (HBV in non-human apes; evidence for recombination between gorilla and chimpanzee variants.

    Directory of Open Access Journals (Sweden)

    Sinéad Lyons

    Full Text Available Hepatitis B virus (HBV infections are widely distributed in humans, infecting approximately one third of the world's population. HBV variants have also been detected and genetically characterised from Old World apes; Gorilla gorilla (gorilla, Pan troglodytes (chimpanzee, Pongo pygmaeus (orang-utan, Nomascus nastusus and Hylobates pileatus (gibbons and from the New World monkey, Lagothrix lagotricha (woolly monkey. To investigate species-specificity and potential for cross species transmission of HBV between sympatric species of apes (such as gorillas and chimpanzees in Central Africa or between humans and chimpanzees or gorillas, variants of HBV infecting captive wild-born non-human primates were genetically characterised. 9 of 62 chimpanzees (11.3% and two from 11 gorillas (18% were HBV-infected (15% combined frequency, while other Old world monkey species were negative. Complete genome sequences were obtained from six of the infected chimpanzee and both gorillas; those from P. t .ellioti grouped with previously characterised variants from this subspecies. However, variants recovered from P. t. troglodytes HBV variants also grouped within this clade, indicative of transmission between sub-species, forming a paraphyletic clade. The two gorilla viruses were phylogenetically distinct from chimpanzee and human variants although one showed evidence for a recombination event with a P.t.e.-derived HBV variant in the partial X and core gene region. Both of these observations provide evidence for circulation of HBV between different species and sub-species of non-human primates, a conclusion that differs from the hypothesis if of strict host specificity of HBV genotypes.

  9. Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

    NARCIS (Netherlands)

    Kummer, J. Alain; Kamp, Angela M.; van Katwijk, Marcel; Brakenhoff, Just P.J.; Radosevic, Katarina; Radosevic, K.; van Leeuwen, Anne Marie; Borst, Jannie; Verweij, Cornelis L.; Hack, C. Erik

    1993-01-01

    The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven

  10. EFFICACY AND TOLERABILITY OF RECOMBINANT HUMAN GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR IN PATIENTS WITH CHEMOTHERAPY-RELATED LEUKOPENIA AND FEVER

    NARCIS (Netherlands)

    BIESMA, B; DEVRIES, EGE; WILLEMSE, PHB; SLUITER, WJ; POSTMUS, PE; LIMBURG, PC; STERN, AC; VELLENGA, E

    1990-01-01

    30 patients with chemotherapy-related leukopenia (white cells 1.0 x 10(9)/l or lower) and fever (temperature 38.5-degrees-C or higher) were treated in double-blind randomised trial with standard antibiotics and 7 days of intravenously administered recombinant human granulocyte-macrophage

  11. Recombinant human activated protein C inhibits local and systemic activation of coagulation without influencing inflammation during Pseudomonas aeruginosa pneumonia in rats

    NARCIS (Netherlands)

    Choi, Goda; Hofstra, Jorrit-Jan H.; Roelofs, Joris J. T. H.; Florquin, Sandrine; Bresser, Paul; Levi, Marcel; van der Poll, Tom; Schultz, Marcus J.

    2007-01-01

    OBJECTIVE: Alveolar fibrin deposition is a hallmark of pneumonia. It has been proposed that recombinant human activated protein C exerts lung-protective effects via anticoagulant and anti-inflammatory pathways. We investigated the role of the protein C system in pneumonia caused by Pseudomonas

  12. Recombinant human activated protein C inhibits local and systemic activation of coagulation without influencing inflammation during Pseudomonas aeruginosa pneumonia in rats

    NARCIS (Netherlands)

    Choi, Goda; Hofstra, Jorrit-Jan H; Roelofs, Joris J T H; Florquin, Sandrine; Bresser, Paul; Levi, Marcel; van der Poll, Tom; Schultz, Marcus J

    OBJECTIVE: Alveolar fibrin deposition is a hallmark of pneumonia. It has been proposed that recombinant human activated protein C exerts lung-protective effects via anticoagulant and anti-inflammatory pathways. We investigated the role of the protein C system in pneumonia caused by Pseudomonas

  13. Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

    Science.gov (United States)

    The ability to rescue an infectious, recombinant, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting enhanced green fluorescent protein (EGFP) gene into the human parainfluenza vi...

  14. Curative Metatarsal Bone Surgery Combined with Intralesional Administration of Recombinant Human Epidermal Growth Factor in Diabetic Neuropathic Ulceration of the Forefoot: A Prospective, Open, Uncontrolled, Nonrandomized, Observational Study

    Directory of Open Access Journals (Sweden)

    Aristides L. Garcia Herrera, MD, PhD

    2017-01-01

    Conclusions: The combination of curative metatarsal bone surgery with intralesional administration of recombinant human EGF resulted in a significant reduction in the re-epithelization time, recidivism, and development of new diabetic lesions. The safety profile was appropriate. However, more randomized, triple-blind, and placebo trials are needed to evaluate the efficacy and safety of this new therapy.

  15. Activation of coagulation by administration of recombinant factor VIIa elicits interleukin 6 (IL-6) and IL-8 release in healthy human subjects

    NARCIS (Netherlands)

    de Jonge, Evert; Friederich, Philip W.; Vlasuk, George P.; Rote, William E.; Vroom, Margaretha B.; Levi, Marcel; van der Poll, Tom

    2003-01-01

    The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in

  16. Enhanced opsonisation of Rhesus D-positive human red blood cells by recombinant polymeric immunoglobulin G anti-G antibodies.

    Science.gov (United States)

    Díaz-Solano, Dylana; Fuenmayor, Jaheli; Montaño, Ramon F

    2018-02-01

    Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. Recombinant polymeric anti-G IgG1 (IgG1μtp) and IgG3 (IgG3μtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. The recombinant IgG1μtp and IgG3μtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. The enhanced opsonic properties of the polymeric anti-G IgG1μtp and IgG3μtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.

  17. In vitro response of human osteoblasts to multi-step sol–gel derived bioactive glass nanoparticles for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Jian Ping, E-mail: jian.fan@ucl.ac.uk [Department of Mechanical Engineering, University College London, London WC1E 7JE (United Kingdom); Kalia, Priya; Di Silvio, Lucy [Biomaterials, Tissue Engineering and Imaging, The Dental Institute, King' s College London, Guy' s Hospital, London SE1 9BT (United Kingdom); Huang, Jie [Department of Mechanical Engineering, University College London, London WC1E 7JE (United Kingdom)

    2014-03-01

    A multi-step sol–gel process was employed to synthesize bioactive glass (BG) nanoparticles. Transmission electron microscopy (TEM) revealed that the BG nanoparticles were spherical and ranged from 30 to 60 nm in diameter. In vitro reactivity of the BG nanoparticles was tested in phosphate buffer saline (PBS), Tris-buffer (TRIS), simulated body fluid (SBF), and Dulbecco's modified Eagle's medium (DMEM), in comparison with similar sized hydroxyapatite (HA) and silicon substituted HA (SiHA) nanoparticles. Bioactivity of the BG nanoparticles was confirmed through Fourier transform infrared spectroscopy (FTIR) analysis. It was found that bone-like apatite was formed after immersion in SBF at 7 days. Solutions containing BG nanoparticles were slightly more alkaline than HA and SiHA, suggesting that a more rapid apatite formation on BG was related to solution-mediated dissolution. Primary human osteoblast (HOB) cell model was used to evaluate biological responses to BG nanoparticles. Lactate dehydrogenase (LDH) cytotoxicity assay showed that HOB cells were not adversely affected by the BG nanoparticles throughout the 7 day test period. Interestingly, MTS assay results showed an enhancement in cell proliferation in the presence of BG when compared to HA and SiHA nanoparticles. Particularly, statistically significant (p < 0.05) alkaline phosphatase (ALP) activity of HOB cells was found on the culture containing BG nanoparticles, suggesting that the cell differentiation might be promoted by BG. Real-time quantitative PCR analysis (qPCR) further confirmed this finding, as a significantly higher level of RUNX2 gene expression was recorded on the cells cultured in the presence of BG nanoparticles when compared to those with HA and SiHA. - Highlights: • Spherical bioactive glass nanoparticles (BG) under 60 nm were synthesized. • An alkali morphological catalyst was used in the synthesis. • Cytotoxicity assays demonstrated that BG was not cytotoxic towards HOB

  18. Recombinant subgroup B human respiratory syncytial virus expressing enhanced green fluorescent protein efficiently replicates in primary human cells and is virulent in cotton rats.

    Science.gov (United States)

    Lemon, Ken; Nguyen, D Tien; Ludlow, Martin; Rennick, Linda J; Yüksel, Selma; van Amerongen, Geert; McQuaid, Stephen; Rima, Bert K; de Swart, Rik L; Duprex, W Paul

    2015-03-01

    Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies. Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with

  19. Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase.

    Science.gov (United States)

    Baldassarre, H; Schirm, M; Deslauriers, J; Turcotte, C; Bordignon, V

    2009-08-01

    The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, beta-lactoglobulin and alpha-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of alpha-lactalbumin was determined using the IDRing assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 +/- 0.97 vs. 2.24 +/- 0.25 mg/ml, P 0.05). We concluded that a decreased/delayed expression of the alpha-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.

  20. Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain.

    Science.gov (United States)

    Sandowski, Yael; Raver, Nina; Gussakovsky, Eugene E; Shochat, Suzan; Dym, Orly; Livnah, Oded; Rubinstein, Menachem; Krishna, Radha; Gertler, Arieh

    2002-11-29

    A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

  1. Study on inter-ethnic human differences in bioactivation and detoxification of estragole using physiologically based kinetic modeling.

    Science.gov (United States)

    Ning, Jia; Louisse, Jochem; Spenkelink, Bert; Wesseling, Sebastiaan; Rietjens, Ivonne M C M

    2017-09-01

    Considering the rapid developments in food safety in the past decade in China, it is of importance to obtain insight into what extent safety and risk assessments of chemicals performed for the Caucasian population apply to the Chinese population. The aim of the present study was to determine physiologically based kinetic (PBK) modeling-based predictions for differences between Chinese and Caucasians in terms of metabolic bioactivation and detoxification of the food-borne genotoxic carcinogen estragole. The PBK models were defined based on kinetic constants for hepatic metabolism derived from in vitro incubations using liver fractions of the two ethnic groups, and used to evaluate the inter-ethnic differences in metabolic activation and detoxification of estragole. The models predicted that at realistic dietary intake levels, only 0.02% of the dose was converted to the ultimate carcinogenic metabolite 1'-sulfooxyestragole in Chinese subjects, whereas this amounted to 0.09% of the dose in Caucasian subjects. Detoxification of 1'-hydroxyestragole, mainly via conversion to 1'-oxoestragole, was similar within the two ethnic groups. The 4.5-fold variation in formation of the ultimate carcinogenic metabolite of estragole accompanied by similar rates of detoxification may indicate a lower risk of estragole for the Chinese population at similar levels of exposure. The study provides a proof of principle for how PBK modeling can identify differences in ethnic sensitivity and provide a more refined risk assessment for a specific ethnic group for a compound of concern.

  2. Functional in vitro studies of recombinant human immunoglobulin G and immunoglobulin A anti-D

    DEFF Research Database (Denmark)

    Nielsen, Leif Kofoed; Green, Trine Hefsgaard; Norderhaug, Lars

    2007-01-01

    The use of anti-D purified from human serum to prevent hemolytic disease of the fetus and newborn due to D is well established. Owing to supply and safety reasons, however, an unlimited and non-plasma-derived source of antibodies for Rhesus prophylaxis is needed.......The use of anti-D purified from human serum to prevent hemolytic disease of the fetus and newborn due to D is well established. Owing to supply and safety reasons, however, an unlimited and non-plasma-derived source of antibodies for Rhesus prophylaxis is needed....

  3. Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1

    OpenAIRE

    Ambadapadi, Sriram; Wang, Peter L.; Palii, Sergiu P.; James, Margaret O.

    2015-01-01

    Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17βestradiol (17β-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3β, 17β-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low μM concentrat...

  4. Effect of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on the growth and radiotherapeutic sensitivity of human lymphoma cell lines

    International Nuclear Information System (INIS)

    Yu Zeyang; Fan Wo; Li Dongqing; Zhu Ran; Wang Yongqing; Wu Jinchang

    2008-01-01

    Objective: To explore the inhibitory effect and radiation sensitization of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on human lymphoma cell lines. Methods: Human lymphoma cell lines Raji and Daudi were treated with rAd-p53, radiation therapy and combined treatment, respectively. The cell growth inhibition was assessed by MTT. The p53 protein expression was detected by Western blotting, and p53 mRNA was detected by BT-PCB. Results: The MTT results showed that the inhibitory effect and radiosensitivity enhancement of rAd-p53 on human lymphoma cell lines were not obvious [Raji: (27.5±4.1)%; Daudi: (28.1±1.6)%]. The results of Western blotting and BT-PCB showed that extrinsic p53 protein and p53 mRNA were expressed to some degree, but not at high-level. In addition, the results didn't demonstrate obvious radiosensitivity enhancement. Conclusions: The role of inhibition and radiosensitivity enhancement of rAd-p53 was not significant on human lymphoma cell lines. (authors)

  5. Variable performance of a human derived Sarcoptes scabiei recombinant antigen ELISA in swine mange diagnosis.

    Science.gov (United States)

    Casais, R; Goyena, E; Martínez-Carrasco, C; Ruiz de Ybáñez, R; Alonso de Vega, F; Ramis, G; Prieto, J M; Berriatua, E

    2013-10-18

    The performance of an indirect ELISA test based on Sarcoptes scabiei var hominis recombinant antigen Ssλ20ΔB3 (rec-ELISA), to diagnose pig mange was investigated in 15 experimentally infected and non-infected pigs and 692 commercial pigs from 16 herds in southeast Spain. These latter animals included 6-7 month old fatteners (13 herds), 11-12 month old replacement sows (1 herd) and ≥24 month old breeding sows (7 herds). All pigs were examined for mites in ear skin scrapings and the presence of S. scabiei-associated macroscopic dermatitis; moreover, fatteners were also tested for antibodies against porcine viruses including: Aujeszky disease virus (ADV), swine influenza virus (SIV), type 2 porcine circovirus (PCV2) and porcine respiratory and reproductive syndrome virus (PRRSV). S. scabiei and chronic hyperkeratotic dermatitis were detected in breeding sows from 6 herds. Mite prevalence in other pigs was 83% in replacement sows, 0% in 7 fattener's herds and 3-82% in other fattener's herds. All fattener herds had pigs with acute hypersensitivity dermatitis and the percentage of affected pigs and lesion area was significantly greater in S. scabiei infected ones. Rec-ELISA relative optical densities (RODs) were greater in older than in young pigs, as well as in infected compared to non-infected pigs. However, RODs differed significantly between infected individuals, regardless of age and origin (commercial or experimental) and the herd prevalence of S. scabiei. Low repeatability between ELISA microtiter plates, suggesting variable specific antibody binding to antigen, are likely partly responsible for ROD variation. Other potential causes of variation were examined in fatteners using random effects logistic regression analysis, after defining a seropositivity threshold value with receiver-operating characteristics (ROC) analysis. The logistic model indicated that seropositivity was associated with large dermatitis areas and with the only herd with low PCV2

  6. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein.

    Science.gov (United States)

    Rahaman, Siti Nurulnabila A; Mat Yusop, Jastina; Mohamed-Hussein, Zeti-Azura; Ho, Kok Lian; Teh, Aik-Hong; Waterman, Jitka; Ng, Chyan Leong

    2016-03-01

    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

  7. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  8. Anthropometric, metabolic, and immunological effects of recombinant human growth hormone in AIDS and AIDS-related complex.

    Science.gov (United States)

    Krentz, A J; Koster, F T; Crist, D M; Finn, K; Johnson, L Z; Boyle, P J; Schade, D S

    1993-03-01

    Metabolic and anthropometric changes induced by "pharmacological" versus "physiological" doses (5.0 vs. 2.5 mg, every other day) of recombinant human growth hormone (rhGH) were compared in 10 human immunodeficiency virus-positive patients with AIDS or AIDS-related complex. Five patients were randomly assigned to each treatment schedule in a 3-month prospective, double-blind clinical trial. Three of the 10 patients, none taking zidovudine and all with low initial CD4 counts, were withdrawn during the study due to acute opportunistic infections. During treatment, insulin-like growth factor-1 (IGF-1) levels increased significantly (p hGH treatment group, whereas no significant change was observed in IGF-1 in the physiological dose rhGH group. In the pharmacological hGH treatment group, weight loss preceding the study was reversed (p hGH group. The pharmacological dose of hGH was associated with minor increments (p fasting plasma glucose, insulin, and C-peptide concentrations, which were of negligible clinical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Long-term intravenous treatment of Pompe disease with recombinant human alpha-glucosidase from milk.

    NARCIS (Netherlands)

    Hout, J.M. van den; Kamphoven, J.H.; Winkel, L.P.; Arts, W.F.M.; Klerk, J.B.C. de; Loonen, M.C.B.; Vulto, A.G.; Cromme-Dijkhuis, A.H.; Weisglas-Kuperus, N.; Hop, W.C.J.; Hirtum, H. van; Diggelen, O.P. van; Boer, M. de; Kroos, M.A.; Doorn, P.A. van; Voort, E.I. van der; Sibbles, B.; Corven, E.J. van; Brakenhoff, J.P.; Hove, J.L. van; Smeitink, J.A.M.; Jong, G. de; Reuser, A.J.J.; Ploeg, A.T. van der

    2004-01-01

    OBJECTIVE: Recent reports warn that the worldwide cell culture capacity is insufficient to fulfill the increasing demand for human protein drugs. Production in milk of transgenic animals is an attractive alternative. Kilogram quantities of product per year can be obtained at relatively low costs,

  10. Long-term intravenous treatment of Pompe disease with recombinant human alpha-glucosidase from milk

    NARCIS (Netherlands)

    J.M.P. van den Hout (Johanna); B. Sibbles (Barbara); J.P. Brakenhoff (Just); A.H. Cromme-Dijkhuis (Adri); N. Weisglas-Kuperus (Nynke); A.J.J. Reuser (Arnold); M.A. Boer (Marijke); J.A.M. Smeitink (Jan); O.P. van Diggelen (Otto); E. van der Voort (Edwin); E.J.J.M. van Corven (Emiel); H. van Hirtum (Hans); J.H.J. Kamphoven (Joep); A.T. van der Ploeg (Ans); J. van Hove (Johan); W.F.M. Arts (Willem Frans); P.A. van Doorn (Pieter); J.B.C. de Klerk (Johannes); M.C.B. Loonen (Christa); A.G. Vulto (Arnold); M.A. Kroos (Marian); W.C.J. Hop (Wim); L.P.F. Winkel (Léon); G. de Jong (Gerard)

    2004-01-01

    textabstractOBJECTIVE: Recent reports warn that the worldwide cell culture capacity is insufficient to fulfill the increasing demand for human protein drugs. Production in milk of transgenic animals is an attractive alternative. Kilogram quantities of product per year can be

  11. Large-scale recombinant expression and purificatoin of human tyrosinase suitabel for structural studies

    NARCIS (Netherlands)

    Lai, X.; Soler-Lopez, M.; Wichers, H.J.; Dijkstra, Bouke

    2016-01-01

    Human tyrosinase (TYR) is a glycoprotein that initiates the first two reactions in the melanin biosynthesis pathway. Mutations in its encoding gene cause Oculocutaneous Albinism type I (OCA1), the most severe form of albinism, which is a group of autosomal recessive disorders characterized by

  12. High-level secretion of native recombinant human calreticulin in yeast

    DEFF Research Database (Denmark)

    Čiplys, Evaldas; Žitkus, Eimantas; Gold, Leslie I.

    2015-01-01

    processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species...

  13. Potential and opportunities for use of recombinant lactic acid bacteria in human health

    Czech Academy of Sciences Publication Activity Database

    Hanniffy, S.; Wiedermann, U.; Repa, A.; Mercenier, A.; Daniel, C.; Fioramonti, J.; Tlaskalová, Helena; Kozáková, Hana; Israelsen, H.; Madsen, S.; Vrang, A.; Hols, P.; Delcour, J.; Bron, P.; Kleerebezem, M.; Wells, J.

    2004-01-01

    Roč. 56, - (2004), s. 1-64 ISSN 0065-2164 Grant - others:EC contract(XE) QLK3-CT-QLK3-CT-2000-00340 Keywords : lactic acid bacteria * human health Subject RIV: EE - Microbiology, Virology Impact factor: 1.156, year: 2004