WorldWideScience

Sample records for human recombinant anti-thyroperoxidase

  1. Human recombinant anti-thyroperoxidase autoantibodies: in vitro cytotoxic activity on papillary thyroid cancer expressing TPO

    OpenAIRE

    Rebuffat, S A; Morin, M.; Nguyen, B; Castex, F; Robert, B.; Péraldi-Roux, S

    2010-01-01

    Background: 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. Results: 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 d...

  2. Anti-thyroperoxidase antibodies from patients with Hashimoto's encephalopathy bind to cerebellar astrocytes.

    Science.gov (United States)

    Blanchin, Stéphanie; Coffin, Christine; Viader, Fausto; Ruf, Jean; Carayon, Pierre; Potier, Francette; Portier, Estelle; Comby, Elisabeth; Allouche, Stéphane; Ollivier, Yann; Reznik, Yves; Ballet, Jean Jacques

    2007-12-01

    A cohort of 10 Hashimoto's encephalopathy (HE) patients, 33 patients with unrelated neurological symptoms, 12 Hashimoto's thyroiditis patients and 4 healthy adult donors was studied to explore the neurological targets of anti-thyroperoxidase (TPO) autoantibodies (aAb) in HE. High levels of anti-TPO aAb were only detected in HE group's cerebrospinal fluids. In immunofluorescence assays on monkey brain cerebellum sections, both HE patients' sera and anti-TPO monoclonal antibodies (mAb) were able to bind cerebellar cells expressing glial fibrillary acid protein. Normal human astrocytes from primary cultures also reacted with anti-TPO mAb. Specific astrocyte binding of anti-TPO aAb suggests a role of these aAb in the HE pathogenesis.

  3. Recombinant human milk proteins.

    Science.gov (United States)

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases.

  4. Recombinant Human Enterovirus 71

    OpenAIRE

    2004-01-01

    Two human enterovirus 71 (HEV71) isolates were identified from hand, foot and mouth disease patients with genome sequences that had high similarity to HEV71 (>93%) at 5´ UTR, P1, and P2 and coxsackievirus A16 (CV-A16, >85%) at P3 and 3´UTR. Intertypic recombination is likely to have occurred between HEV71 and CV-A16 or an as-yet to be described CV-A16-like virus.

  5. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  6. Human Insulin from Recombinant DNA Technology

    Science.gov (United States)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

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

    African Journals Online (AJOL)

    Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen ... three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). ..... characterization of a polyol- responsive monoclonal.

  12. Quadrivalent Human Papillomavirus recombinant vaccine associated lipoatrophy.

    Science.gov (United States)

    Ojaimi, Samar; Buttery, Jim P; Korman, Tony M

    2009-08-06

    Involutional lipoatrophy, a loss of subcutaneous fat, may be idiopathic, associated with inflammatory skin conditions, or trauma, and has also been reported following injections of medications including insulin, corticosteroids and penicillin. There have also been reports in association with Diptheria Pertussis Tetanus (DPT) vaccine. We report on two cases of lipoatrophy associated with the new Quadrivalent Human Papillomavirus (HPV) recombinant vaccine (Gardasil).

  13. Population-specific recombination sites within the human MHC region

    OpenAIRE

    2013-01-01

    Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European an...

  14. Feasibility of a recombinant human apolipoprotein E reference material

    NARCIS (Netherlands)

    Schiele, F.; Barbier, A.; Visvikis, A.; Aggerbeck, L.; Rosseneu, M.; Havekes, L.; Huttinger, M.; Profilis, C.; Siest, G.

    1998-01-01

    The aim of this work was to prepare a recombinant apo E material and to determine its suitability as a reference material. We produced human apo E3 using recombinant DNA technology. The cDNA of human apo E3 was cloned in the pARHS bacterial expression vector and used to transfect E. Coli BL21 (DE3)

  15. [Preparation of recombinant human insulin--study of downstream process].

    Science.gov (United States)

    Yu, Rong; Li, Xiaohong; Yang, Jiyu; Wu, Wutong

    2004-10-01

    This study was intended to establish a method of preparation of recombinant human insulin, with (His)6-Arg-Arg-human proinsulin (RRhPI) expressed by Escherichia coli. After DEAE-Sepharose Fast Flow ion-exchange chromatography, Sephadex G-25 chromatography and refolding, enzyme cleavage and Superdex 75 size exclusion chromatography,the RRhPI expressed by Escherichia coli in inclusion body form was converted to human insulin. The obtained recombinant human insulin was analyzed by SDS-PAGE, HPLC, amino acid composition analysis and bioidentity test (mouse convulsion test). The results indicate that our obtained preparation is highly purified, active recombinant human insulin.

  16. Recombinant human thrombopoietin in myelosuppressive chemotherapy.

    Science.gov (United States)

    Vadhan-Raj, S

    2001-07-01

    Recombinant human thrombopoietin (rhTPO) is a full-length glycosylated molecule that has been under evaluation in the setting of chemotherapy-induced myelosuppression. It has been shown to be a potent stimulator of platelet production in cancer patients when administered prior to chemotherapy. The peak platelet response to a single dose of rhTPO is observed around day 12, and is accompanied by a significant increase in the number of mature megakaryocytes in bone marrow. Consistent with this biologic effect, rhTPO administered postchemotherapy has been shown to be effective in attenuating severe thrombocytopenia induced by carboplatin, which produces a late platelet nadir. Early clinical experience with a regimen that produces an early nadir, however, such as AI (doxorubicin [Adriamycin] and ifosfamide [Ifex]), suggests that administration of rhTPO both prior to and following chemotherapy might be important to reduce thrombocytopenia severity. Treatment with rhTPO in these clinical trials has been well tolerated with a favorable safety profile. Randomized clinical trials have been initiated to determine further the importance of schedule in the prevention and treatment of severe thrombocytopenia in cancer patients.

  17. Age-dependent recombination rates in human pedigrees.

    Directory of Open Access Journals (Sweden)

    Julie Hussin

    2011-09-01

    Full Text Available In humans, chromosome-number abnormalities have been associated with altered recombination and increased maternal age. Therefore, age-related effects on recombination are of major importance, especially in relation to the mechanisms involved in human trisomies. Here, we examine the relationship between maternal age and recombination rate in humans. We localized crossovers at high resolution by using over 600,000 markers genotyped in a panel of 69 French-Canadian pedigrees, revealing recombination events in 195 maternal meioses. Overall, we observed the general patterns of variation in fine-scale recombination rates previously reported in humans. However, we make the first observation of a significant decrease in recombination rates with advancing maternal age in humans, likely driven by chromosome-specific effects. The effect appears to be localized in the middle section of chromosomal arms and near subtelomeric regions. We postulate that, for some chromosomes, protection against non-disjunction provided by recombination becomes less efficient with advancing maternal age, which can be partly responsible for the higher rates of aneuploidy in older women. We propose a model that reconciles our findings with reported associations between maternal age and recombination in cases of trisomies.

  18. Recombinant human antithrombin III: rhATIII.

    Science.gov (United States)

    2004-01-01

    GTC Biotherapeutics (formerly Genzyme Transgenics Corporation) is developing a transgenic form of antithrombin III known as recombinant human antithrombin III [rhATIII]. It is produced by inserting human DNA into the cells of goats so that the targeted protein is excreted in the milk of the female offspring. The transgenic goats have been cloned in collaboration with the Louisiana State University Agriculture Center. GTC Biotherapeutics is conducting clinical trials of rhATIII in coagulation disorders. rhATIII is believed to be both safer and more cost-effective than the currently available plasma-derived product. rhATIII is also being investigated in cancer and acute lung injury. Genzyme Transgenics Corporation, originally a subsidiary of Genzyme Corporation, changed its name to GTC Biotherapeutics in June 2002; it is no longer a subsidiary of Genzyme Corporation. GTC Biotherapeutics is seeking partners for the commercialisation of rhATIII. Restructuring of GTC Biotherapeutics to support its commercialisation programmes was announced in February 2004. Genzyme Transgenics Corporation was developing rhATIII in association with Genzyme General (Genzyme Corporation) in the ATIII LLC joint venture, but in November 2000 a letter of intent was signed for the reacquisition of the rights by Genzyme Transgenics Corporation. It was announced in February 2001 that this reacquisition was not going to be completed and that the development of rhATIII was to continue with ATIII LLC. However, in July 2001, Genzyme Transgenics Corporation reacquired all the rights in the transgenic antithrombin III programme. SMI Genzyme Ltd, a joint venture between Sumitomo Metal Industries, Japan, and Genzyme Transgenics Corporation, USA, was set up to fund development of transgenic antithrombin III in Asia. However, in October 2000, Genzyme Transgenics Corporation reacquired, from Sumitomo Metal Industries, the rights to its technology for production of medicines from milk in 18 Asian countries

  19. Absence of the TAP2 human recombination hotspot in chimpanzees.

    Directory of Open Access Journals (Sweden)

    Susan E Ptak

    2004-06-01

    Full Text Available Recent experiments using sperm typing have demonstrated that, in several regions of the human genome, recombination does not occur uniformly but instead is concentrated in "hotspots" of 1-2 kb. Moreover, the crossover asymmetry observed in a subset of these has led to the suggestion that hotspots may be short-lived on an evolutionary time scale. To test this possibility, we focused on a region known to contain a recombination hotspot in humans, TAP2, and asked whether chimpanzees, the closest living evolutionary relatives of humans, harbor a hotspot in a similar location. Specifically, we used a new statistical approach to estimate recombination rate variation from patterns of linkage disequilibrium in a sample of 24 western chimpanzees (Pan troglodytes verus. This method has been shown to produce reliable results on simulated data and on human data from the TAP2 region. Strikingly, however, it finds very little support for recombination rate variation at TAP2 in the western chimpanzee data. Moreover, simulations suggest that there should be stronger support if there were a hotspot similar to the one characterized in humans. Thus, it appears that the human TAP2 recombination hotspot is not shared by western chimpanzees. These findings demonstrate that fine-scale recombination rates can change between very closely related species and raise the possibility that rates differ among human populations, with important implications for linkage-disequilibrium based association studies.

  20. 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.

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

    African Journals Online (AJOL)

    ajl yemi

    2011-11-14

    Nov 14, 2011 ... 1The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi ... might play a role in a broad range of molecular and ... The resulting recombinant expression vector pPIC9K/ HSA-CP was.

  2. The influence of recombination on human genetic diversity.

    Directory of Open Access Journals (Sweden)

    Chris C A Spencer

    2006-09-01

    Full Text Available In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.

  3. Evidence of recombination within human alpha-papillomavirus

    Directory of Open Access Journals (Sweden)

    Carvajal-Rodríguez Antonio

    2007-03-01

    Full Text Available Abstract Background Human papillomavirus (HPV has a causal role in cervical cancer with almost half a million new cases occurring each year. Presence of the carcinogenic HPV is necessary for the development of the invasive carcinoma of the genital tract. Therefore, persistent infection with carcinogenic HPV causes virtually all cervical cancers. Some aspects of the molecular evolution of this virus, as the putative importance of recombination in its evolutionary history, are an opened current question. In addition, recombination could also be a significant issue nowadays since the frequency of co-infection with more than one HPV type is not a rare event and, thus, new recombinant types could be currently being generated. Results We have used human alpha-PV sequences from the public database at Los Alamos National Laboratory to report evidence that recombination may exist in this virus. A model-based population genetic approach was used to infer the recombination signal from the HPV DNA sequences grouped attending to phylogenetic and epidemiological information, as well as to clinical manifestations. Our results agree with recently published ones that use a different methodology to detect recombination associated to the gene L2. In addition, we have detected significant recombination signal in the genes E6, E7, L2 and L1 at different groups, and importantly within the high-risk type HPV16. The method used has recently been shown to be one of the most powerful and reliable procedures to detect the recombination signal. Conclusion We provide new support to the recent evidence of recombination in HPV. Additionally, we performed the recombination estimation assuming the best-fit model of nucleotide substitution and rate variation among sites, of the HPV DNA sequence sets. We found that the gene with recombination in most of the groups is L2 but the highest values were detected in L1 and E6. Gene E7 was recombinant only within the HPV16 type. The

  4. 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.

  5. In vivo metabolism of recombinant human erythropoietin in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Spivak, J.L.; Hogans, B.B.

    1989-01-01

    We compared the in vivo plasma clearance and organ accumulation in anesthetized rats of 125I-labeled, recombinant human erythropoietin and 125I-labeled, desialylated recombinant erythropoietin. The immediate volume of distribution of 125I-labeled, recombinant erythropoietin approximated that of the plasma volume. Its plasma clearance was multiexponential, with an initial rapid distribution phase (t1/2 = 53 minutes) and a slower elimination phase (t1/2 = 180 minutes). Organ accumulation of labeled recombinant erythropoietin, as compared with 125I-labeled human albumin, was negligible until 30 minutes after injection when small amounts appeared in the kidneys and bone marrow. Only 24% of the 125I-labeled, desialylated recombinant erythropoietin was recovered immediately after injection, and 96% of the hormone was cleared from the plasma with a t1/2 of 2.0 minutes. The bulk of the desialylated hormone accumulated in the liver where it was rapidly catabolized and its breakdown products released back into the plasma. Significantly, in contrast to unmodified erythropoietin, there was also early accumulation of desialylated hormone in the kidneys, marrow, and spleen. Desialylated orosomucoid but not orosomucoid, yeast mannan, or dextran sulfate 500 inhibited the rapid plasma clearance and hepatic accumulation of desialylated erythropoietin. Oxidation of the desialylated hormone restored its plasma recovery and clearance to normal but rendered it biologically inactive, and accumulation in organs other than the kidney was negligible.

  6. Recombinant Human Erythropoietin in the Treatment of Acute Ischemic Stroke

    NARCIS (Netherlands)

    Ehrenreich, Hannelore; Weissenborn, Karin; Prange, Hilmar; Schneider, Dietmar; Weimar, Christian; Wartenberg, Katja; Schellinger, Peter D.; Bohn, Matthias; Becker, Harald; Wegrzyn, Martin; Jaehnig, Peter; Herrmann, Manfred; Knauth, Michael; Baehr, Mathias; Heide, Wolfgang; Wagner, Armin; Schwab, Stefan; Reichmann, Heinz; Schwendemann, Guenther; Dengler, Reinhard; Kastrup, Andreas; Bartels, Claudia

    2009-01-01

    Background and Purpose-Numerous preclinical findings and a clinical pilot study suggest that recombinant human erythropoietin (EPO) provides neuroprotection that may be beneficial for the treatment of patients with ischemic stroke. Although EPO has been considered to be a safe and well-tolerated dru

  7. Production und characterization of recombinant human lactoferrin

    NARCIS (Netherlands)

    Veen, Henricus Antonius van

    2008-01-01

    Human lactoferrin (hLF) is an iron-binding glycoprotein of Mr 77,000 that belongs to the transferrin family. Based on extensive research showing antimicrobial, anti-inflammatory and immunomodulatory activities of hLF, the molecule is postulated to be involved in the innate host defence against infec

  8. Recombinant human thrombopoietin: basic biology and evaluation of clinical studies.

    Science.gov (United States)

    Kuter, David J; Begley, C Glenn

    2002-11-15

    Thrombocytopenia is a common medical problem for which the main treatment is platelet transfusion. Given the increasing use of platelets and the declining donor population, identification of a safe and effective platelet growth factor could improve the management of thrombocytopenia. Thrombopoietin (TPO), the c-Mpl ligand, is the primary physiologic regulator of megakaryocyte and platelet development. Since the purification of TPO in 1994, 2 recombinant forms of the c-Mpl ligand--recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)--have undergone extensive clinical investigation. Both have been shown to be potent stimulators of megakaryocyte growth and platelet production and are biologically active in reducing the thrombocytopenia of nonmyeloablative chemotherapy. However, neither TPO has demonstrated benefit in stem cell transplantation or leukemia chemotherapy. Other clinical studies have investigated the use of TPO in treating chronic nonchemotherapy-induced thrombocytopenia associated with myelodysplastic syndromes, idiopathic thrombocytopenic purpura, thrombocytopenia due to human immunodeficiency virus, and liver disease. Based solely on animal studies, TPO may be effective in reducing surgical thrombocytopenia and bleeding, ex vivo expansion of pluripotent stem cells, and as a radioprotectant. Ongoing and future studies will help define the clinical role of recombinant TPO and TPO mimetics in the treatment of chemotherapy- and nonchemotherapy-induced thrombocytopenia.

  9. A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA).

    Science.gov (United States)

    Kaufman, H; Schlom, J; Kantor, J

    1991-07-30

    Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.

  10. Posttranslational modifications in human plasma MBL and human recombinant MBL

    DEFF Research Database (Denmark)

    Jensen, Pia Hønnerup; Laursen, Inga; Matthiesen, Finn

    2007-01-01

    the intact protein in its active conformation. For the first time, positions and occupation frequency of partial hydroxylations and partial glycosylations are reported in MBL. Hydroxylation and glycosylation patterns of both recombinant and plasma derived MBL were determined, using a combination of mass......(202)) was modified in trace amounts to dehydroalanine, as detected by a 34 Da mass loss. This impairs the formation of a disulphide bond in the carbohydrate recognition domain. The dehydroalanine was identified in similar small amounts in both recombinant and plasma-derived MBL....

  11. 75 FR 18548 - In the Matter of Certain Products and Pharmaceutical Compositions Containing Recombinant Human...

    Science.gov (United States)

    2010-04-12

    ... From the Federal Register Online via the Government Publishing Office INTERNATIONAL TRADE COMMISSION In the Matter of Certain Products and Pharmaceutical Compositions Containing Recombinant Human... pharmaceutical compositions containing recombinant human erythropoietin by reason of infringement of claims 1...

  12. Variation in human recombination rates and its genetic determinants.

    Directory of Open Access Journals (Sweden)

    Adi Fledel-Alon

    Full Text Available BACKGROUND: Despite the fundamental role of crossing-over in the pairing and segregation of chromosomes during human meiosis, the rates and placements of events vary markedly among individuals. Characterizing this variation and identifying its determinants are essential steps in our understanding of the human recombination process and its evolution. STUDY DESIGN/RESULTS: Using three large sets of European-American pedigrees, we examined variation in five recombination phenotypes that capture distinct aspects of crossing-over patterns. We found that the mean recombination rate in males and females and the historical hotspot usage are significantly heritable and are uncorrelated with one another. We then conducted a genome-wide association study in order to identify loci that influence them. We replicated associations of RNF212 with the mean rate in males and in females as well as the association of Inversion 17q21.31 with the female mean rate. We also replicated the association of PRDM9 with historical hotspot usage, finding that it explains most of the genetic variance in this phenotype. In addition, we identified a set of new candidate regions for further validation. SIGNIFICANCE: These findings suggest that variation at broad and fine scales is largely separable and that, beyond three known loci, there is no evidence for common variation with large effects on recombination phenotypes.

  13. Recombinant anti-human melanoma antibodies are versatile molecules.

    Science.gov (United States)

    Neri, D; Natali, P G; Petrul, H; Soldani, P; Nicotra, M R; Vola, R; Rivella, A; Creighton, A M; Neri, P; Mariani, M

    1996-08-01

    The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

  14. 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

    BACKGROUND: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human...... 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...... glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary...

  15. 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......) concentrations. DESIGN: Randomized, controlled, assessor-blinded, AMH-stratified (low: 5.0-14.9 pmol/L [0.7-infertility centers in four countries. PATIENT(S): Two hundred sixty-five women aged ≤37 years. INTERVENTION(S): Controlled...

  16. [Use of human recombinant erythropoietin in children with cancer].

    Science.gov (United States)

    Guyot, D; Margueritte, G

    2005-09-01

    Eighty percent of children with cancer suffer from anemia at the time of diagnosis. The physiopathology of anemia is complex. Although anemia can be life threatening, its consequences on the physical, psychological and social state of the child are often minimized. Blood transfusion is the main treatment of anemia: its efficacy is immediate but shortlasting, and it involves infectious and hemolytic risks. The human recombinant erythropoietin has been used for more than 25-years, and is often prescribed to adults with cancer and anemia. The human recombinant erythropoietin rHuEPO is nowadays used when blood transfusion is contra-indicated because of religious or cultural considerations, although several promising studies have been conducted about rHuEPO and children with cancer since 1996: it might be soon the preferential alternative treatment to anemia in children with cancer.

  17. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  18. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    Science.gov (United States)

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  19. Recombinant expression of hydroxylated human collagen in Escherichia coli

    OpenAIRE

    Rutschmann, Christoph; Baumann, Stephan; Cabalzar, Jürg; Luther, Kelvin B.; Hennet, Thierry

    2014-01-01

    Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveal...

  20. Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure

    OpenAIRE

    Kucuk, Nurten; Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

    2015-01-01

    Objectives In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase i...

  1. 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 effort...... of developing the world’s first fully recombinant antivenom based on human IgGs targeting the key toxins from the notorious black mamba (Dendroaspis polylepis)....

  2. Recombinant Functional Human Lactoferrin Expressed in Baculovirus System

    Institute of Scientific and Technical Information of China (English)

    Tao LIU; Yao-Zhou ZHANG; Xiang-Fu WU

    2006-01-01

    Human lactoferrin (hLf) is a multifunctional iron-binding glycoprotein. In this study, we amplified hLfcDNA by reverse transcription-polymerase chain reaction from normal human mammary gland.The nucleotide sequence of the hLf was identical to the known hLf. We constructed a recombinant virus,vBm-hLf, harboring the hLfgene and exploited the BmN cells as host to produce recombinant human lactoferrin(rhLf). It was found that a recombinant protein with a molecular mass of approximately 78 kDa was expressed.Approximately 13.5 μg rhLf was purified from 1-2× 105 BmN cells infected by vBm-hLf and the rhLf proved to be biologically active. This method established in our study will pave the way for efficient production of rhLf for further application of this protein in the future.

  3. Construction of human artificial chromosome vectors by recombineering.

    Science.gov (United States)

    Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare

    2005-05-23

    Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

  4. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    Science.gov (United States)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  5. [Clinical use of recombinant human thrombopoietin. Status and perspectives].

    Science.gov (United States)

    Hansen, P B; Hasselbalch, H C

    2001-05-07

    Thrombopoietin (TPO) is primarily produced by hepatocytes and regulates the production and differentiation of megakaryocytes and platelets in the bone marrow. The endogenous TPO level is increased when the megakaryocyte count is low, and high in aplastic anaemia and after myeloablative chemotherapy. TPO is cloned and manufactured by a recombinant technique for clinical use. Treatment with recombinant human TPO (rhTPO) after intensive chemotherapy may reduce the need for platelet transfusions. Administration of granulocyte colony-stimulating factor in combination with rhTPO has enhanced the mobilisation and harvest product of haematopoietic stem cells. Whether rhTPO is effective in the treatment of the myelodysplastic syndrome, aplastic anaemia, and other conditions with bone marrow insufficiency (including AIDS) is not yet known. In liver cirrhosis, the endogenous TPO level rapidly increases after liver transplantation. Accordingly, substitution of rhTPO may be indicated in advanced liver failure complicated by thrombocytopenia and bleeding.

  6. Multinodular goiter treatment with radioiodine aided by recombinant human TSH in different doses: a randomized, double-blind, placebo-controlled study;Administracao previa do TSH humano recombinante, em diferentes doses, no tratamento do bocio multinodular com iodo radioativo: um estudo randomizado, duplo cego, controlado com placebo

    Energy Technology Data Exchange (ETDEWEB)

    Albino, Claudio Cordeiro

    2009-07-01

    Background: There is not an optimal treatment for multinodular goiter (MNG). Surgery is the main therapeutic option because it decreases thyroid volume, reduces compression symptoms and provide histological diagnosis. Radioiodine ({sup 131}I) is an efficient therapeutic option for the treatment of MNG mainly when surgery is not indicated or when the patient refused it. However, high activities of {sup 131}I are frequently required for clinically significant results. This procedure increases the body radiation exposure and the hospitalization costs. Recombinant human TSH (rh TSH) allows a reduction in the administered activity of {sup 131}I with effective thyroid volume (TV) reduction. However, this combination therapeutic can increase collateral effects. Objective: To evaluate the efficacy and safety of low and intermediate doses of rh TSH compared to placebo, associated with a fixed activity of {sup 131}I in MNG treatment. Patients and Methods: Thirty patients with MNG received 0.1 mg of rh TSH (group I, n=10), 0.01 mg of rh TSH (group II, n=10), or placebo (control group, n=10). After 24 hours, 30 mCi of {sup 131}I was given to all patients. Radioactive iodine uptake (RAIU) was determined before and 24 hours after rh TSH. Before and 2, 7, 180 and 360 days after the TV was measured by magnetic resonance image (MRI). The smallest cross-sectional area of tracheal lumen (Scat) was also measured with MRI before, 2 and 7 days after treatment. Antithyroid antibodies, TSH, T3 and free T4 were assessed regularly. Results: After 6 months, the decrease in TV was more significant in groups I (30.3 +- 16.5%) and II (22.6 +- 14.5%), than in control group (5.0 +- 14.6%; p=0.01). After 12 months, TV decreased more in group I (39.2 +- 16.9%) and group II (38.8 +- 24.4%) than in group III (23.4 +- 23.59%) but it was not statistically significant (p=0.205). During the first 30 days,total T3 and free T4 increased, without reaching thyrotoxic levels and TSH decreased. After 12 months

  7. Biotinylated recombinant human erythropoietins: Bioactivity and utility as receptor ligand

    Energy Technology Data Exchange (ETDEWEB)

    Wojchowski, D.M.; Caslake, L. (Pennsylvania State Univ., University Park (USA))

    1989-08-15

    Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.

  8. Broad-scale recombination patterns underlying proper disjunction in humans.

    Directory of Open Access Journals (Sweden)

    Adi Fledel-Alon

    2009-09-01

    Full Text Available Although recombination is essential to the successful completion of human meiosis, it remains unclear how tightly the process is regulated and over what scale. To assess the nature and stringency of constraints on human recombination, we examined crossover patterns in transmissions to viable, non-trisomic offspring, using dense genotyping data collected in a large set of pedigrees. Our analysis supports a requirement for one chiasma per chromosome rather than per arm to ensure proper disjunction, with additional chiasmata occurring in proportion to physical length. The requirement is not absolute, however, as chromosome 21 seems to be frequently transmitted properly in the absence of a chiasma in females, a finding that raises the possibility of a back-up mechanism aiding in its correct segregation. We also found a set of double crossovers in surprisingly close proximity, as expected from a second pathway that is not subject to crossover interference. These findings point to multiple mechanisms that shape the distribution of crossovers, influencing proper disjunction in humans.

  9. Vancouver Experience of Recombinant Human Platelet-Derived Growth Factor.

    Science.gov (United States)

    Younger, Alistair; Penner, Murray; Montijo, Harvey E

    2016-12-01

    Joint arthrodesis utilizing autogenous bone graft remains the gold standard of treatment in fusion procedures of the foot and ankle. Graft harvest, however, has been associated with increased morbidity to patients as well as increased costs. With this in mind, multiple clinical studies have evaluated the efficacy of recombinant human platelet-derived growth factor (rh-PDGF-BB) with beta-tricalcium phosphate (B-TCP) to augment in foot and ankle arthrodesis with favorable results. These factors have led to the increased use of rh-PDGF-BB with B-TCP in Vancouver with good clinical results. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Recombinant Human Erythropoietin for Treating Treatment-Resistant Depression

    DEFF Research Database (Denmark)

    Miskowiak, Kamilla W; Vinberg, Maj; Christensen, Ellen M;

    2014-01-01

    Pharmacological treatments for depression have insufficient efficacy in 30-40% of patients and fail to reverse cognitive deficits. Erythropoietin (EPO) has neurotrophic actions and aids neurocognitive function. The aim of this exploratory study was to determine whether recombinant human EPO...... improves mood and memory in treatment-resistant depression. Forty treatment-resistant depressed unipolar patients with Hamilton Depression Rating Scale-17 (HDRS-17) score ≥ 17 were randomized to eight weekly EPO (Eprex; 40,000 IU) or saline infusions in a double-blind, placebo-controlled, parallel...

  11. Mineralization of Hydroxyapatite Regulated by Recombinant Human-like Collagen

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We reported recombinant human-like type I collagen inducing growth of hydroxyapatite crystals in vitro in the form of self-assembly of nano-fibrils of mineralized collagen resembling extracellular matrix, which obey the same rules, but is superior to the collagen derived from animal tissues because the latter may carry diseases of animals and cause immunological reactions. The mineralized collagen fibrils aligned parallel to each other to form mineralized collagen fibers. Hydroxyapatite nanocrystals grew on the surface of these collagen fibrils with the c-axis of nanocrystals of HA orienting along the longitudinal axis of the fibrils.

  12. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    OpenAIRE

    Meng-Hwan Lee; Yin-Shen Lin; Ching-Fu Tu; Chon-Ho Yen

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitat...

  13. Effect of recombinant human thrombopoietin on exsanguine thrombocytopenia mice

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM:To investigate effect of recombinant human thrombopoietin on exsanguine thrombocytopenia mice. METHODS:Normal peripheral platelet counts were performed on sample obtained from the tail vein of purebred Babl/c mice including experimental and control groups before experimentation. rhTPO was injected into the mice by intraperitoneal injection once a day for 7 days. On the seventh and the fourteenth day, the mice were phlebotomized from the supra-obitalis vein in order to make exsanguine thrombocytopenia animal model. At the same time, we observed the biological activity of recombinant human thrombopoietin in vivo and the mice's death rate. RESULTS: On the seventh day and the fourteenth day, platelet counts of mice treated by rhTPO were higher than those by PBS (P<0.05). Moreover the platelet counts of mice in experimental group of rhTPO showed increasing tendency following experimental days. In addition, death happened in two groups after those mice were phlebotomized from the supra-obitalis vein, but the death rate in negative control group was evidently higher than that in experimental group (P<0.05). CONCLUSION:rhTPO had obvious biological activity in increasing platelet production, which resulted in the drop in thrombocytopenia mice's death rate.

  14. Prevalence of lipodystrophy associated with human recombinant insulin

    Directory of Open Access Journals (Sweden)

    S. Akbarzadeh

    2008-01-01

    Full Text Available AbstractBackground and Purpose: Lipodystrophy is potentially a clinical adverse effect, associated with insulin therapy and is believed that usage of human recombinant insulin’s is associated with decreasing prevalence of Lipodystrophy. The objective of this study was to determine the prevalence of insulin induced Lipodystrophy, among diabetic out-patients referred to Imam Khomeini Hospital, in Sari during 2007.Materials and Methods: In this cross sectional descriptive study, 220 diabetic patients referred to the Diabetes Center at Imam Khomeini Hospital, in Sari, who under treatment by insulin at least three months prior to referral was evaluated.First, the demographic and clinical characteristics of the patients were recorded in a questionnaire; then all patients were examined clinically to evaluate lipodystrophy. In all subjects, glycated hemoglobin (HbA1C was measured to assess the range of blood glucose level control. Recorded data were analyzed by statistical methods, such as descriptive T-test and X².Results: Of 220 diabetic patients studied, thirty-five (15.9% showed clinical evidences of insulin induced Lipodystrophy; 32 out of 35 cases of Lipodystrophic patients (14.5% had Lipohypertrophy, while 3 cases (1.4% had Lipoatrophy.The factors included Age, Sex, Education, BMI (Body mass index, type of Diabetes, The duration of insulin consumption and injection site had statistically significant effects on development of insulin induced Lipodystrophy (P<0.05.Conclusion: The results of this study demonstrated that despite using human recombinant insulin’s, the prevalence of insulin induced lipodystrophy, especially Lipohypertrophy, has remained high up to present. Therefore, regular examination of patients for this side effect is necessary, especially in subjects without good control of blood glucose level.Prevalence of lipodystrophy associated with human recombinant insulinZ. Kashi, M.D. + Z. Hajheydari, M.D.* O. Akha, M.D. * S

  15. Properties of purified recombinant human polyamine oxidase, PAOh1/SMO.

    Science.gov (United States)

    Wang, Yanlin; Murray-Stewart, Tracy; Devereux, Wendy; Hacker, Amy; Frydman, Benjamin; Woster, Patrick M; Casero, Robert A

    2003-05-16

    The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.

  16. Effects of recombinant humant erythropoietin in normal humans

    DEFF Research Database (Denmark)

    Lundby, Carsten; Olsen, Niels Vidiendal

    2011-01-01

    , and although it has been speculated that non-erythropoietic effects of EPO (angiogenesis, shift in muscle fibre types, cognitive effects) may be responsible for the increase in exercise performance, this has not been confirmed. EPO induced haemodynamic effects call for careful monitoring during......This review describes some of the physiological effects of recombinant human erythropoietin (EPO) in healthy humans. At the blood level EPO increases the arterial O2 content not only by increasing red blood cell volume, but also by an equally important decrease in plasma volume. Well before that...... result in suppression of endogenous EPO production through a decrease in intrarenal oxygen consumption. EPO elevates the arterial blood pressure even in healthy subjects. The receptor for EPO is present in many tissues. However, the functional effects of EPO in the skeletal muscle seem limited...

  17. Retrieval of human DNA from rodent-human genomic libraries by a recombination process.

    Science.gov (United States)

    Neve, R L; Bruns, G A; Dryja, T P; Kurnit, D M

    1983-09-01

    Human Alu repeat ("BLUR") sequences have been cloned into the mini-plasmid vector piVX. The resulting piBLUR clones have been used to rescue selectively, by recombination, bacteriophage carrying human DNA sequences from genomic libraries constructed using DNA from rodent-human somatic cell hybrids. piBLUR clones are able to retrieve human clones from such libraries because at least one Alu family repeat is present on most 15 to 20 kb fragments of human DNA and because of the relative species-specificity of the sequences comprising the Alu family. The rapid, selective plaque purification achieved results in the construction of a collection of recombinant phage carrying diverse human DNA inserts from a specific subset of the human karyotype. Subfragments of two recombinants rescued from a mouse-human somatic cell hybrid containing human chromosomes X, 10, 13, and 22 were mapped to human chromosomes X and 13, respectively, demonstrating the utility of this protocol for the isolation of human chromosome-specific DNA sequences from appropriate somatic cell hybrids.

  18. 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

  19. Expression and purification of recombinant human hemangiopoietin in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Ren Qian; Ma Fengxia; Chen Zhong; Lu Shihong; Han Zhibo; Liu Yongiun; Xu Bin; Zhang Xiangyu; Han Zhongchao

    2008-01-01

    Objective: To express the soluble recombinant hemangiopoietin protein in E. coli BL21(DE3). Methods: Using human fetal live cDNA as a template, a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99, pQE60 and pET32c to construct different recombinant prokaryotic expression systems. After selecting,the soluble rhHAPO fusion protein was expressed stably in E. coli BL21 (DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid (NTA) affinity chromatography. After cleavage with enterokinase, the rhHAPO protein was applied to Fast Flow SP sepharose column. Results: The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells. Conclusion: We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.

  20. Recombinant Human Prolactin Protects against Irradiation Induced Myelosuppression

    Institute of Scientific and Technical Information of China (English)

    Weici Zhang; Rui Sun; Jianhua Zhang; Jian Zhang; Zhigang Tian

    2005-01-01

    Prolactin is a multifunctional hormone that exerts many separate functions and acts as an important connection between the endocrine and immune systems. There are increasing researches implicating the role of prolactin in hematopoiesis. Enhanced erythropoiesis in pregnant women and direct erythropoietic effects in vitro of plasma either from pregnant or lactating mice have been reported. Furthermore, regression of erythroblastic leukemia has been observed in a significant number of rats after hypophysectomy. In this study, the effects of recombinant human prolactin (rhPRL) on hematopoiesis were assessed in irradiated mice. Mice were treated with rhPRL for five consecutive days after exposure to a lethal dose or a sub-dose irradiation. Prolonged survival rate and increased erythropoiesis were observed in the irradiation-induced myelosuppressive mice. It was concluded that rhPRL might act on erythropoiesis and could be a potential candidate for the treatment of irradiation-induced myelosuppresion in clinic. Cellular & Molecular Immunology.

  1. Recombinant human-like collagen directed growth of hydroxyapatite nanocrystals

    Science.gov (United States)

    Zhai, Y.; Cui, F. Z.

    2006-05-01

    Bones are biocomposites with hierarchical structure that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Type I collagen proteins, acidic extracellular matrix proteins, play a critical role in mineral formation and many researches on artificial bones have been made inspired by nature using type I collagen derived from animal tissues. Here we report that recombinant human-like type I collagen, an acidic protein, can direct growth of hydroxyapatite (HA) nanocrystals in vitro in the form of self-assembly of nano-fibrils of mineralized collagen resembling extracellular matrix. The mineralized collagen fibrils aligned parallel to each other to form mineralized collagen fibers. HA nanocrystals grew on the surface of these collagen fibrils with the c-axis of nanocrystals of HA orienting along the longitudinal axis of the fibrils. These artificial analogs of bone have a potential clinical application in bone repair.

  2. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  3. Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

    OpenAIRE

    Ser Huy Teh; Mun Yik Fong; Zulqarnain Mohamed

    2011-01-01

    The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The...

  4. 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...

  5. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

    Institute of Scientific and Technical Information of China (English)

    ZOU Hong-yun; MA Li; MENG Min-jie; YAO Xin-sheng; LIN Ying; WU Zhen-qiang; HE Xiao-wei; WANG Ju-fang; WANG Xiao-ning

    2007-01-01

    Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor (TCR) gene recombination.Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVβ chain was examined by the TCR GeneScan technique.Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ25' and Dβ 23' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation.Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire. However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  6. Recombinant production of human interleukin 6 in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Henrik Nausch

    Full Text Available In this study, we compared basic expression approaches for the efficient expression of bioactive recombinant human interleukin-6 (IL6, as an example for a difficult-to-express protein. We tested these approaches in a laboratory scale in order to pioneer the commercial production of this protein in Escherichia coli (E. coli. Among the various strategies, which were tested under Research and Development (R&D conditions, aggregation-prone IL6 was solubilized most effectively by co-expressing cytoplasmic chaperones. Expression of a Glutathion-S-Transferase (GST fusion protein was not efficient to increase IL6 solubility. Alteration of the cultivation temperature significantly increased the solubility in both cases, whereas reduced concentrations of IPTG to induce expression of the T7lac-promotor only had a positive effect on chaperone-assisted expression. The biological activity was comparable to that of commercial IL6. Targeting the expressed protein to an oxidizing environment was not effective in the generation of soluble IL6. Taken together, the presence of chaperones and a lowered cultivation temperature seem effective to isolate large quantities of soluble IL6. This approach led to in vivo soluble, functional protein fractions and reduces purification and refolding requirements caused by downstream purification procedures. The final yield of soluble recombinant protein averaged approximately 2.6 mg IL6/liter of cell culture. These findings might be beneficial for the development of the large-scale production of IL6 under the conditions of current good manufacturing practice (cGMP.

  7. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line.

    Science.gov (United States)

    Zou, Hong-yun; Ma, Li; Meng, Min-jie; Yao, Xin-sheng; Lin, Ying; Wu, Zhen-qiang; He, Xiao-wei; Wang, Ju-fang; Wang, Xiao-ning

    2007-03-05

    Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether thebreceptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkathuman T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of Tcell receptor (TCR) gene recombination. TCR Dbeta-Jbeta signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVbeta chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVbeta chain was examined by the TCR GeneScan technique. RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dbeta2-Jbeta2 signal joints and ds RSS breaks associated with the Dbeta2 5' and Dbeta 2 3' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVbeta chain did not change during cell proliferation. RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  8. Plant-derived recombinant human serum transferrin demonstrates multiple functions.

    Science.gov (United States)

    Brandsma, Martin E; Diao, Hong; Wang, Xiaofeng; Kohalmi, Susanne E; Jevnikar, Anthony M; Ma, Shengwu

    2010-05-01

    Human serum transferrin (hTf) is the major iron-binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high-quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 microg/g fresh leaf weight). Furthermore, plant-derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum-free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell-specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes.To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon-like peptide 1 (GLP-1) or its derivative in plants. Here, we show that plant-derived hTf-GLP-1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro.

  9. Similarity in recombination rate estimates highly correlates with genetic differentiation in humans.

    Directory of Open Access Journals (Sweden)

    Hafid Laayouni

    Full Text Available Recombination varies greatly among species, as illustrated by the poor conservation of the recombination landscape between humans and chimpanzees. Thus, shorter evolutionary time frames are needed to understand the evolution of recombination. Here, we analyze its recent evolution in humans. We calculated the recombination rates between adjacent pairs of 636,933 common single-nucleotide polymorphism loci in 28 worldwide human populations and analyzed them in relation to genetic distances between populations. We found a strong and highly significant correlation between similarity in the recombination rates corrected for effective population size and genetic differentiation between populations. This correlation is observed at the genome-wide level, but also for each chromosome and when genetic distances and recombination similarities are calculated independently from different parts of the genome. Moreover, and more relevant, this relationship is robustly maintained when considering presence/absence of recombination hotspots. Simulations show that this correlation cannot be explained by biases in the inference of recombination rates caused by haplotype sharing among similar populations. This result indicates a rapid pace of evolution of recombination, within the time span of differentiation of modern humans.

  10. 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.

  11. 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.

  12. Human recombinant RNASET2: A potential anti-cancer drug

    Science.gov (United States)

    Roiz, Levava; Smirnoff, Patricia; Lewin, Iris; Shoseyov, Oded; Schwartz, Betty

    2016-01-01

    The roles of cell motility and angiogenetic processes in metastatic spread and tumor aggressiveness are well established and must be simultaneously targeted to maximize antitumor drug potency. This work evaluated the antitumorigenic capacities of human recombinant RNASET2 (hrRNASET2), a homologue of the Aspergillus niger T2RNase ACTIBIND, which has been shown to display both antitumorigenic and antiangiogenic activities. hrRNASET2 disrupted intracellular actin filament and actin-rich extracellular extrusion organization in both CT29 colon cancer and A375SM melanoma cells and induced a significant dose-dependent inhibition of A375SM cell migration. hrRNASET2 also induced full arrest of angiogenin-induced tube formation and brought to a three-fold lower relative HT29 colorectal and A375SM melanoma tumor volume, when compared to Avastin-treated animals. In parallel, mean blood vessel counts were 36.9% lower in hrRNASET2-vs. Avastin-treated mice and survival rates of hrRNASET2-treated mice were 50% at 73 days post-treatment, while the median survival time for untreated animals was 22 days. Moreover, a 60-day hrRNASET2 treatment period reduced mean A375SM lung metastasis foci counts by three-fold when compared to untreated animals. Taken together, the combined antiangiogenic and antitumorigenic capacities of hrRNASET2, seemingly arising from its direct interaction with intercellular and extracellular matrices, render it an attractive anticancer therapy candidate. PMID:27014725

  13. A recombinant human enzyme for enhanced interstitial transport of therapeutics.

    Science.gov (United States)

    Bookbinder, L H; Hofer, A; Haller, M F; Zepeda, M L; Keller, G-A; Lim, J E; Edgington, T S; Shepard, H M; Patton, J S; Frost, G I

    2006-08-28

    Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

  14. Recombinant human factor IX produced from transgenic porcine milk.

    Science.gov (United States)

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

  15. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    Directory of Open Access Journals (Sweden)

    Meng-Hwan Lee

    2014-01-01

    Full Text Available Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa. The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX. The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

  16. Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

    Institute of Scientific and Technical Information of China (English)

    JIE-BO MI; JIN YAN; XIAO-JIE DING; ZHEN-QUAN GUO; MEI-PING ZHAO; WEN-BAO CHANG

    2007-01-01

    Objective To produce specific monoclonal antibody(mAb)against recombinant human erythropoietin(rHuEPO)for development of higmy efficient methods for erythropoietin detection in biological fluids.Methods rHuEPO was covalently coupled with bovine serum albumin(BSA)and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology.The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA),SDS-PAGE and Western blot.Results The isotype of F3-mAb Was found to be IgM with an affinity constant of 2.1x108 L/mol.The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

  17. Human recombinant RNASET2: A potential anti-cancer drug.

    Science.gov (United States)

    Roiz, Levava; Smirnoff, Patricia; Lewin, Iris; Shoseyov, Oded; Schwartz, Betty

    2016-01-01

    The roles of cell motility and angiogenetic processes in metastatic spread and tumor aggressiveness are well established and must be simultaneously targeted to maximize antitumor drug potency. This work evaluated the antitumorigenic capacities of human recombinant RNASET2 (hrRNASET2), a homologue of the Aspergillus niger T2RNase ACTIBIND, which has been shown to display both antitumorigenic and antiangiogenic activities. hrRNASET2 disrupted intracellular actin filament and actin-rich extracellular extrusion organization in both CT29 colon cancer and A375SM melanoma cells and induced a significant dose-dependent inhibition of A375SM cell migration. hrRNASET2 also induced full arrest of angiogenin-induced tube formation and brought to a three-fold lower relative HT29 colorectal and A375SM melanoma tumor volume, when compared to Avastin-treated animals. In parallel, mean blood vessel counts were 36.9% lower in hrRNASET2-vs. Avastin-treated mice and survival rates of hrRNASET2-treated mice were 50% at 73 days post-treatment, while the median survival time for untreated animals was 22 days. Moreover, a 60-day hrRNASET2 treatment period reduced mean A375SM lung metastasis foci counts by three-fold when compared to untreated animals. Taken together, the combined antiangiogenic and antitumorigenic capacities of hrRNASET2, seemingly arising from its direct interaction with intercellular and extracellular matrices, render it an attractive anticancer therapy candidate.

  18. Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives.

    Science.gov (United States)

    Rossi, Elena J; Sim, Lyann; Kuntz, Douglas A; Hahn, Dagmar; Johnston, Blair D; Ghavami, Ahmad; Szczepina, Monica G; Kumar, Nag S; Sterchi, Erwin E; Nichols, Buford L; Pinto, B M; Rose, David R

    2006-06-01

    Inhibitors targeting pancreatic alpha-amylase and intestinal alpha-glucosidases delay glucose production following digestion and are currently used in the treatment of Type II diabetes. Maltase-glucoamylase (MGA), a family 31 glycoside hydrolase, is an alpha-glucosidase anchored in the membrane of small intestinal epithelial cells responsible for the final step of mammalian starch digestion leading to the release of glucose. This paper reports the production and purification of active human recombinant MGA amino terminal catalytic domain (MGAnt) from two different eukaryotic cell culture systems. MGAnt overexpressed in Drosophila cells was of quality and quantity suitable for kinetic and inhibition studies as well as future structural studies. Inhibition of MGAnt was tested with a group of prospective alpha-glucosidase inhibitors modeled after salacinol, a naturally occurring alpha-glucosidase inhibitor, and acarbose, a currently prescribed antidiabetic agent. Four synthetic inhibitors that bind and inhibit MGAnt activity better than acarbose, and at comparable levels to salacinol, were found. The inhibitors are derivatives of salacinol that contain either a selenium atom in place of sulfur in the five-membered ring, or a longer polyhydroxylated, sulfated chain than salacinol. Six-membered ring derivatives of salacinol and compounds modeled after miglitol were much less effective as MGAnt inhibitors. These results provide information on the inhibitory profile of MGAnt that will guide the development of new compounds having antidiabetic activity.

  19. Recombinant human erythropoietin for repair of white matter damage

    Institute of Scientific and Technical Information of China (English)

    Wei Zhou; Xiao Rong; Li Tao; Weineng Lu

    2011-01-01

    Erythropoietin has been shown to exhibit neuroprotective effects in animal models. A neonatal rat model of hypoxic-ischemic white matter damage was established via bilateral carotid artery ligation in 4-day-old Sprague-Dawley rats. The rats were subsequently treated with recombinant human erythropoietin to observe pathological changes in the brain and long-term neurobehavioral functions before and after intervention. Results showed that the number of myelin basic protein-positive cells, which reflected myelin/oligodendrocyte damage, significantly increased, although the number of amyloid precursor protein-positive cells, which reflected axonal injury, significantly decreased in periventricular white matter at 72 hours and 7 days following erythropoietin intervention. The number of glial fibrillary acidic protein-positive cells, indicating astrocytic damage, significantly decreased in periventricular white matter of erythropoietin-treated rats at 48 hours, 72 hours, 7 days, and 26 days. Following erythropoietin intervention in the 30-day-old rats, head-turning time in the slope test was shortened and open-field test scores increased. These results suggested that erythropoietin promoted repair of white matter damage, as well as improved neurobehavioral functions in a rat model of hypoxic-ischemic injury.

  20. The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

    OpenAIRE

    2009-01-01

    Summary: The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination—a copy choice mechanism involving the migration of reverse transcr...

  1. Expression of adrenomedullin in rats after spinal cord injury and intervention effect of recombinant human erythropoietin

    Science.gov (United States)

    Zhao, Liang; Jing, Yu; Qu, Lin; Meng, Xiangwei; Cao, Yang; Tan, Huibing

    2016-01-01

    The expression of adrenomedullin (ADM) in injured tissue of rat spinal cord was observed and the effect of recombinant human erythropoietin was analyzed. A total of 45 Sprague-Dawley rats were selected and divided into 3 equal groups including, a sham-operation group in which rats received an excision of vertebral plate; a spinal cord injury model group and a recombinant human erythropoietin group in which rats with spinal cord injury received a caudal vein injection of 300 units recombinant human erythropoietin after injury. Hematoxylin and eosin staining was performed to observe the spinal cord injury conditions. Immunohistochemical staining was performed to observe the expression of ADM. Pathologic changes in the group of recombinant human erythropoietin at various times were significantly less severe than those in the group of spinal cord injury model. The expression of ADM was increased particularly in the group of recombinant human erythropoietin (P<0.01). The improved Tarlov scores of the group of spinal cord injury model and the group of recombinant human erythropoietin were lower than those of the sham-operation group at 3, 6 and 9 days (P<0.01). Thus, the recombinant human erythropoietin is capable of alleviating the secondary injury of spinal cord. One of the mechanisms may be achieved by promoting the increase of ADM expression. PMID:28101163

  2. Human recombinant erythropoietin in the prevention and treatment of anemia of prematurity.

    Science.gov (United States)

    Ohls, Robin K

    2002-01-01

    Human recombinant erythropoietin has been studied extensively as treatment for a variety of anemias. Since in vitro studies showed the primary etiology of the anemia of prematurity to be insufficient serum erythropoietin concentrations, clinical trials have evaluated the administration of human recombinant erythropoietin to preterm infants to treat this indication. These studies were followed by pharmacokinetic determinations in animal models and preterm infants, which revealed that preterm infants required greater doses of human recombinant erythropoietin because of a more rapid clearance and greater volume of distribution. Recent studies have focused on the administration of human recombinant erythropoietin in the first weeks of life to alleviate the anemia caused by excessive phlebotomy losses, and to prevent the anemia of prematurity. In addition, human recombinant erythropoietin has been tried clinically in a variety of neonatal populations in an attempt to decrease or eliminate transfusions. Although much information has been accumulated about the clinical use of human recombinant erythropoietin in preterm infants over the last 15 years, many questions remain unanswered. The evolution of clinical practice in the care of extremely low birthweight infants continues to affect the number of transfusions. It is likely that human recombinant erythropoietin administration in combination with instituting rigorous transfusion guidelines and decreasing phlebotomy losses will have the greatest impact in decreasing transfusion requirements in all preterm and term neonates, regardless of the etiology of their anemia.

  3. Involvement of BDNF and NGF in the mechanism of neuroprotective effect of human recombinant erythropoietin nanoforms.

    Science.gov (United States)

    Solev, I N; Balabanyan, V Yu; Volchek, I A; Elizarova, O S; Litvinova, S A; Garibova, T L; Voronina, T A

    2013-06-01

    Human recombinant erythropoietin adsorbed on poly(butyl)cyanoacrylate nanoparticles and administered intraperitoneally in a dose of 0.05 mg/kg exhibited a neuroprotective effect in experimental intracerebral posttraumatic hematomas (hemorrhagic stroke) and reduced animal mortality. Human recombinant erythropoietin, native and adsorbed on lactic and glycolic acid copolymer-based nanoparticles, exhibited no antistroke effect on this model. Analysis of reverse transcription PCR products showed that human recombinant erythropoietin adsorbed on poly(butyl)cyanoacrylate nanoparticles more than 2-fold increased the expression of BDNF and NGF neurotrophins in the rat brain frontal cortex and hippocampus.

  4. Comparative pharmacology of a new recombinant FSH expressed by a human cell Line

    DEFF Research Database (Denmark)

    Koechling, Wolfgang; Plaksin, Daniel; Croston, Glenn

    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...

  5. Analysis of carbohydrate residues on recombinant human thyrotropin receptor.

    Science.gov (United States)

    Oda, Y; Sanders, J; Roberts, S; Maruyama, M; Kiddie, A; Furmaniak, J; Smith, B R

    1999-06-01

    An investigation of the sugar groups on recombinant human TSH receptors (TSHR) expressed in CHO-K1 cells and solubilized with detergents is described. Western blotting studies with TSHR monoclonal antibodies showed that the receptor was present principally as two bands with approximate molecular masses of 120 and 100 kDa. Further blotting studies using lectins and/or involving treatment with different glycosidases indicated that the 100-kDa band contained about 16 kDa of high mannose-type sugars, and the 120-kDa band contained about 33 kDa of complex-type sugars. It was possible to separate the 120- and 100-kDa components of the TSHRs by lectin affinity chromatography. In particular, Galanthus nivalis lectin, which binds high mannose-type sugars, bound the 100-kDa band, but not the 120-kDa band, whereas Datura stramonium lectin, which binds complex-type sugars, bound the 120-kDa band, but not the 100-kDa band. 125I-Labeled TSH binding studies with the various lectin column fractions showed that TSH-binding activity was principally associated with the complex-type sugar containing the 120-kDa form of the receptor rather than the high mannose-containing 100-kDa form. During peptide chain glycosylation, high mannose-type sugar residues are attached first and then modified by the formation of complex type structures to form the mature glycoprotein. Our data suggest that in the case of the TSH receptor, this type of posttranslational processing has an important role in forming the TSH-binding site.

  6. [Metabolism, Distribution and Excretion of Recombinant Human Thrombopoietin in Mice

    Science.gov (United States)

    Liu, Xiu-Wen; Tang, Zhong-Ming; Song, Hai-Feng; Dou, Gui-Fang

    2001-12-01

    The metabolism, distribution and excretion profiles of recombinant human thrombopoietin (rhTPO) in mice were studied by means of (125)I-labeled rhTPO ((125)I-rhTPO) combined with size exclusive high performance liquid chromatography (SHPLC) or trichloroacetic acid (TCA) precipitation analysis. (125)I-rhTPO was prepared by iodogen method. Purification was performed on Sephacryl S-200 HR gel. Radioactive-purity of (125)I-rhTPO identified by SHPLC was (96.9 +/- 1.5)% (n = 3). The proliferation effect of TPO dependent cell line (TD-3) and the increase of peripheral platelet counts in mouse by (125)I-rhTPO demonstrated that (125)I-labeled protein maintained the biological activities of TPO both in vitro and in vivo. SHPLC analysis of serum and urine samples taken after sc 1 micro g/mouse (345 kBq/mouse) of (125)I-rhTPO revealed that there were two lower molecular weight (125)I-degradation metabolites ((125)I-MI and (125)I-MII) other than parent molecule. (125)I-MI was mainly found in urine, and (125)I-MII was detected both in serum and in urine. The maximal concentration of (125)I-rhTPO was reached at 2 hours after injection. The terminal half-life was 10.8 hours, which was much longer than those of other peptides. TCA precipitable radioactivity in tissue showed that the radioactivity in bone marrow was rather high. The highest level was found in urinary system. Levels in adrenals, lymph nodes, and fat were near to that in serum. Lowest was found in brain. The main excretion route was urinary system and (98 +/- 5.6)% of (125)I-rhTPO was excreted within 72 hours after dosing.

  7. Recombinant human erythropoietin improves neurological outcomes in very preterm infants

    Science.gov (United States)

    Song, Juan; Sun, Huiqing; Xu, Falin; Kang, Wenqing; Gao, Liang; Guo, Jiajia; Zhang, Yanhua; Xia, Lei; Wang, Xiaoyang

    2016-01-01

    Objective To evaluate the efficacy and safety of repeated low‐dose human recombinant erythropoietin (rhEPO) in the improvement of neurological outcomes in very preterm infants. Methods A total of 800 infants of ≤32‐week gestational age who had been in an intensive care unit within 72 hours after birth were included in the trial between January 2009 and June 2013. Preterm infants were randomly assigned to receive rhEPO (500IU/kg; n = 366) or placebo (n = 377) intravenously within 72 hours after birth and then once every other day for 2 weeks. The primary outcome was death or moderate to severe neurological disability assessed at 18 months of corrected age. Results Death and moderate/severe neurological disability occurred in 91 of 338 very preterm infants (26.9%) in the placebo group and in 43 of 330 very preterm infants (13.0%) in the rhEPO treatment group (relative risk [RR] = 0.40, 95% confidence interval [CI] = 0.27–0.59, p < 0.001) at 18 months of corrected age. The rate of moderate/severe neurological disability in the rhEPO group (22 of 309, 7.1%) was significantly lower compared to the placebo group (57 of 304, 18.8%; RR = 0.32, 95% CI = 0.19–0.55, p < 0.001), and no excess adverse events were observed. Interpretation Repeated low‐dose rhEPO treatment reduced the risk of long‐term neurological disability in very preterm infants with no obvious adverse effects. Ann Neurol 2016;80:24–34 PMID:27130143

  8. Effect of recombinant human endostatin on endometriosis in mice

    Institute of Scientific and Technical Information of China (English)

    JIANG Hong-qing; LI Ya-li; ZOU Jie

    2007-01-01

    Background Direct and indirect evidences have suggested that angiogenesis is a prerequisite for the development of endometriosis. Aiming at offering experimental evidences for anti-angiogenesis therapy, we transplanted the eutopic endometrium from patient with endometriosis into the severe combined immunodeficiency disease (SCID) mice, to evaluate the effect of the endostatin on the growth and angiogenesis of the established endometriosis lesions in SCID mice model.Methods Eutopic endometrium of women with endometriosis was transplanted into the SCID mice. The mice were randomized into treatment (n=10) and control groups (n=10). Two weeks after the implantation of endometrium fragment,whereas the control group received equivalent volume of PBS (200 μl/d). The volume of endometriotic lesions in SCID mice was measured every three days, and all the treatment lasted for 14 days. Immunohistochemistry was used to determine microvessel density (MVD) and the expression of VEGF. The results were analyzed by t test and X2 test to value the treating effect.Results Compared with the control group, growth of endometriosis lesion was reduced in the mice treated with YH-16.Statistically significant differences in the volume and weight of the ectopic lesions were observed between the treatment and the control groups (P<0.05). Microscopical examination showed that after being treated with YH-16, the volume of the endometrial tissues decreased, the glands depauperated, and the glandular epithelium partially degenerated.Necrotic debris was observed in the endometrial stroma. MVD and expression of VEGF in the treatment group were significantly lower than those in the control group (P<0.05).Conclusions Recombinant human endostatin affects the maintenance and growth of endometriotic tissues by inhibiting angiogenesis and reducing the expression of VEGF in ectopic lesion. The angiostatic agent may be promising as a therapy for endometriosis.

  9. Cardiovascular effects of recombinant human erythropoietin in predialysis patients.

    Science.gov (United States)

    Portolés, J; Torralbo, A; Martin, P; Rodrigo, J; Herrero, J A; Barrientos, A

    1997-04-01

    Treatment with recombinant human erythropoietin (rHuEPO) has solved the problem of anemia in patients on dialysis. However, its application to predialysis patients has raised some doubts about its effects on the progression of renal disease and on blood pressure (BP) and hemodynamic regulation. We have prospectively studied over at least 6 months a group of 11 predialysis patients receiving rHuEPO treatment (initial dose, 1,000 U subcutaneously three times a week). Clinical assessment and biochemical and hematologic measurements were made once every 2 weeks. Twenty-four-hour ambulatory BP monitoring, echocardiography, and determination of neurohumoral mediators of hemodynamics were performed once every 3 months. An adequate hematologic response was found (hemoglobin, 11.7 +/- 0.4 g/dL v 9 +/- 0.3 g/dL) without changes in the progression of renal disease. A decrease in cardiac output and an increase in total peripheral resistance was seen as anemia improved. A trend toward decreased left ventricular (LV) thickness and a significant decrease in LV mass index (from 178.2 +/- 20.6 g/m2 to 147.3 +/- 20.6 g/m2) were observed. Blood pressure control did not improve; moreover, in some patients an increase in systolic values was detected by ambulatory BP. Casual BP remained seemingly stable. Sequential determinations of neurohumoral mediators of hemodynamic substances (endothelin, renin, norepinephrine, epinephrine, dopamine) failed to explain these results. Ambulatory BP reveals a worse control in some patients who were previously hypertensive and confirms the utility of this technique in the assessment of patients under erythropoietin treatment. The trend toward LV hypertrophy regression without improved BP control confirms the role of anemia among the multiple factors leading to LV hypertrophy in end-stage renal disease (ESRD), and opens therapeutic possibilities. Better control of BP may avoid a potential offsetting of beneficial effects that correcting anemia would

  10. Protection against cisplatin-induced nephrotoxicity by recombinant human erythropoietin.

    Science.gov (United States)

    Yalcin, Suayib; Müftüoğlu, Sevda; Cetin, Eren; Sarer, Banu; Yildirim, Berna Akkuş; Zeybek, Dilara; Orhan, Bülent

    2003-01-01

    Cisplatin (CDDP) is a potent nephrotoxin, and nephrotoxicity is its most important dose-limiting toxicity. In this study, we aimed to investigate the role of recombinant human erythropoietin (rhEPO) in the protection of cisplatin-induced nephrotoxicity and compare its efficacy with the cell-protective agent amifostine. All experiments were conducted on female Wistar albino rats. Animals were randomly assigned to four groups, each including six rats. Group A received only CDDP, group B received CDDP plus rhEPO, group C received CDDP plus amifostine, and group D received only rhEPO. At the end of 7 wk, hemoglobin (Hgb), hematocrite (Htc), blood urea nitrogen (BUN), and creatinine (Cr) levels were determined and kidneys of the rats were removed. The weights of the kidneys were measured and sent for histopathological examination. Proximal tubules from four areas of the kidney (outer cortex, inner cortex, the medullary ray, and outer stripe of outer medulla [OSOM]) were evaluated. There were statistically significant differences among the groups in terms of tubular scores, including overall renal tubular score, cortex, inner cortex, OSOM, and medullary ray tubular scores, and Htc levels. Group A rats had the worse tubular scores in all categories when compared to group D rats. When the results of groups B and C were compared, there were no differences in terms of BUN, Cr levels, and tubular scores, but the Htc level was significantly higher in group B. Group B rats had better overall and OSOM tubular scores when compared to group A. Group C also had better overall and OSOM tubular scores compared to group A. The present study showed for the first time that rhEPO plays an important role in the prevention of cisplatin-induced nephrotoxicity and it is as effective as amifostine.

  11. Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene

    Science.gov (United States)

    Greenwald, David L.; Cashman, Siobhan M.

    2010-01-01

    Purpose. Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Methods. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Results. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. Conclusions. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination. PMID:20671268

  12. Recombinant human DNase in children with airway malacia and lower respiratory tract infection.

    NARCIS (Netherlands)

    Boogaard, R.; Jongste, J.C. de; Vaessen-Verberne, A.A.; Hop, W.C.J.; Merkus, P.J.F.M.

    2009-01-01

    BACKGROUND: Children with airway malacia often have protracted courses of airway infections, because dynamic airway collapse during coughing results in impaired mucociliary clearance. The aim of this study was to determine the effect of the mucolytic drug recombinant human deoxyribonuclease

  13. Factors that contribute to the immmunogenicity of therapeutic recombinant human proteins.

    Science.gov (United States)

    Mukovozov, Ilya; Sabljic, Thomas; Hortelano, Gonzalo; Ofosu, Frederick A

    2008-05-01

    Use of recombinant human proteins has revolutionized medicine by providing over 200 highly purified hormones and proteins that effectively treat many inherited and acquired peptide hormone and protein deficiencies. With the exception of therapeutic monoclonal antibodies, these biological medicines are synthesized by cultured cells using DNA sequences that would yield proteins with identical amino acid sequences as endogenous human proteins. Therefore, there was the broad expectation that recombinant human biological medicines would be non-immunogenic in patients capable of synthesizing even sub-optimal levels of these therapeutic proteins to which they are innately tolerant. However, the widespread clinical use of recombinant human proteins has demonstrated that nearly all of them are immunogenic. This observation suggests that factors additional to differences in amino acid sequences of endogenous and biotherapeutic proteins contribute to the immunogenicity of therapeutic proteins. The main aim of this review is to summarize some of the factors that are known to contribute to the immunogenicity of recombinant therapeutic proteins.

  14. Mecasermin (recombinant human insulin-like growth factor I).

    Science.gov (United States)

    Rosenbloom, Arlan L

    2009-01-01

    Growth hormone (GH) exercises its growth effects by stimulating insulin-like growth factor I (IGF-I) synthesis in the liver (endocrine IGF-I) and by inducing chondrocyte differentiation/replication and local production of IGF-I (paracrine/autocrine IGF-I). Injectable recombinant human (rh)IGF-I (mecasermin) has been available for nearly 20 years for treatment of the rare instances of GH insensitivity caused by GH receptor defects or GH-inhibiting antibodies. Full restoration of normal growth, as occurs with rhGH replacement of GH deficiency, is not seen, presumably because only the endocrine deficiency is addressed. RhIGF-I has also been effective as an insulin-sensitizing agent in severe insulin-resistant conditions. Although the insulin-sensitizing effect may benefit both type 1 and type 2 diabetes, there are no ongoing clinical trials because of concern about risk of retinopathy and other complications. Promotion of rhIGF-I for treatment of idiopathic short stature has been intensive, with neither data nor rationale suggesting that there might be a better response than has been documented with rhGH. Other applications that have either been considered or are undergoing clinical trial are based on the ubiquitous tissue-building properties of IGF-I and include chronic liver disease, cystic fibrosis, wound healing, AIDS muscle wasting, burns, osteoporosis, Crohn's disease, anorexia nervosa, Werner syndrome, X-linked severe combined immunodeficiency, Alzheimer's disease, muscular dystrophy, amyotrophic lateral sclerosis, hearing loss prevention, spinal cord injury, cardiovascular protection, and prevention of retinopathy of prematurity. The most frequent side effect is hypoglycemia, which is readily controlled by administration with meals. Other common adverse effects involve hyperplasia of lymphoid tissue, which may require tonsillectomy/adenoidectomy, accumulation of body fat, and coarsening of facies. The anti-apoptotic properties of IGF-I are implicated in cancer

  15. Factors influencing recombination frequency and distribution in a human meiotic crossover hotspot.

    Science.gov (United States)

    Jeffreys, Alec J; Neumann, Rita

    2005-08-01

    Little is known about the factors that influence the frequency and distribution of meiotic recombination events within human crossover hotspots. We now describe the detailed analysis of sperm recombination in the NID1 hotspot. Like the neighbouring MS32 hotspot, the NID1 hotspot is associated with a minisatellite, suggesting that hotspots predispose DNA to tandem repetition. Unlike MS32, crossover resolution breakpoints in NID1 avoid the minisatellite, producing a cold spot within the hotspot. This avoidance may be related to the palindromic nature of the minisatellite interfering with the generation and/or processing of recombination intermediates. The NID1 hotspot also contains a single nucleotide polymorphism (SNP) close to the centre, which appears to directly influence the frequency of crossover initiation. Quantitative gene conversion assays show that this SNP affects the frequency of gene conversion and crossover to a very similar extent, providing evidence that conversions and crossovers are triggered by the same recombination initiating events. The recombination-suppressing allele is over-transmitted to recombinant progeny, and provides the most dramatic example to date of recombination-mediated meiotic drive, of a magnitude sufficient to virtually guarantee that the recombination suppressor will eventually replace the more active allele in human populations.

  16. Human imprinted chromosomal regions are historical hot-spots of recombination.

    Directory of Open Access Journals (Sweden)

    Ionel Sandovici

    2006-07-01

    Full Text Available Human recombination rates vary along the chromosomes as well as between the two sexes. There is growing evidence that epigenetic factors may have an important influence on recombination rates, as well as on crossover position. Using both public database analysis and wet-bench approaches, we revisited the relationship between increased rates of meiotic recombination and genome imprinting. We constructed metric linkage disequilibrium (LD maps for all human chromosomal regions known to contain one or more imprinted genes. We show that imprinted regions contain significantly more LD units (LDU and have significantly more haplotype blocks of smaller sizes than flanking nonimprinted regions. There is also an excess of hot-spots of recombination at imprinted regions, and this is likely to do with the presence of imprinted genes, per se. These findings indicate that imprinted chromosomal regions are historical "hot-spots" of recombination. We also demonstrate, by direct segregation analysis at the 11p15.5 imprinted region, that there is remarkable agreement between sites of meiotic recombination and steps in LD maps. Although the increase in LDU/Megabase at imprinted regions is not associated with any significant enrichment for any particular sequence class, major sequence determinants of recombination rates seem to differ between imprinted and control regions. Interestingly, fine-mapping of recombination events within the most male meiosis-specific recombination hot-spot of Chromosome 11p15.5 indicates that many events may occur within or directly adjacent to regions that are differentially methylated in somatic cells. Taken together, these findings support the involvement of a combination of specific DNA sequences and epigenetic factors as major determinants of hot-spots of recombination at imprinted chromosomal regions.

  17. Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

    Science.gov (United States)

    Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

    2010-10-01

    Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.

  18. DNA induces conformational changes in a recombinant human minichromosome maintenance complex.

    Science.gov (United States)

    Hesketh, Emma L; Parker-Manuel, Richard P; Chaban, Yuriy; Satti, Rabab; Coverley, Dawn; Orlova, Elena V; Chong, James P J

    2015-03-20

    ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.

  19. International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

    Science.gov (United States)

    An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

  20. Translational mixed-effects PKPD modelling of recombinant human growth hormone - from hypophysectomized rat to patients

    DEFF Research Database (Denmark)

    Thorsted, A; Thygesen, P; Agersø, H;

    2016-01-01

    BACKGROUND AND PURPOSE: We aimed to develop a mechanistic mixed-effects pharmacokinetic (PK)-pharmacodynamic (PD) (PKPD) model for recombinant human growth hormone (rhGH) in hypophysectomized rats and to predict the human PKPD relationship. EXPERIMENTAL APPROACH: A non-linear mixed-effects model...

  1. 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

  2. Topological Data Analysis Generates High-Resolution, Genome-wide Maps of Human Recombination.

    Science.gov (United States)

    Camara, Pablo G; Rosenbloom, Daniel I S; Emmett, Kevin J; Levine, Arnold J; Rabadan, Raul

    2016-07-01

    Meiotic recombination is a fundamental evolutionary process driving diversity in eukaryotes. In mammals, recombination is known to occur preferentially at specific genomic regions. Using topological data analysis (TDA), a branch of applied topology that extracts global features from large data sets, we developed an efficient method for mapping recombination at fine scales. When compared to standard linkage-based methods, TDA can deal with a larger number of SNPs and genomes without incurring prohibitive computational costs. We applied TDA to 1,000 Genomes Project data and constructed high-resolution whole-genome recombination maps of seven human populations. Our analysis shows that recombination is generally under-represented within transcription start sites. However, the binding sites of specific transcription factors are enriched for sites of recombination. These include transcription factors that regulate the expression of meiosis- and gametogenesis-specific genes, cell cycle progression, and differentiation blockage. Additionally, our analysis identifies an enrichment for sites of recombination at repeat-derived loci matched by piwi-interacting RNAs.

  3. 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......, 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...... human growth hormone group increased after 3 (p growth factor-I during the treatment period was expressed as area under the curve (AUC). The AUCIGF-I was significantly larger...

  4. In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes.

    Science.gov (United States)

    Maul, Ronald; Warth, Benedikt; Schebb, Nils Helge; Krska, Rudolf; Koch, Matthias; Sulyok, Michael

    2015-06-01

    The mycotoxin deoxynivalenol (DON), formed by Fusarium species, is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon ingestion, the majority of the toxin is excreted by humans and animal species as glucuronide conjugate. First in vitro data indicated that DON phase II metabolism is strongly species dependent. However, kinetic data on the in vitro metabolism as well as investigations on the specific enzymes responsible for DON glucuronidation in human are lacking. In the present study, the DON metabolism was investigated using human microsomal fractions and uridine-diphosphoglucuronyltransferases (UGTs) as well as liver microsomes from five animal species. Only two of the twelve tested human recombinant UGTs led to the formation of DON glucuronides with a different regiospecificity. UGT2B4 predominantly catalyzed the formation of DON-15-O-glucuronide (DON-15GlcA), while for UGT2B7 the DON-3-O-glucuronide (DON-3GlcA) metabolite prevailed. For human UGTs, liver, and intestinal microsomes, the glucuronidation activities were low. The estimated apparent intrinsic clearance (Clapp,int) for all human UGT as well as tissue homogenates was microsomes, moderate Clapp,int between 1.5 and 10 mL/min mg protein were calculated for carp, trout, and porcine liver. An elevated glucuronidation activity was detected for rat and bovine liver microsomes leading to Clapp,int between 20 and 80 mL/min mg protein. The obtained in vitro data points out that none of the animal models is suitable for estimating the human DON metabolism with respect to the metabolite pattern and formation rate.

  5. Production of the Polyclonal Anti-human Metallothionein 2A Antibody with Recombinant Protein Technology

    Institute of Scientific and Technical Information of China (English)

    Faiz M.M.T.MARIKAR; Qi-Ming SUN; Zi-Chun HUA

    2006-01-01

    Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the eDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

  6. Immunogenicity analysis following human immunodeficiency virus recombinant DNA and recombinant vaccinia virus Tian Tan prime-boost immunization.

    Science.gov (United States)

    Liu, Cunxia; Du, Shouwen; Li, Chang; Wang, Yuhang; Wang, Maopeng; Li, Yi; Yin, Ronglan; Li, Xiao; Ren, Dayong; Qin, Yanqing; Ren, Jingqiang; Jin, Ningyi

    2013-06-01

    This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of recombinant DNA (pCCMp24) and recombinant attenuated vaccinia virus Tian Tan (rddVTT-CCMp24). Intramuscular immunization was performed on days 0 (prime) and 21 (boost). The immunogenicity of the vaccine schedules was determined by measuring human immunodeficiency virus (HIV)-specific binding antibody levels and cytokine (interleukin-2 and interleukin-4) concentrations in peripheral blood, analyzing lymphocyte proliferation capacity against HIV epitopes and CD4(+)/CD8(+) cell ratio, and monitoring interferon-gamma levels at different times post-immunization. The results showed that pCCMp24, rddVTT-CCMp24 and their prime-boost immunization induced humoral and cellular immune responses. The pCCMp24/rddVTT-CCMp24 immunization strategy increased CD8(+) T cells and induced more IFN-γ-secreting cells compared with single-shot rDNA. The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides. These results demonstrated that prime-boost immunization with rDNA and rddVTT-CCMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations, especially IFN-γ response, providing a basis for further studies.

  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. 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

  9. Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

    Institute of Scientific and Technical Information of China (English)

    CHEN Qingyong; WANG Chunyou; CHEN Daoda; CHEN Jianying; JIANG Chunfang; ZHENG Hai

    2006-01-01

    The recombinant defective adenovirus vector carrying human PTEN tumor suppres sor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector AdPTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×1010 pfu/mL, and about 70 % breast cancer cells were infected with Ad PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

  10. Crystallization And Preliminary Crystallographic Analysis of Recombinant Human Galectin-1

    Energy Technology Data Exchange (ETDEWEB)

    Scott, S.A.; Scott, K.; Blanchard, H.

    2009-06-04

    Human galectin-1 has been cloned, expressed in E. coli, purified and crystallized in the presence of both lactose (ligand) and {beta}-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.

  11. 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...... 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...

  12. Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Ser Huy Teh

    2011-01-01

    Full Text Available The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.

  13. 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

    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...... 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...... prevented Aquaporin1 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...

  14. Recombinant expression of human nerve growth factor beta in rabbit bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Fan, Bo-Sheng; Lou, Ji-Yu

    2010-12-01

    Nerve growth factor (NGF) is required for the differentiation and maintenance of sympathetic and sensory neurons. In the present study, the recombinant expression of human nerve growth factor beta (hNGF-β) gene in rabbit bone marrow mesenchymal stem cells (rMSCs) was undertaken. Recombinant vector containing hNGF-β was constructed and transferred into rMSCs, the expressions of the exogenous in rMSCs were determined by reverse transcriptase PCR (RT-PCR), ELISA and Western blot, whereas the biological activity of recombinant hNGF-β was confirmed using PC12 cells and cultures of dorsal root ganglion neurons from chicken embryos. The results showed that the hNGF-β gene expressed successfully in the rMSCs, a polypeptide with a molecular weight of 13.2 kDa was detected. The maximal expression level of recombinant hNGF-β in rMSCs reached 126.8012 pg/10(6) cells, the mean concentration was 96.4473 pg/10(6) cells. The recombinant hNGF-β in the rMSCs showed full biological activity when compared to commercial recombinant hNGF-β.

  15. Characterization of ATPase Activity of Recombinant Human Pif1

    Institute of Scientific and Technical Information of China (English)

    Yu HUANG; Deng-Hong ZHANG; Jin-Qiu ZHOU

    2006-01-01

    Saccharomyces cerevisiae Pif1p helicase is the founding member of the Pif1 subfamily that is conserved from yeast to human. The potential human homolog of the yeast PIF1 gene has been cloned from the cDNA library of the Hek293 cell line. Here, we described a purification procedure of glutathione Stransferase (GST)-fused N terminal truncated human Pif1 protein (hPif1△N) from yeast and characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and single-stranded DNA. Km for ATP for the ATPase activity is approximately 200 μM. As the ATPase activity is essential for hPif1's helicase activity, these results will facilitate the further investigation on hPif1.

  16. Robotics for recombinant DNA and human genetics research

    Energy Technology Data Exchange (ETDEWEB)

    Beugelsdijk, T.J.

    1990-01-01

    In October of 1989, molecular biologists throughout the world formally embarked on ultimately determining the set of genetic instructions for a human being. Called by some the Manhattan Project'' a molecular biology, pursuit of this goal is projected to require approximately 3000 man years of effort over a 15-year period. The Humane Genome Initiative is a worldwide research effort that has the goal of analyzing the structure of human deoxyribonucleic acid (DNA) and determining the location of all human genes. The Department of Energy (DOE) has designated three of its national laboratories as centers for the Human Genome Project. These are Los Alamos National Laboratory (LANL), Lawrence Livermore National Laboratory (LLNL), and Lawrence Berkeley Laboratory (LBL). These laboratories are currently working on different, but complementary technology development areas in support of the Human Genome Project. The robotics group at LANL is currently working at developing the technologies that address the problems associated with physical mapping. This article describes some of these problems and discusses some of the robotics approaches and engineering tolls applicable to their solution. 7 refs., 8 figs., 1 tab.

  17. Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells.

    Science.gov (United States)

    Hanada, Katsuhiro; Yamaoka, Yoshio

    2014-10-01

    Helicobacter pylori is a gram-negative pathogenic bacterium that colonises the human stomach. The chronic infection it causes results in peptic ulcers and gastric cancers. H. pylori can easily establish a chronic infection even if the immune system attacks this pathogen with oxidative stress agents and immunoglobulins. This is attributed to bacterial defence mechanisms against these stresses. As a defence mechanism against oxidative stresses, in bacterial genomes, homologous recombination can act as a repair pathway of DNA's double-strand breaks (DSBs). Moreover, homologous recombination is also involved in the antigenic variation in H. pylori. Gene conversion alters genomic structures of babA and babB (encoding outer membrane proteins), resulting in escape from immunoglobulin attacks. Thus, homologous recombination in bacteria plays an important role in the maintenance of a chronic infection. In addition, H. pylori infection causes DSBs in human cells. Homologous recombination is also involved in the repair of DSBs in human cells. In this review, we describe the roles of homologous recombination with an emphasis on the maintenance of a chronic infection.

  18. High-resolution recombination patterns in a region of human chromosome 21 measured by sperm typing.

    Directory of Open Access Journals (Sweden)

    Irene Tiemann-Boege

    2006-05-01

    Full Text Available For decades, classical crossover studies and linkage disequilibrium (LD analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in "hot spots." Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region.

  19. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    Science.gov (United States)

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  20. 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...

  1. 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 effe

  2. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    Energy Technology Data Exchange (ETDEWEB)

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M. (National Institutes of Health, Bethesda, MD (USA))

    1990-03-06

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by ({sup 3}H)azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the ({sup 3}H)azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.

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

    NARCIS (Netherlands)

    Halim, Liem Andhyk; Brinks, Vera; Jiskoot, Wim; Romeijn, Stefan; Praditpornsilpa, Kearkiat; Assawamakin, Anunchai; Schellekens, Huub

    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

  4. Strategy to tether organometallic ruthenium-arene anticancer compounds to recombinant human serum albumin.

    Science.gov (United States)

    Ang, Wee Han; Daldini, Elisa; Juillerat-Jeanneret, Lucienne; Dyson, Paul J

    2007-10-29

    In order to utilize macromolecules for drug targeting and delivery, a strategy to tether organometallic ruthenium-arene drugs to carrier protein molecules was developed. The approach involves the design of a drug fragment capable of conjugating to linker molecules on a modified carrier protein via hydrazone bond formation. The proof-of-concept using recombinant human serum albumin is described.

  5. Improvement of lymphocyte proliferation in human immunodeficiency virus infection after recombinant interleukin-2 treatment

    DEFF Research Database (Denmark)

    Afzelius, P; Nielsen, S D; Nielsen, Jens Ole

    1999-01-01

    In this study, the effect of recombinant interleukin-2 (rIL-2) on the function of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients was examined. Using polymerase chain reaction (PCR), an impaired ability of PBMC from 8 patients to respond upon mi...

  6. B-1 cells and naturally occuring antibodies: influencing the immunogenicity of recombinant human therapeutic proteins

    NARCIS (Netherlands)

    Sauerborn, M.S.; Schellekens, H.

    2009-01-01

    Recombinant human therapeutic proteins are increasingly being used to treat serious and life-threatening diseases like multiple sclerosis, diabetes mellitus, and cancer. An important side effect of these proteins is the development of antidrug antibodies, which can be neutralizing and thus interfere

  7. Pharmacoeconomic review of recombinant human DNase in the management of cystic fibrosis

    NARCIS (Netherlands)

    Zijlstra, Gerrit; Boersma, Cornelis; Frijlink, Henderik W.; Postma, Maarten J.

    2004-01-01

    For the treatment of patients with cystic fibrosis, recombinant human deoxyribonuclease I is widely used. Deoxyribonuclease I has a positive effect on lung function and the number of hospitalizations. Deoxyribonuclease I is currently administered by nebulization, which is an inefficient administrati

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

    NARCIS (Netherlands)

    Halim, Liem Andhyk; Brinks, Vera; Jiskoot, Wim; Romeijn, Stefan; Praditpornsilpa, Kearkiat; Assawamakin, Anunchai; Schellekens, Huub

    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

  9. Recombinant human interleukin 6 in metastatic renal cell cancer : A phase II trial

    NARCIS (Netherlands)

    Stouthard, JML; Goey, H; deVries, EGE; deMulder, PH; Groenewegen, A; Stoter, G; Sauerwein, HP; Bakker, PJM; Veenhof, CHN

    A phase II trial investigating the anti-tumour effects of recombinant human interleukin 6 (rhlL-6) in patients with metastatic renal cell cancer was carried out. RhIL-6 (150 mu g) was administered as a daily subcutaneous injection for 42 consecutive days on an outpatient basis. Forty-nine patients

  10. 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

  11. Prevention of murine experimental autoimmune orchitis by recombinant human interleukin-6

    DEFF Research Database (Denmark)

    Li, Lu; Itoh, Masahiro; Ablake, Maila;

    2002-01-01

    We studied the effect of exogenously administered recombinant human interleukin (IL)-6 on the development of experimental autoimmune orchitis (EAO) in C3H/Hej mice. IL-6 significantly reduced histological signs of EAO and appearance of delayed type hypersensitivity against the immunizing testicul...

  12. 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...

  13. 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

  14. Recombinant human interleukin 6 in metastatic renal cell cancer : A phase II trial

    NARCIS (Netherlands)

    Stouthard, JML; Goey, H; deVries, EGE; deMulder, PH; Groenewegen, A; Stoter, G; Sauerwein, HP; Bakker, PJM; Veenhof, CHN

    1996-01-01

    A phase II trial investigating the anti-tumour effects of recombinant human interleukin 6 (rhlL-6) in patients with metastatic renal cell cancer was carried out. RhIL-6 (150 mu g) was administered as a daily subcutaneous injection for 42 consecutive days on an outpatient basis. Forty-nine patients w

  15. Improvement of lymphocyte proliferation in human immunodeficiency virus infection after recombinant interleukin-2 treatment

    DEFF Research Database (Denmark)

    Afzelius, P; Nielsen, S D; Nielsen, Jens Ole

    1999-01-01

    In this study, the effect of recombinant interleukin-2 (rIL-2) on the function of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients was examined. Using polymerase chain reaction (PCR), an impaired ability of PBMC from 8 patients to respond upon...

  16. 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...

  17. 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

  18. Antiproliferative activity of recombinant human interferon-λ2 ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... His- hIFN-λ2 vector, the stably transformed BmN cells expressing hIFN-λ2 gene were selected using. Zeocin. Following ... of human cells and tissues, after IFN-λs and its receptors binding .... observed under a microscope to visualize green fluore- scence. .... –related cytokines: from structure to function. Eur.

  19. 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.

  20. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

    OpenAIRE

    Gil, Geun-Cheol; Velander, William H.; Van Cott, Kevin E

    2008-01-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan stru...

  1. Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis.

    Science.gov (United States)

    Schweiger, M; Hennig, K; Lerner, F; Niere, M; Hirsch-Kauffmann, M; Specht, T; Weise, C; Oei, S L; Ziegler, M

    2001-03-09

    Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.

  2. 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.

  3. Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

    Science.gov (United States)

    Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

    2012-09-01

    Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus.

  4. 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.

  5. Recombinant human lymphotoxin: Expression in Escherichia coli, purification, and some properties

    Energy Technology Data Exchange (ETDEWEB)

    Denisov, A.A.; Torskii, S.P.; Nikolaeva, O.G.; Volozhantseva, I.Y. [State Research Institute of Applied Microbiology, Obolensk (Russian Federation)

    1995-09-01

    Biosynthesis of human lymphotoxin lacking 21 amino acid residues was studied in the recombinant strain Escherichia coli SG20050/pLT21. The main content of the protein was found to be soluble and predominantly located in the cytoplasm of E. coli. Synthesis of soluble recombinant lymphotoxin was maximal under cultivation in Luria broth at 32{degrees}C for 24 h. A method for isolation and purification of the recombinant protein from E. coli was elaborated. The procedure includes gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE- and CM-Sephadex. The protein was obtained with 97-fold purification and final yield of 62%. The specific activity was 10{sup 8} units per mg protein. Some physicochemical properties of the protein were investigated. 24 refs., 5 figs., 2 tabs.

  6. 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...... on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses...

  7. Selection of LNA-containing DNA aptamers against recombinant human CD73

    DEFF Research Database (Denmark)

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G

    2015-01-01

    tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences......LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX...... with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several...

  8. [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.

  9. Paroxetine suppresses recombinant human P2X7 responses

    OpenAIRE

    Dao-Ung, Phuong; Skarratt, Kristen; Fuller, Stephen; Stokes, Leanne

    2015-01-01

    P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part vi...

  10. Analysis of human B cell response to recombinantLeishmaniaLPG3

    Institute of Scientific and Technical Information of China (English)

    Mostafa Haji Fatahaliha; Maryam Hosseini; Sanaz Rasolzadeh; Dariush Shane Bandi; Behzad Baradaran; Farhad Jadidi-Niaragh; Mehdi Yousefi

    2015-01-01

    Objective:To evaluate the capability of recombinant Leishmania LPG3 and its fragments in the activation of B cells.Methods:In the present study, human B cells were purified from peripheral blood of 10 adult healthy subjects using magnetic-activated cell sorting technique. Subsequently, purified B cells were treated with recombinant LPG3, and itsN-terminal and C-terminal fragments at different concentrations (2, 10 and 20 μg/mL). B cell activation was assessed through expression of CD69 molecule by flowcytometry and secretion of IL-6, TNF-αα and IL-10 cytokines via enzyme-linked immunosorbent assay following treatment with recombinant antigens.Results:Our results showed that while the recombinant LPG-3 could significantly increase the production of IL-6 and TNF-α (P<0.05) in B cells, it had no effect on the secretion of IL-10 by B cells.Conclusions: Our study indicated that recombinant LPG-3 and especially itsN-terminal fragment could stimulate B cell response as an important immune response component against leishmaniasis. Thus, it seems that it can be considered as an effective adjuvant in vaccine developments against leishmaniasis.

  11. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    Science.gov (United States)

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  12. Production of Recombinant Vector Containing the Coding Sequence of Human Hepcidin

    Directory of Open Access Journals (Sweden)

    Keyhanvar, N.

    2013-01-01

    Full Text Available Background and objective: Hepcidin is a cystein-rich antimicrobial peptide,which is secreted by the liver. It fights against wide spectrum of bacteria, virusesand fungi and it is a major regulator of iron homeostasis. Today, scientists havemade many efforts on the production of hepcidin. Baculovirus expression systemis one of the best eukaryotic expression systems for production of recombinanthepcidin and production of the recombinant vector is one of the most importantsteps in this expression system.Material & Methods: First, the total RNA was separated from HepG2 cell lineas a source of hepcidin expression. Then, after synthesis of total cDNA, humanhepcidin sequence was amplified, using specific primers by PCR method. Next,hepcidin sequence was cloned into pTZ57R/T vector. After digestion ofrecombinant vector using ECoRI and BamHI restriction enzymes, recombinantpFastBac HT B vector containing human hepcidin cDNA was produced.Results: Coding sequence of human hepcidin is correctly cloned into pTZ57R/Tvector and sub cloning into pFastBac HT B vector is performed successfully. Thepresence of a clear band near 274 bp resulted from PCR amplification andrestriction enzyme are the confirmation of the cloning of human hepcidin.Conclusion: According to our knowledge, the present study is the first work thatfocuses on recombinant vector containing coding sequence of human prohepcidin.This recombinant vector can be used for human hepcidin production.Keywords: Vector; Hepcidin; Iron

  13. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Institute of Scientific and Technical Information of China (English)

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

  14. Expression of GPI anchored human recombinant erythropoietin in CHO cells is devoid of glycosylation heterogeneity.

    Science.gov (United States)

    Singh, Pankaj Kumar; Devasahayam, Mercy; Devi, Sobita

    2015-04-01

    Erythropoietin is a glycohormone involved in the regulation of the blood cell levels. It is a 166 amino acid protein having 3 N-glycosylation and one O-linked glycosylation sites, and is used to treat anaemia related illness. Though human recombinant erythropoietin (rEPO) is produced in CHO cells, the loss in quality control is 80% due to incomplete glycosylation of the rEPO with low levels of fully glycosylated active rEPO. Here, we describe the expression from CHO cells of fully glycosylated human rEPO when expressed as a GPI anchored molecule (rEPO-g). The results demonstrated the production of a homogenous completely glycosylated human rEPO-g as a 42 kD band without any low molecular weight glycoform variants as shown by affinity chromatography followed by SDS-PAGE and anti-human EPO specific western blot. The western blot using specific monoclonal antibody is the available biochemical technique to prove the presence of homogeneity in the expressed recombinant protein. The GPI anchor can be removed during the purification process to yield a therapeutically relevant recombinant erythropoietin molecule cells with a higher in vivo biological activity due to its high molecular weight of 40 kD. This is possibly the first report on the production of a homogenous and completely glycosylated human rEPO from CHO cells for efficient therapy.

  15. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

    Science.gov (United States)

    Barbas, C F; Björling, E; Chiodi, F; Dunlop, N; Cababa, D; Jones, T M; Zebedee, S L; Persson, M A; Nara, P L; Norrby, E

    1992-01-01

    A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies. PMID:1384050

  16. Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

    Institute of Scientific and Technical Information of China (English)

    PAN Xin-ting; ZHU Qing-yun; LI De-chun; YANG Ji-cheng; ZHANG Zi-xiang; ZHU Xing-guo; ZHAO Hua

    2008-01-01

    Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhlL-24) on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n=10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0x1010 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P <0.05). The intratumoral MVD decreased significantly in the treated tumors (P <0.05).Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

  17. Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Davis, J.M.; Arakawa, T.; Strickland, T.W.; Yphantis, D.A.

    1987-05-05

    Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I/sup -/ and acrylamide but not by Cs/sup +/. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability.

  18. No increase in female recombination frequency in the distal part of the human pseudoautosomal region

    Energy Technology Data Exchange (ETDEWEB)

    Vergnaud, G. [Institut de Biologie, Nantes (France)

    1994-12-01

    In human, the genetic map is larger in female than in male. However, for unknown reasons, a reversed situation is observed toward at least some telomeres, where recombination in males can be 10-fold that observed in females. Henke et al. report that male and female recombination rates are equal in the very distal part of the human pseudoautosomal region (PAR), thus suggesting that the telomeric excess of male recombination, when observed, may not exist right to the tip of chromosomes. Such an observation is of importance for the genetic mapping of chromosome ends, for both practical and biological reasons. Thus, I have examined in detail our own data for this region and failed to confirm the previous report. I will explain here the origin of the discrepancy, in the CEPH family K1333 presented in detail in the Henke et al. report, which accounts for half (3/6) of the maternal recombination events detected in the most distal interval of the PAR. 9 refs., 1 fig.

  19. Does recombinant human Epo increase exercise capacity by means other than augmenting oxygen transport?

    DEFF Research Database (Denmark)

    Lundby, C; Robach, P; Boushel, R;

    2008-01-01

    This study was performed to test the hypothesis that administration of recombinant human erythropoietin (rHuEpo) in humans increases maximal oxygen consumption by augmenting the maximal oxygen carrying capacity of blood. Systemic and leg oxygen delivery and oxygen uptake were studied during...... before rHuEpo treatment). Blood buffer capacity remained unaffected by rHuEpo treatment and hemodilution. The augmented hematocrit did not compromise peak cardiac output. In summary, in healthy humans, rHuEpo increases maximal oxygen consumption due to augmented systemic and muscular peak oxygen delivery....

  20. Intrachromosomal recombination between highly diverged DNA sequences is enabled in human cells deficient in Bloom helicase.

    Science.gov (United States)

    Wang, Yibin; Li, Shen; Smith, Krissy; Waldman, Barbara Criscuolo; Waldman, Alan S

    2016-05-01

    Mutation of Bloom helicase (BLM) causes Bloom syndrome (BS), a rare human genetic disorder associated with genome instability, elevation of sister chromatid exchanges, and predisposition to cancer. Deficiency in BLM homologs in Drosophila and yeast brings about significantly increased rates of recombination between imperfectly matched sequences ("homeologous recombination," or HeR). To assess whether BLM deficiency provokes an increase in HeR in human cells, we transfected an HeR substrate into a BLM-null cell line derived from a BS patient. The substrate contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI, as well as a functional tk gene to serve as a potential recombination partner for the tk-neo gene. The two tk sequences on the substrate displayed 19% divergence. A double-strand break was introduced by expression of I-SceI and repair events were recovered by selection for G418-resistant clones. Among 181 events recovered, 30 were accomplished via HeR with the balance accomplished by nonhomologous end-joining. The frequency of HeR events in the BS cells was elevated significantly compared to that seen in normal human fibroblasts or in BS cells complemented for BLM expression. We conclude that BLM deficiency enables HeR in human cells.

  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

    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-terminally tag......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...... 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...

  2. 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.

  3. Treatment of type I and II hereditary angioedema with Rhucin, a recombinant human C1 inhibitor.

    Science.gov (United States)

    Varga, Lilian; Farkas, Henriette

    2008-11-01

    Hereditary and acquired angioedema are of outstanding clinical importance, as edematous attacks associated with these conditions can thrust afflicted patients into mortal danger. Currently, C1 inhibitor concentrate - a human blood product - is available as a replacement therapy. In view of the limited number of donors, as well as the risk of transmission of blood-borne infections, it is a reasonable expectation to develop a therapeutic alternative based on recombinant technology, which would eliminate all these shortcomings. Pharming (Leiden, The Netherlands) has developed Rhucin, a recombinant human C1 inhibitor, as a proprietary product, which is currently being evaluated in Phase III clinical trials. Ongoing studies conducted within the framework of the development program are almost complete and their interim findings are reassuring. This should facilitate successful regulatory approval in the near future, which is indispensable in order to make Rhucin available for patients with hereditary angioedema or other disorders amenable to C1 inhibitor replacement.

  4. Recombinant human erythropoietin increases cerebral cortical width index and neurogenesis following ischemic stroke

    Institute of Scientific and Technical Information of China (English)

    Zhongmin Wen; Peiji Wang

    2012-01-01

    The cerebral cortical expansion index refers to the ratio between left and right cortex width and is recognized as an indicator for cortical hyperplasia. Cerebral ischemia was established in CB-17 mice in the present study, and the mice were subsequently treated with recombinant human erythropoietin via subcutaneous injection. Results demonstrated that cerebral cortical width index significantly increased. Immunofluorescence detection showed that the number of nuclear antigen antibody/5-bromodeoxyuridine-positive cells at the infarction edge significantly increased. Correlation analysis revealed a negative correlation between neurological scores and cortical width indices in rats following ischemic stroke. These experimental findings suggested that recombinant human erythropoietin promoted cerebral cortical hyperplasia, increased cortical neurogenesis, and enhanced functional recovery following ischemic stroke.

  5. Coordination study of recombinant human-like collagen and zinc (II)

    Science.gov (United States)

    Yu, Yuan-Yuan; Fan, Dai-Di

    2011-10-01

    In the present investigation, the complex of recombinant human-like collagen (r-HLC) with zinc (II) has been synthesized in aqueous solution and was analyzed by UV-vis spectroscopy, FTIR spectroscopy, circular dichroism (CD) spectroscopy, thermo gravimetric (TG) and differential scanning calorimetry (DSC) analysis. It can be concluded from UV-vis spectra that there exists interaction between r-HLC and zinc, and the complex is a new chemical compound different from pure r-HLC. In the complex of Zn, recombinant human-like collagen acts as ligand, linking the zinc ion via both groups of C dbnd O and N-H. Besides, the results of TG and DSC confirm that the complex was significantly different from ligand, and the former is more thermally stable in comparison with the latter. The results obtained from the current investigation are of crucial importance to understand the r-HLC-Zn complex and provide theoretical evidence for the further study.

  6. 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

    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 thro...

  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

    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...... an increase in very low levels of insulin-like growth factor-I, even in patients with cirrhosis with advanced disease, but the clinical benefits remain to be demonstrated....

  8. Recombinant Human Thrombopoietin Treatment Promotes Hematopoiesis Recovery in Patients with Severe Aplastic Anemia Receiving Immunosuppressive Therapy

    OpenAIRE

    2015-01-01

    Objective. To assess the effectiveness of recombinant human thrombopoietin (rhTPO) in severe aplastic anemia (SAA) patients receiving immunosuppressive therapy (IST). Methods. Eighty-eight SAA patients receiving IST from January 2007 to December 2012 were included in this retrospective analysis. Of these, 40 subjects received rhTPO treatment (15000 U, subcutaneously, three times a week). rhTPO treatment was discontinued when the platelet count returned to normal range. Hematologic response, b...

  9. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2

    OpenAIRE

    Zhang, Zheng; WANG, GUOXIAN; Li, Chen; Liu, Danping

    2013-01-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2+ ...

  10. Comparison of Ehrlichia chaffeensis Recombinant Proteins for Serologic Diagnosis of Human Monocytotropic Ehrlichiosis

    OpenAIRE

    Yu, Xue-Jie; Patricia A Crocquet-Valdes; Cullman, Louis C.; Popov, Vsevolod L.; Walker, David H.

    1999-01-01

    Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients’ sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated t...

  11. PDI-, PPI- and chaperone-catalyzed refolding of recombinant human IL-2 and GM-CSF

    Institute of Scientific and Technical Information of China (English)

    徐明波; 孟文华; 马贤凯

    1995-01-01

    The studies on PDI-, PP1- and chaperone-catalyzed refolding of recombinant human IL-2 and GM-CSF show that PDI can prevent the mismatch of disalfide bonds and formation of aggregates by interchains linkage, furthermore, PDI can correct, the mismatching of disulflde bonds in IL-2 isomers. PPI can increase the rate of folding reaction while chaperone can prevent the aggregation during the folding process. In addition, there is a synergistic effect between them.

  12. Effect of recombinant erythropoietin on functional activity of cultured human cells.

    Science.gov (United States)

    Emel'yanova, E A; Kosykh, A V; Sukhanov, Yu V; Vorotelyak, E A; Vasil'ev, A V

    2012-08-01

    We studied the effect of recombinant human erythropoietin on functional activity of skin cells in vitro. It was found that erythropoietin stimulated proliferation of mesenchymal and epithelial cells and effectively protected epidermal HaCaT cells from apoptosis. Insignificant effect of erythropoietin on contraction of collagen gel by mesenchymal cells was revealed. These findings suggest that erythropoietin can be a promising component of wound-healing preparations.

  13. [Measurement of human thyroid peroxidase autoantibodies by enzyme immunoassay using recombinant human TPO].

    Science.gov (United States)

    Inoue, T; Ishiguro, R; Takenouchi, H; Umeki, K; Matsumoto, K; Yagihashi, S; Kato, H; Kotani, T; Ohtaki, S

    1994-03-01

    An EIA for measuring anti-TPO autoantibodies (rhTPO-EIA) was developed using recombinant human TPO expressed in CHO cells and was compared with MC-HA generally used in laboratory routine work. rhTPO-EIA showed a satisfactory reproducibility in the intra-assay test and did not have an accidental error of lots. Almost equal number of healthy females and males were measured for their IgG binding to TPO to define a normal range of anti-TPO autoantibodies. After setting 20 IU/ml as an upper limit of normal range, sera from patient with thyroid disorders were measured for their anti-TPO autoantibodies. Chronic thyroiditis and Graves' disease were highly positive, while adenoma, thyroid cancer, SLE, and RA were low in their positivity. The positive rate of anti-TPO autoantibodies was compatible to those of previous reports in each disorder. Seventy-two sera from patients with chronic thyroiditis or Graves' disease were measured for their autoantibodies by both rhTPO-EIA and MC-HA and the results were compared between both methods. A correlation coefficient was 0.486. Following absorption with thyroglobulin, sera were measured again and as the results, the correlation coefficient increased to 0.723. Therefore, MC-HA was thought to be influenced in the presence of anti-thyroglobulin autoantibodies. Since rhTPO-EIA is excellent in quality and not affected by anti-thyroglobulin antibodies, it is useful and applicable to clinical diagnosis and observation of thyroid disorders.

  14. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk.

    Science.gov (United States)

    Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

    2008-07-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.

  15. Production of recombinant human bile salt stimulated lipase and its variant in Pichia pastoris.

    Science.gov (United States)

    Sahasrabudhe, A V; Solapure, S M; Khurana, R; Suryanarayan, V; Ravishankar, S; deSousa, S M; Das, G

    1998-12-01

    hBSSL and its truncated variant hBSSL-C cDNA clones were expressed in Pichia pastoris using two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45-50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized in P. pastoris and was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.

  16. Genetic recombination within the human T-cell receptor. cap alpha. -chain gene complex

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-12-01

    Genetic analyses of the human T-cell receptor (TCR) ..cap alpha..-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 TCR..cap alpha.. 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 TCR..cap alpha.. constant region gene were observed in this study. A high recombination frequency for the TCR..cap alpha.. gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCR..cap alpha.. haplotypes.

  17. Selection of LNA-containing DNA aptamers against recombinant human CD73.

    Science.gov (United States)

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G; Larsen, Niels; Nielsen, Ronni; Derbyshire, Nicola; Mandrup, Susanne; Ditzel, Henrik J; Wengel, Jesper

    2015-05-01

    LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.

  18. Expression of Human Sperm Membrane Protein in the Recombinant Salmonella Typhimurium Vaccine

    Institute of Scientific and Technical Information of China (English)

    匡颖; 胡菁华; 翟玉梅; 缨时英; 王琳芳; 严缘昌

    1999-01-01

    A 550 bp cDNA fragment of HSD-Ⅰ coding for an extracellular domain of hu-man sperm membrane protein(hSMP-1)was ligated with an Adapter containing the universal stop codon,and the ligated fragment cDNA was then cloned into the MAS of pUC19.The desired plasmid with correct open reading frame was obtained, and was cut with EcoR Ⅰ.The insert was purified and then cloned into the two asd+ Salmonella expression vectors(the low copy number plasmid-pYA292 and the high copy number plasmid-pYA3137).The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recom-binant plasmids were transferred into the non-pathogenic Salmonella typhimurium X4550,which was deletion of the △cya, △crp and △asd genes.Western-blot analysis of the whole cell lysate of the two recombinants of S.typhimurium showed a predomi-nant protein band at 21 KD,which reacted with the anti-hSMP-1 antiserum. The re-sult indicated that two recombinants of S.typhimurium containing the 550 bp cDNA of HSD-Ⅰ were constructed and the characteristics of their growth in vitro were deter-mined. They may be used as new potential mucosalanti fertility.

  19. 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.

  20. 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.

  1. 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.

  2. One-step purification of soluble recombinant human 6-phosphogluconate dehydrogenase from Escherichia coli.

    Science.gov (United States)

    Chan, Barden; Sukhatme, Vikas P

    2013-11-01

    6-Phosphogluconate dehydrogenase (6PGD), the third enzyme in the pentose phosphate pathway, was recently identified as a novel target in human lung cancer. In this report, we present an expression and purification scheme of recombinant human 6PGD from Escherichia coli. Using a DE3 derivative strain expressing tRNAs for seven rare codons in E. coli called Rosetta2 (DE3), a large quantity of soluble human 6PGD can be expressed with an N-terminal histidine tag and purified by a one-step purification procedure to near homogeneity without denaturants or refolding. Three to seven milligrams of purified protein could be obtained from 100 ml of culture. This recombinant human 6PGD follows classic Michaelis-Menton saturation kinetics with respect to both substrates NADP(+) and 6-phosphogluconate. The respective k(cat) and K(m) were comparable to those of 6PGDs purified from mammalian tissues. Using this purified 6PGD enzyme, we devised an endpoint colorimetric assay suitable for high-throughput screening for human 6PGD inhibitors.

  3. 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.

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

    Science.gov (United States)

    Martínez, José L; Liu, Lifang; Petranovic, Dina; Nielsen, Jens

    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 the generation of a set of plasmids to produce functional human hemoglobin in Saccharomyces cerevisiae, with final titers of active hemoglobin exceeding 4% of the total cell protein. In this study, we propose a strategy for further engineering S. cerevisiae by altering the oxygen sensing pathway by deleting the transcription factor HAP1, which resulted in an increase of the final recombinant active hemoglobin titer exceeding 7% of the total cellular protein.

  5. Recombination networks as genetic markers in a human variation study of the Old World.

    Science.gov (United States)

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

    2012-04-01

    We have analyzed human genetic diversity in 33 Old World populations including 23 populations obtained through Genographic Project studies. A set of 1,536 SNPs in five X chromosome regions were genotyped in 1,288 individuals (mostly males). We use a novel analysis employing subARG network construction with recombining chromosomal segments. Here, a subARG is constructed independently for each of five gene-free regions across the X chromosome, and the results are aggregated across them. For PCA, MDS and ancestry inference with STRUCTURE, the subARG is processed to obtain feature vectors of samples and pairwise distances between samples. The observed population structure, estimated from the five short X chromosomal segments, supports genome-wide frequency-based analyses: African populations show higher genetic diversity, and the general trend of shared variation is seen across the globe from Africa through Middle East, Europe, Central Asia, Southeast Asia, and East Asia in broad patterns. The recombinational analysis was also compared with established methods based on SNPs and haplotypes. For haplotypes, we also employed a fixed-length approach based on information-content optimization. Our recombinational analysis suggested a southern migration route out of Africa, and it also supports a single, rapid human expansion from Africa to East Asia through South Asia.

  6. Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.

    OpenAIRE

    Hellerman, J G; Cone, R C; Potts, J. T.; Rich, A; Mulligan, R C; Kronenberg, H M

    1984-01-01

    In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recomb...

  7. Subcutaneous administration of human C1 inhibitor with recombinant human hyaluronidase in patients with hereditary angioedema.

    Science.gov (United States)

    Riedl, Marc A; Lumry, William R; Li, H Henry; Banerji, Aleena; Bernstein, Jonathan A; Ba, Murat; Bjrkander, Janne; Magerl, Markus; Maurer, Marcus; Rockich, Kevin; Chen, Hongzi; Schranz, Jennifer

    2016-11-01

    The currently approved method of C1 inhibitor (C1 INH) administration for patients with hereditary angioedema with C1 INH deficiency (HAE) is by intravenous injection. A C1 INH subcutaneous formulation may provide an attractive mode of administration for some patients. To evaluate efficacy and safety of two doses of subcutaneous, plasma-derived C1 INH with the dispersing agent, recombinant human hyaluronidase (rHuPH20) to prevent angioedema attacks in patients with HAE. A randomized, double-blind, dose-ranging, crossover study, patients 12 years of age (n = 47) with a confirmed diagnosis of HAE were randomly assigned to receive subcutaneous injections of 1000 U C1 INH with 24,000 U rHuPH20 or 2000 U C1 INH with 48,000 U rHuPH20 every 3 or 4 days for 8 weeks and then crossed-over for another 8-week period. The primary efficacy end point was the number of angioedema attacks during each treatment period. The study was terminated early as a precaution related to non-neutralizing antibodies to rHuPH20 in 45% of patients. The mean standard deviation number of angioedema attacks during the 8-week treatment periods were 1.58 1.59 with 1000 U C1 INH and 0.97 1.26 with 2000 U. The mean (95% confidence interval [CI]) within-patient difference (2000 U-1000 U, respectively) was 0.61 (95% CI, 1.23 to 0.01) attacks per month (p = 0.0523), and 0.56 (95% CI, 1.06 to 0.05) attacks that required acute treatment, (p = 0.0315). No deaths or other serious adverse events were reported. Injection-site reaction was the most common adverse event. Despite early termination, this study demonstrated a clinically and statistically significant difference in burden of disease, which favored 2000 U C1 INH, without associated serious adverse events.

  8. 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

    machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use...... underlying BRCA1’s function in maintaining genome stability and tumor suppression but may also point to a previously unrecognized role of BRCA1 in B-cell lymphomagenesis....... of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein ligase RNF168-deficient, Fanconi anemia group J protein (FACJ, BRIP1)-deficient, or DNA endonuclease RBBP8 (CtIP)-compromised cells, whereas an increased frequency of S-region inversions was observed in breast...

  9. 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.

  10. Increase Renaturation Yield of Reteplase Using the Recombinant Human Protein Disulfide Isomerase

    Institute of Scientific and Technical Information of China (English)

    Zhao Youchun(赵友春); Wang Ge; Kong Yang; Wang Yanbing; Zhang Changkai; Chen Chao; Liang Bufeng

    2004-01-01

    Reteplase, the recombinant type of novel tissue plasminogen activator (t-PA) variant, is a promising thrombolytics in clinics. Expressed in the form of an inclusion body, reteplase consists of about 40 % of the total intracellular proteins of Escherichia coli. The recombinant human protein disulfide isomerase (rhPDI) is used to increase the chance for the correct matching of the 18 hydrosulfide groups of the reteplase molecule in the renaturation process and it increase is the reteplase renaturation yield from 1%~2% to 15%~20% with a the purity aboue 99% and the specific activity of 5(105 IU/mg is reached. This novel method can reduce significantly the cost of production.

  11. Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

    Institute of Scientific and Technical Information of China (English)

    Faiz MMT Marikar; Dingyuan Ma; Jianqiang Ye; Bo Tang; Weijuan Zheng; Jing Zhang; Min Lu; Zichun Hua

    2008-01-01

    The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichla coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refoided and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD不得 protein was allowed the production of high titre polycional antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.Cellular & Molecular Immunology. 2008;5(6):471-474.

  12. Human olfactory receptors: recombinant expression in the baculovirus/Sf9 insect cell system, functional characterization, and odorant identification.

    Science.gov (United States)

    Matarazzo, Valéry; Ronin, Catherine

    2013-01-01

    Cell surface expression of recombinant olfactory receptors (ORs) is a major limitation in characterizing their functional nature. We have shown that the recombinant expression of a human OR, OR 17-210, in the baculovirus/Sf9 insect cell system allows this protein to be expressed at the cell surface. We used Ca(2+) imaging to demonstrate that recombinant OR 17-210 produces cellular activities upon odorant stimulation with ketones. Furthermore, this expression and functional system has been used to show that the preincubation of Human Odorant Binding Protein 2A decrease the calcium response of OR 17-210 following stimulation by acetophenone and beta ionone.

  13. 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.

  14. Immunomodulation by α(1)-proteinase inhibitor: lack of chemotactic effects of recombinant human α(1)-proteinase inhibitor from yeast on human peripheral blood granulocytes

    OpenAIRE

    Mosheimer, Birgit; Alzner, Reinhard; Wiedermann, Christian J.

    2007-01-01

    Introduction: Recombinant α(1)-proteinase inhibitor, clinically developed for inhalative augmentation therapy in patients with α(1)-proteinase inhibitor deficiency or cystic fibrosis, may directly contribute to leukocyte accumulation as it may function as a chemoattractant. The migratory effects of yeast-derived human recombinant α(1)-proteinase inhibitor on human peripheral blood neutrophils and eosinophils were therefore tested in vitro. Materials and Methods: Human peripheral blood leukocy...

  15. 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.

  16. Translational mixed-effects PKPD modelling of recombinant human growth hormone - from hypophysectomized rat to patients

    DEFF Research Database (Denmark)

    Thorsted, Anders; Thygesen, Peter; Agersø, Henrik

    2016-01-01

    BACKGROUND AND PURPOSE: We aimed to develop a mechanistic mixed-effects pharmacokinetic (PK)-pharmacodynamic (PD) (PKPD) model for recombinant human growth hormone (rhGH) in hypophysectomized rats and to predict the human PKPD relationship. EXPERIMENTAL APPROACH: A non-linear mixed-effects model...... was developed from experimental PKPD studies of rhGH and effects of long-term treatment as measured by insulin-like growth factor 1 (IGF-1) and bodyweight gain in rats. Modelled parameter values were scaled to human values using the allometric approach with fixed exponents for PKs and unscaled for PDs...... a clinically relevant biomarker, IGF-1, to a primary clinical end-point, growth/bodyweight gain. Scaling of the model parameters provided robust predictions of the human PKPD in growth hormone-deficient patients including variability....

  17. 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.

  18. 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

  19. 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.

  20. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    Science.gov (United States)

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. 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

    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 PMID:23776617

  2. Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

    Directory of Open Access Journals (Sweden)

    Sina Mirzaahmadi

    2011-01-01

    Full Text Available The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

  3. Production and characterisation of a monoclonal antibody to human papillomavirus type 16 using recombinant vaccinia virus.

    Science.gov (United States)

    McLean, C S; Churcher, M J; Meinke, J; Smith, G L; Higgins, G; Stanley, M; Minson, A C

    1990-06-01

    A monoclonal antibody was raised against the major capsid protein L1 of human papillomavirus type 16, using a recombinant vaccinia virus that expresses the L1 protein, as a target for screening. This antibody, designated CAMVIR-1, reacted with a 56 kilodalton protein in cells infected with L1-vaccinia virus, and the protein was present in a predominantly nuclear location. The antibody also detects the HPV-16 L1 antigen in formalin fixed, paraffin wax embedded biopsy specimens and on routine cervical smears. The antibody reacts strongly and consistently with biopsy specimens containing HPV-16 or HPV-33, but very weak reactions were occasionally observed with biopsy specimens or smears containing HPV-6 or HPV-11. The potential advantages of using a vaccinia recombinant are (i) the target protein is synthesised in a eukoryotic cell so that its "processing" and location are normal; (ii) cells infected with vaccinia recombinants can be subjected to various fixing procedures similar to those used for routine clinical material. This greatly increases the probability that an identified antibody will be useful in a clinical setting.

  4. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    Directory of Open Access Journals (Sweden)

    Kalpana Dulal

    2012-01-01

    Full Text Available Bacterial artificial chromosome (BAC technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV. The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  5. PRM-151 (recombinant human serum amyloid P/pentraxin 2) for the treatment of fibrosis.

    Science.gov (United States)

    Duffield, Jeremy S; Lupher, Mark L

    2010-06-01

    Serum amyloid P or pentraxin 2 (PTX2) is a highly phylogenetically conserved, naturally circulating plasma protein and a soluble pattern recognition receptor of the innate immune system. The unique binding activities of PTX2 suggest that it may localize specifically to sites of injury and function to aid in the removal of damaged tissue. The recent discovery of its ability to regulate certain monocyte differentiation states has identified PTX2 as a novel and potentially powerful antifibrotic agent. A fully recombinant form of the human PTX2 protein, designated PRM-151, has recently initiated human clinical trials. Here we review the molecular, cellular and structural biology of PRM-151/PTX2 in vitro and in several in vivo preclinical models of fibrotic disease that demonstrate its potential as a first-in-class natural modulator of fibrotic pathology with significant potential to treat a wide variety of human diseases.

  6. A chemical compound that stimulates the human homologous recombination protein RAD51.

    Science.gov (United States)

    Jayathilaka, Krishanthi; Sheridan, Sean D; Bold, Tyler D; Bochenska, Katarzyna; Logan, Hillary L; Weichselbaum, Ralph R; Bishop, Douglas K; Connell, Philip P

    2008-10-14

    RAD51 and other members of the RecA family of strand exchange proteins assemble on ssDNA to form presynaptic filaments, which carry out the central steps of homologous recombination. A microplate-based assay was developed for high-throughput measurement of hRAD51 filament formation on ssDNA. With this method, a 10,000 compound library was screened, leading to the identification of a small molecule (RS-1) that enhances hRAD51 binding in a wide range of biochemical conditions. Salt titration experiments showed that RS-1 can enhance filament stability. Ultrastructural analysis of filaments formed on ssDNA showed that RS-1 can increase both protein-DNA complex lengths and the pitch of helical filament turns. RS-1 stimulated hRAD51-mediated homologous strand assimilation (D-loop) activity by at least 5- to 11-fold, depending on the condition. This D-loop stimulation occurred even in the presence of Ca(2+) or adenylyl-imidodiphosphate, indicating that the mechanism of stimulation was distinct from that conferred by Ca(2+) and/or inhibition of ATPase. No D-loop activity was observed in the absence of a nucleotide triphosphate cofactor, indicating that the compound does not substitute for this requirement. These results indicate that RS-1 enhances the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments. Cell survival assays in normal neonatal human dermal fibroblasts demonstrated that RS-1 promotes a dose-dependent resistance to the cross-linking chemotherapeutic drug cisplatin. Given that RAD51-dependent recombination is a major determinant of cisplatin resistance, RS-1 seems to function in vivo to stimulate homologous recombination repair proficiency. RS-1 has many potential applications in both research and medical settings.

  7. Generation of biologically active multi-sialylated recombinant human EPOFc in plants.

    Directory of Open Access Journals (Sweden)

    Alexandra Castilho

    Full Text Available Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO. Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i branching (ii β1,4-galactosylation as well as for the (iii synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.

  8. Biologically active recombinant human progastrin(6-80) contains a tightly bound calcium ion.

    Science.gov (United States)

    Baldwin, G S; Hollande, F; Yang, Z; Karelina, Y; Paterson, A; Strang, R; Fourmy, D; Neumann, G; Shulkes, A

    2001-03-16

    Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

  9. Optimizing conditions for production of high levels of soluble recombinant human growth hormone using Taguchi method.

    Science.gov (United States)

    Savari, Marzieh; Zarkesh Esfahani, Sayyed Hamid; Edalati, Masoud; Biria, Davoud

    2015-10-01

    Human growth hormone (hGH) is synthesized and stored by somatotroph cells of the anterior pituitary gland and can effect on body metabolism. This protein can be used to treat hGH deficiency, Prader-Willi syndrome and Turner syndrome. The limitations in current technology for soluble recombinant protein production, such as inclusion body formation, decrease its usage for therapeutic purposes. To achieve high levels of soluble form of recombinant human growth hormone (rhGH) we used suitable host strain, appropriate induction temperature, induction time and culture media composition. For this purpose, 32 experiments were designed using Taguchi method and the levels of produced proteins in all 32 experiments were evaluated primarily by ELISA and dot blotting and finally the purified rhGH protein products assessed by SDS-PAGE and Western blotting techniques. Our results indicate that media, bacterial strains, temperature and induction time have significant effects on the production of rhGH. The low cultivation temperature of 25°C, TB media (with 3% ethanol and 0.6M glycerol), Origami strain and a 10-h induction time increased the solubility of human growth hormone.

  10. Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LCs0 value of 18.4 uM and 0.70 μM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 μM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3μM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

  11. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

    Science.gov (United States)

    Schwarz, Harald; Schmittner, Maria; Duschl, Albert; Horejs-Hoeck, Jutta

    2014-01-01

    Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

  12. 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...... in joint space width (JSW) seen on radiographs, and pain scores on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Results. One hundred ninety-two patients were randomized and evaluated for safety, 180 completed the trial, and 168 were evaluated for the primary efficacy end...

  13. Crystallization and molecular-replacement solution of a truncated form of human recombinant tetranectin

    DEFF Research Database (Denmark)

    Nielsen, Betina Bryde; Kastrup, Jette Sandholm Jensen; Rasmussen, Hanne B.;

    2000-01-01

    The two C-terminal domains, TN23 (residues 17-181), of human recombinant tetranectin, a plasminogen kringle 4 binding C-type lectin, have been crystallized in two different space groups. Using PEG 8000 as precipitant and at a pH of 8.5, crystals belonging to the monoclinic space group C2 are obta.......5 A has been collected from the monoclinic crystals. Using the structure of full-length tetranectin, a molecular-replacement solution has been obtained. The crystal packing shows that TN23 crystallizes as a trimer, with one trimer in the asymmetric unit....

  14. New insights for identification of doping with recombinant human erythropoietin micro-doses after high hydration

    DEFF Research Database (Denmark)

    Martin, L.; Ashenden, M; Bejder, Jacob

    2016-01-01

    To minimize the chances of being caught after doping with recombinant human erythropoietins (rhEPO), athletes have turned to new practices using micro-doses and excess fluid ingestion to accelerate elimination and decrease the probability of detection. Our objective was to test the sensitivity...... subjects. After an injection in the evening, urine and plasma samples were collected the following morning. Half of the subjects then drank a bolus of water and new samples were collected 80 min later. Interestingly, rhEPO was detected in 100% of the samples even after water ingestion. A second similar...

  15. Recombinant human erythropoietin, and myocardial and cerebral acute ischemia: implications for clinical use.

    Directory of Open Access Journals (Sweden)

    Rafael Alejandro Gómez Baute

    2010-07-01

    Full Text Available Recombinant human erythropoietin has been used for more than two decades in clinical practice with promising results in the treatment of anemia associated with chronic renal insufficiency and in patients with cancer. Recent evidence has uncovered new nonhematopoietic functions of this protein and have brought new hope in the treatment of diseases with ischemic component. In the present review is rife with detail about these new features in the light of new discoveries and explores the therapeutic opportunities offered by these new scientific evidence.

  16. Expression and Characterization of Truncated Recombinant Human Cytochrome P450 2J2

    OpenAIRE

    Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Kim, Donghak

    2014-01-01

    The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a Ni2+-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this trunca...

  17. 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......-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...

  18. High-expression of recombinant human concensus interferon-α by Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    HAO Yuyou; SHI Qiqi; HE Yun; ZHUANG Yingping; WANG Yonghong; ZHANG Siliang; CHU Ju; LIU Zhimin

    2007-01-01

    The present work focused on the high expression of recombinant human consensus interferon-α (cIFN) by Pichia pastoris.The cycle of glycerol feeding,the strategy of methanol feeding and the optimum pH for protein induction were studied.The optimized strategies were a 4-h glycerol-feeding period,induction pH being kept at 5.0 and methanol concentration being kept under 5 g/L.The maximum dry cell weight,cIFN production and bioactivity obtained were 168,1.24 g/L and 5.4 × 107 U/mL,respectively.

  19. Effects of recombinant human insulin-like growth factor I on glomerular dynamics in the rat.

    OpenAIRE

    Hirschberg, R; Kopple, J D; Blantz, R C; Tucker, B J

    1991-01-01

    This study was undertaken to investigate the mechanisms by which an infusion of recombinant human insulin-like growth factor I (rhIGF-I) increases GFR and renal plasma flow (RPF) in rats. Glomerular micropuncture studies were carried out in 14 nonstarved Munich Wistar rats and in 12 rats deprived of food for 60-72 h. Animals were given an intravenous injection and infusion of either rhIGF-I or vehicle. In both nonstarved and starved animals, the IGF-I injection and infusion increased the seru...

  20. 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

    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...... expression in brain tissue was measured by real time PCR. RESULTS: Treatment with rhEpo increased survival in mice with CM in a dose- and time-dependent manner and reduced apoptotic cell death of neurons as well as the expression of pro-inflammatory cytokines in the brain. This neuroprotective effect...

  1. Targeting of human aFGF gene into silkworm,Bombyx mori L. through homologous recombination

    Institute of Scientific and Technical Information of China (English)

    吴小锋; 曹翠平

    2004-01-01

    The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

  2. Targeting of human aFGF gene into silkworm, Bombyx mori L.Through homologous recombination

    Institute of Scientific and Technical Information of China (English)

    吴小锋; 曹翠平

    2004-01-01

    The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

  3. Expression of recombinant human lysozyme in the milk of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    YU Zhengquan; FAN Baoliang; DAI Yunping; ZHENG Ming; NIU Huiling; WANG Meili; WANG Lili; FEI Jing; LI Ning

    2003-01-01

    Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal ofimproving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic micewas 0.2 mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.

  4. Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

    Institute of Scientific and Technical Information of China (English)

    戴五星; 梁靓; 高红; 黄海浪; 陈智浩; 程继忠; 皇甫永穆

    2004-01-01

    Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69 % in total bacterial protein and 74.09 % in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M.tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.

  5. The synaptonemal complex and meiotic recombination in humans: new approaches to old questions.

    Science.gov (United States)

    Vallente, Rhea U; Cheng, Edith Y; Hassold, Terry J

    2006-06-01

    Meiotic prophase serves as an arena for the interplay of two important cellular activities, meiotic recombination and synapsis of homologous chromosomes. Synapsis is mediated by the synaptonemal complex (SC), originally characterized as a structure linked to pairing of meiotic chromosomes (Moses (1958) J Biophys Biochem Cytol 4:633-638). In 1975, the first electron micrographs of human pachytene stage SCs were presented (Moses et al. (1975) Science 187:363-365) and over the next 15 years the importance of the SC to normal meiotic progression in human males and females was established (Jhanwar and Chaganti (1980) Hum Genet 54:405-408; Pathak and Elder (1980) Hum Genet 54:171-175; Solari (1980) Chromosoma 81:315-337; Speed (1984) Hum Genet 66:176-180; Wallace and Hulten (1985) Ann Hum Genet 49(Pt 3):215-226). Further, these studies made it clear that abnormalities in the assembly or maintenance of the SC were an important contributor to human infertility (Chaganti et al. (1980) Am J Hum Genet 32:833-848; Vidal et al. (1982) Hum Genet 60:301-304; Bojko (1983) Carlsberg Res Commun 48:285-305; Bojko (1985) Carlsberg Res Commun 50:43-72; Templado et al. (1984) Hum Genet 67:162-165; Navarro et al. (1986) Hum Reprod 1:523-527; Garcia et al. (1989) Hum Genet 2:147-53). However, the utility of these early studies was limited by lack of information on the structural composition of the SC and the identity of other SC-associated proteins. Fortunately, studies of the past 15 years have gone a long way toward remedying this problem. In this minireview, we highlight the most important of these advances as they pertain to human meiosis, focusing on temporal aspects of SC assembly, the relationship between the SC and meiotic recombination, and the contribution of SC abnormalities to human infertility.

  6. 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.

  7. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  8. Inhibitory effects of pomegranate extracts on recombinant human maltase-glucoamylase.

    Science.gov (United States)

    Kawakami, Kayoko; Li, Peng; Uraji, Misugi; Hatanaka, Tadashi; Ito, Hideyuki

    2014-09-01

    α-Glucosidase inhibitors are currently used in the treatment of type 2 diabetes. In this study, we investigated the inhibitory activities of aril and pericarp extracts from pomegranates obtained various regions against recombinant human maltase-glucoamylase (MGAM). The inhibitory activities of the aril extracts tended to be stronger than those of the pericarp extracts. The Iranian aril extract was the most effective inhibitor. We investigated the polyphenol content of the pomegranate extracts using the Folin-Ciocalteu method. Among the aril extracts, the Iranian aril extract showed the highest polyphenol content. We further evaluated inhibitory activity against α-glucosidase from the rat small intestine. Pomegranate extract used in this study showed slightly different inhibitory activities according to α-glucosidase origin. Iranian aril extract was the most effective inhibitor of α-glucosidases, especially recombinant human MGAM. Bioassay-guided fractionation of the pomegranate arils led to identification of punicalagin and oenothein B as potent inhibitors of α-glucosidase. Oenothein B showed inhibitory activity with a half-maximal inhibitory concentration (IC(50)) value of 174 μM. Its potency was comparable to that of the α-glucosidase inhibitor acarbose with an IC(50) value of 170 μM. Dixon plot kinetic analysis of oenothein B showed a noncompetitive inhibition with a K(i) value of 102 μM. These results suggest that pomegranate arils would be useful for suppressing postprandial hyperglycemia.

  9. Crystallization and molecular-replacement solution of a truncated form of human recombinant tetranectin

    DEFF Research Database (Denmark)

    Nielsen, B B; Kastrup, J S; Rasmussen, H;

    2000-01-01

    The two C-terminal domains, TN23 (residues 17-181), of human recombinant tetranectin, a plasminogen kringle 4 binding C-type lectin, have been crystallized in two different space groups. Using PEG 8000 as precipitant and at a pH of 8.5, crystals belonging to the monoclinic space group C2 are obta......The two C-terminal domains, TN23 (residues 17-181), of human recombinant tetranectin, a plasminogen kringle 4 binding C-type lectin, have been crystallized in two different space groups. Using PEG 8000 as precipitant and at a pH of 8.5, crystals belonging to the monoclinic space group C2...... are obtained, with unit-cell parameters a = 160.4, b = 44.7, c = 107.5 A, beta = 127.6 degrees. Using sodium formate as precipitant and at a pH of 5.0, TN23 crystallizes in a rhombohedral space group, with unit-cell parameters a = b = c = 107.4 A, alpha = beta = gamma = 78.3 degrees. A full data set to 4...

  10. In vitro evaluation of Bacopa monniera extract and individual constituents on human recombinant monoamine oxidase enzymes.

    Science.gov (United States)

    Singh, Rajbir; Ramakrishna, Rachumallu; Bhateria, Manisha; Bhatta, Rabi Sankar

    2014-09-01

    Bacopa monniera is a traditional Ayurvedic medicinal plant that has been used worldwide for its nootropic action. Chemically standardized extract of B. monniera is now available as over the counter herbal remedy to enhance memory in children and adults. Considering the nootropic action of B. monniera, we evaluated the effect of clinically available B. monniera extract and six of B. monniera constituents (bacoside A3, bacopaside I, bacopaside II, bacosaponin C, bacosine, and bacoside A mixture) on recombinant human monoamine oxidase (MAO) enzymes. The effect of B. monniera extract and individual constituents on human recombinant MAO-A and MAO-B enzymes was evaluated using MAO-Glo(TM) assay kit (Promega Corporation, USA), following the instruction manual. IC50 and mode of inhibition were measured for MAO enzymes. Bacopaside I and bacoside A mixture inhibited the MAO-A and MAO-B enzymes. Bacopaside I exhibited mixed mode of inhibition with IC50 and Ki values of 17.08 ± 1.64 and 42.5 ± 3.53 µg/mL, respectively, for MAO-A enzyme. Bacopaside I is the major constituent of B. monniera, which inhibited the MAO-A enzyme selectively.

  11. Ability of recombinant human catalase to suppress inflammation of the murine lung induced by influenza A.

    Science.gov (United States)

    Shi, Xunlong; Shi, Zhihui; Huang, Hai; Zhu, Hongguang; Zhou, Pei; Zhu, Haiyan; Ju, Dianwen

    2014-06-01

    Influenza A virus pandemics and emerging antiviral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and inflammation of the lung. We have previously investigated the therapeutic efficacy of recombinant human catalase (rhCAT) against viral pneumonia in mice, but the protection mechanisms involved were not explored. In the present study, we have performed a more in-depth analysis covering survival, lung inflammation, immune cell responses, production of cytokines, and inflammation signaling pathways in mice. Male imprinting control region mice were infected intranasally with high pathogenicity (H1N1) influenza A virus followed by treatment with recombinant human catalase. The administration of rhCAT resulted in a significant reduction in inflammatory cell infiltration (e.g., macrophages and neutrophils), inflammatory cytokine levels (e.g., IL-2, IL-6, TNF-α, IFN-γ), the level of the intercellular adhesion molecule 1 chemokine and the mRNA levels of toll-like receptors TLR-4, TLR-7, and NF-κB, as well as partially maintaining the activity of the antioxidant enzymes system. These findings indicated that rhCAT might play a key protective role in viral pneumonia of mice via suppression of inflammatory immune responses.

  12. Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon.

    Science.gov (United States)

    Pfefferkorn, E R; Guyre, P M

    1984-01-01

    The growth of Toxoplasma gondii in cultured human fibroblasts was inhibited by recombinant human gamma interferon at concentrations of 8 to 16 U/ml. The interferon was titrated by observing a total inhibition of parasite plaque formation 7 days after infection. Inhibition of the growth of T. gondii in the early days after infection was measured by marked reductions in the incorporation of radioactive uracil, a precursor that can only be used by the parasites. This assay showed that when cells were pretreated with gamma interferon for 1 day and then infected, inhibition of T. gondii growth could be readily detected 1 or 2 days after infection. When the pretreatment was omitted and parasites and gamma interferon were added at the same time, no inhibition of parasite growth could be detected 1 day later, although it was apparent after 2 days. Cultures from which the gamma interferon had been removed by washing after a 1-day treatment showed inhibition of T. gondii growth. Gamma interferon had no effect on the viability of extracellular parasites, but it did inhibit the synthesis of host cell RNA and protein by ca. 50% 3 days after treatment. This degree of inhibition is unlikely, of itself, to compromise the growth of T. gondii. Recombinant alpha and beta interferons had no effect on the growth of T. gondii. Images PMID:6425215

  13. 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.

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2015-11-01

    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. Optimisation of recombinant production of active human cardiac SERCA2a ATPase.

    Science.gov (United States)

    Antaloae, Ana V; Montigny, Cédric; le Maire, Marc; Watson, Kimberly A; Sørensen, Thomas L-M

    2013-01-01

    Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.

  18. Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV.

    Science.gov (United States)

    Juozapaitienė, Vaida; Bartkutė, Brigita; Michailovienė, Vilma; Zakšauskas, Audrius; Baranauskienė, Lina; Satkūnė, Sandra; Matulis, Daumantas

    2016-12-20

    Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E. coli strains, and the length of recombinant CA XIV protein construct were analyzed for the production CA XIV in large scale by using affinity purification. Active site titration by inhibitors and the isothermal titration calorimery revealed over 96% purity of the protein. Enzymatic activity of the purified CA XIV was determined by following the CO2 hydration using the stopped-flow technique. Several inhibitors were discovered that exhibited selectivity towards CA XIV over other CA isoforms and could be developed as drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Human Jk recombination signal binding protein gene (IGKJRB): Comparison with its mouse homologue

    Energy Technology Data Exchange (ETDEWEB)

    Amakawa, Ryuichi; Jing, Wu; Matsunami, Norisada; Hamaguchi, Yasushi; Matsuda, Fumihiko; Kawaichi, Masashi; Honjo, Tasuku (Kyoto Univ., Sakyo-ku, Kyoto (Japan)); Ozawa, Kazuo (Tsukuba Life Science Center, Tsukuba, Ibraraki (Japan))

    1993-08-01

    The mouse Igkjrb protein specifically binds to the immunoglobulin Jk recombination signal sequence. The IGKJRB gene is highly conserved among many species such as human, Xenopus, and Drosophila. Using cDNA fragments of the mouse Igkjrb gene, the authors isolated its human counterpart, IGKJRB. The human genome contains one functional IGKJRB gene and two types of processed pseudogenes. In situ chromosome hybridization analysis demonstrated that the functional gene is localized at chromosome 3q25, and the pseudogenes (IGKJRBP1 and IGKJRBP2, respectively) are located at chromosomes 9p13 and 9q13. The functional gene is composed of 13 exons spanning at least 67 kb. Three types of cDNA with different 5[prime] sequences were isolated by rapid amplification of cDNA ends, suggesting the presence of three proteins. The aPCR-1 protein, which possessed the exon 1 sequence, was the counterpart of the mouse RBP-2 type protein. The aPCR-2 and 3 proteins may be specific to human cells because the mouse counterparts were not detected. The amino acid sequences of the human and mouse IGKJRB genes were 98% homologous in exons 2-11, whereas the homology of the human and mouse exon 1 sequences was 75%. 40 refs., 7 figs.

  20. 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

  1. Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin

    OpenAIRE

    1997-01-01

    A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low Mr peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the pr...

  2. Pharmacokinetics of human recombinant tissue-type plasminogen activator, administered intra-abdominally, in a rat peritonitis model

    NARCIS (Netherlands)

    van Goor, Harry; Bom, VJJ; van der Meer, J; Sluiter, WJ; Geerards, S; de Graaf, JS; Bleichrodt, RP; van der Schaaf, W

    1996-01-01

    Human recombinant tissue-type plasminogen activator (rtPA), administered intraperitoneally, may promote intraabdominal fibrinolysis in peritonitis, thereby preventing adhesion and abscess formation. The pharmacokinetics of a single intraperitoneal dose of 0.5 or 2.0 mg/ml human rtPA were assessed in

  3. Pharmacokinetics of human recombinant tissue-type plasminogen activator, administered intra-abdominally, in a rat peritonitis model

    NARCIS (Netherlands)

    van Goor, Harry; Bom, VJJ; van der Meer, J; Sluiter, WJ; Geerards, S; de Graaf, JS; Bleichrodt, RP; van der Schaaf, W

    1996-01-01

    Human recombinant tissue-type plasminogen activator (rtPA), administered intraperitoneally, may promote intraabdominal fibrinolysis in peritonitis, thereby preventing adhesion and abscess formation. The pharmacokinetics of a single intraperitoneal dose of 0.5 or 2.0 mg/ml human rtPA were assessed in

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

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available 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.

  5. Bioactive recombinant human lactoferrin, derived from rice, stimulates mammalian cell growth.

    Science.gov (United States)

    Huang, N; Bethell, D; Card, C; Cornish, J; Marchbank, T; Wyatt, D; Mabery, K; Playford, R

    2008-01-01

    Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [(3)H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.

  6. Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma.

    Science.gov (United States)

    Achilonu, Ikechukwu; Siganunu, Thendo P; Dirr, Heini W

    2014-07-01

    The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, β and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the β subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) μM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03μmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.

  7. Biotransformation of chlorpyrifos and diazinon by human liver microsomes and recombinant human cytochrome P450s (CYP).

    Science.gov (United States)

    Sams, C; Cocker, J; Lennard, M S

    2004-10-01

    The cytochrome P450 (CYP)-mediated biotransformation of the organophosphorothioate insecticides chlorpyrifos and diazinon was investigated. Rates of desulphuration to the active oxon metabolite (chlorpyrifos-oxon and diazinon-oxon) and dearylation to non-toxic hydrolysis products were determined in human liver microsome preparations from five individual donors and in recombinant CYP enzymes. Chlorpyrifos and diazinon underwent desulphuration in human liver microsome with mean Km = 30 and 45 microM and V(max) = 353 and 766 pmol min(-1) mg(-1), respectively. Dearylation of these compounds by human liver microsome proceeded with Km = 12 and 28 microM and V(max) = 653 and 1186 pmol min(-1) mg(-1), respectively. The apparent intrinsic clearance (V(max)/Km) of dearylation was 4.5- and 2.5-fold greater than desulphuration for chlorpyrifos and diazinon, respectively. Recombinant human CYP2B6 possessed the highest desulphuration activity for chlorpyrifos, whereas CYP2C19 had the highest dearylation activity. In contrast, both desulphuration and dearylation of diazinon were catalysed at similar rates, in the rank order CYP2C19 > CYP1A2 > CYP2B6 > CYP3A4. Both organophosphorothioates were more readily detoxified (dearylation) than bioactivated (desulphuration) in all human liver microsome preparations. However, the role of individual CYP enzymes in these two biotransformation pathways varied according to the structure of the organophosphorothioate, which was reflected in different activation/detoxification ratios for chlorpyrifos and diazinon. Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon.

  8. 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.

  9. Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech.

    Science.gov (United States)

    Baranzini, Nicolò; Pedrini, Edoardo; Girardello, Rossana; Tettamanti, Gianluca; de Eguileor, Magda; Taramelli, Roberto; Acquati, Francesco; Grimaldi, Annalisa

    2017-01-09

    In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68(+) and HmAIF-1(+) macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

  10. Anti-apoptotic effects of recombinant human granulocyte colony-stimulating factor in focal cerebral ischemic rats

    Institute of Scientific and Technical Information of China (English)

    Xia Yuan; Shiming Zhang; Wanli Dong; Qi Fang

    2011-01-01

    The neuroprotective effects of granulocyte colony-stimulating factor in cerebral ischemia/reperfusion injury are currently contentious. The present study examined the effects of subcutaneous injection of recombinant human granulocyte colony-stimulating factor (50 μg/kg) over 5 days in a model of cerebral ischemia/reperfusion with intraluminal filament occlusion in rats. The results indicated that recombinant human granulocyte colony-stimulating factor reduced brain infarct volume following cerebral ischemia/reperfusion injury in rats, down-regulated the expression of caspase-3 mRNA (a key protease for apoptosis in the cerebral ischemia zone), lowered the rate of neuronal apoptosis in the cerebral ischemia zone, and notably ameliorated neurological function. These results indicate that recombinant human granulocyte colony-stimulating factor has anti-apoptotic effects on neurons following focal cerebral ischemia/reperfusion injury, and exerts neuroprotective effects.

  11. Expression and purification of recombinant human chemokine SDF-1βin E. coli

    Institute of Scientific and Technical Information of China (English)

    ZHENG Hong; Stephen C PEIPER; ZHU Xi-hua

    2002-01-01

    Objective: To obtain recombinant human SDF-1β expressed in E. coli and purify SDF-1β with biological activity from the bacterium. Methods: A thioredoxin-SDF-1β fusion protein (26 × 103) composed of230 amino acid residues was expressed in E. coli AD494 (DE3)pLysS under the induction of IPTG when pET32a(+)-SDF-1β was used as an expression vector. Purified SDF-1β was produced through following procedures: Bacteria lysis, metal-chelated affinity chromatography (MAC), enterokinase digestion to separate SDF-1β from fusion protein, cation exchange chromatography (CEC) and reverse-phase high performance liquid chromatography (RP-HPLC). Western blot with anti-SDF-1β monoclonal antibody (mAb), N-terminal amino acid sequencing, ligand-binding assay and cytosensor/microphysiometry were used to investigate the biochemical characters and biological activities of the purified SDF-1β. Results: From 10% to 15% of total bacterium protein was expressed as fusion protein. Approximately 400μg purified SDF-1β (7. 8 × 103) consisting of 71 amino acid residues were produced from 1 L of fermented bacteria. Western blot showed that anti-SDF-1β mAb bound with the purified SDF-1β specifically. N-terminal amino acid sequencing indicates that N-terminus of purified SDF-1β possessed as the same amino acid sequence as nature one. Purified SDF-1β not only had the binding activity with CXCR4 expressing cells [Kd = ( 12.20± 2. 99) nmol/L ], but also activated CXCR4 expressing cell signaling specifically in a dose-dependence manner. Conclusion: The purified recombinant human SDF-1β produced with this method possesses biochemical characters and biological activities as same as those nature human SDF-1β.

  12. Antitumor effects of recombinant human adenovirus-p53 against human cutaneous squamous cell carcinoma in mice.

    Science.gov (United States)

    Li, Yuanchao; He, Wei; Wang, Rupeng; Yang, Libin; Zhou, Chunli; Zhang, Bin

    2016-12-01

    The present study was conducted to identify the anti-tumor effects of rAd/p53, which is a recombinant human serotype 5 adenovirus, in cutaneous squamous cell carcinoma (cSCC). Mouse models of human cSCC were constructed by injecting human cutaneous squamous cell carcinoma cells into both flanks of nude mice. Subsequently, the 75 nude mice with cSCC xenograft tumors were randomly divided into recombinant human serotype 5 adenovirus (rAd)/p53, rAd/p53 + 5-fluorouracil (5-Fu) and 5-Fu groups. One side of the tumors was administered the therapeutic agents as the therapeutic group, whereas the remaining side was treated with medical saline as the control. At 24, 48, 72, 120 and 168 h post-intratumoral injection, alterations in tumor volume, tumor necrosis and the expression of several tumor-associated genes, including Smad4, Brca1 and matrix metalloproteinase (MMP-2), were analyzed. Compared with its control group, the rAd/P53 group exhibited a significantly increased tumor necrosis ratio. In addition, Smad4 and Brca1 expression levels increased significantly at various time points (Pp53 + 5-Fu group, the tumor necrosis ratio, and Smad4 and Brca1 expression levels also significantly increased at various time points (PP53 group. In addition, p53 expression exhibited a positive correlation with the tumor necrosis ratio and Smad4 expression, and showed a negative correlation with MMP-2 gene transcription (Pp53 has a potent anti-tumor effect in cSCC via the promotion of tumor necrosis and regulating the expression of various tumor-associated genes.

  13. Construction of a recombinant adenovirus Vector of human papillomavirus type 16 L1_E7c

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 L1 proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7c. Methods: Human papillomavirus type 16 L1 open reading frame without terminator codon (TAA) (5559- 7152) and E7c (682- 855) were amplified using PCR. The L1 and E7c fragments were inserted into pGEM-T easy vectors by T- A strategy, named pTAL1 and pTAE7c. pTAL1 was cut with Hind III and BglII, the pTAE7c with BamHI and ClaI. The L1 DNA fragment, E7c and pBluesscript SK were ligated together using T4 DNA ligase. pBSL1-E7c and pBSL1-E7c was digested with Hind III and Xhol. The L1-E7c fragment was inserted into adenovirus shuttle plasmids pCAl4, named pCAl4L1-E7c. DNA sequence results indicated that The L1-E7c DNA fragment can encode the HPV16L1-E7 fusion protein correctly. Results: The L1 and E7c DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7c, pBSL1-E7c and pCA14L1-E7c were constructed correctly. The pCAl4L1-E7c can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10. Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCALl-E7c for the further research.

  14. 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.

  15. 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.

  16. 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.

  17. Effects of recombinant human GM-CSF on proliferation of clonogenic cells in acute myeloblastic leukemia.

    Science.gov (United States)

    Griffin, J D; Young, D; Herrmann, F; Wiper, D; Wagner, K; Sabbath, K D

    1986-05-01

    Proliferation of acute myeloblastic leukemia (AML) cells in vitro is limited in most cases to a small subset of blasts that have several properties of stem cells. These leukemic colony-forming cells (AML-CFU) generally require addition of exogenous growth factors for proliferation in agar or methylcellulose. These factors can be supplied by media conditioned by phytohemagglutinin-stimulated normal leukocytes or by CSF-secreting tumor cell lines. However, the exact factor or factors required for stimulation of AML-CFU growth have not been defined. We compared the AML-CFU stimulatory activity of a human recombinant GM-CSF with that of GCT-CM, Mo-CM, and the PHA-leukocyte feeder system in 15 cases of AML. In each of the 12 cases that required exogenous growth factors for maximum AML-CFU growth, recombinant GM-CSF could replace either GM-CSF or Mo-CM, and could partially replace the PHA-leukocyte feeder system. These results indicate that this GM-CSF is a growth promoter of AML-CFU in these culture systems.

  18. Overexpression of c-fos increases recombination frequency in human osteosarcoma cells.

    Science.gov (United States)

    van den Berg, S; Rahmsdorf, H J; Herrlich, P; Kaina, B

    1993-05-01

    We have shown previously that overexpression of c-Ha-ras, v-mos or c-fos increases the spontaneous level of chromosomal aberrations and gene mutations in NIH 3T3 cells, and that reduction of the Fos protein level inhibits aberration induction by c-Ha-ras and v-mos and also by irradiation with ultraviolet light (van den Berg et al., Mol. Carcinogenesis, 4, 460-466). In order to examine whether fos is also involved in DNA recombination, thymidine kinase (tk) deficient human osteosarcoma cells containing two versions of the herpes simplex virus tk gene inactivated by base insertion were either transiently or stably transfected with various fos expression plasmids. The frequency of tk+ revertants was significantly enhanced both upon transient transfection with RSV-promoter-fos gene constructs and by stimulation of Fos synthesis in stably transfected cells harbouring an inducible metallothionein promoter-fos construct. No such increases were observed in cells transfected with plasmids containing a truncated version of c-fos. The data indicate that c-fos is involved in generating various types of genetic changes including homologous recombination; a role of c-fos in genetic instability may contribute to its action in tumor promotion and progression.

  19. Recombinant human arginase I immobilized on gold and silver nanoparticles: preparation and properties

    Directory of Open Access Journals (Sweden)

    Natalia Stasyuk

    2011-07-01

    Full Text Available Metal nanoparticles (NPs, such as gold (Au and silver (Ag, are important for chemistry, physics, and biology due to their unique optical, electrical, and photothermal properties. Such NPs are widely used for immobilization of various bioactive substances, including peptides, enzymes, antibodies and DNA. The synthesis of silver and gold nanoparticles was carried out by reduction of silver nitrate by glucose and reduction of tetrachloroauric acid by sodium citrate, respectively. The size and structure of the AgNPs and AuNPs were characterized using TEM, AFM and XRD methods. The average size of the AgNPs and AuNPs was between 8 and 15 nm. Recombinant arginase I was immobilized using the carbodiimidepentafluorophenol method on the surface of NPs functionalized with ω-mercaptohexadecanoic acid. It was shown that recombinant human liver arginase I isolated from the yeast Hansenula polymorpha maintains satisfactory stability after immobilization on both NPs. The immobilized arginase retained 40% of its activity on the surface of AuNPs and 25% on AgNPs compared to the free arginase after storage at +4 ºC during 25 days. The immobilized enzyme can be used for assay of arginine in pharmaceuticals, in food products and in blood.

  20. Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

    Science.gov (United States)

    Chen, Zhen; Dai, Huixia; Liu, Zhenwei; Zhang, Liping; Pang, Jianlei; Ou, Jiquan; Yang, Daichang

    2014-04-01

    Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

  1. 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

  2. Evolutionary Dynamics and Temporal/Geographical Correlates of Recombination in the Human Enterovirus Echovirus Types 9, 11, and 30▿

    Science.gov (United States)

    McWilliam Leitch, E. C.; Cabrerizo, M.; Cardosa, J.; Harvala, H.; Ivanova, O. E.; Kroes, A. C. M.; Lukashev, A.; Muir, P.; Odoom, J.; Roivainen, M.; Susi, P.; Trallero, G.; Evans, D. J.; Simmonds, P.

    2010-01-01

    The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis. PMID:20610722

  3. Evolutionary dynamics and temporal/geographical correlates of recombination in the human enterovirus echovirus types 9, 11, and 30.

    Science.gov (United States)

    McWilliam Leitch, E C; Cabrerizo, M; Cardosa, J; Harvala, H; Ivanova, O E; Kroes, A C M; Lukashev, A; Muir, P; Odoom, J; Roivainen, M; Susi, P; Trallero, G; Evans, D J; Simmonds, P

    2010-09-01

    The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.

  4. Purification and characterization of biologically active recombinant human Eppin expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    ZHU Qing-yi; GU Xiao-jian; YANG Jin; WANG Jun-hong; TANG Bo; WU Hong-fei

    2008-01-01

    Background Eppin(epididymis protease inhibitor)appears to play an important role in primate fertility.However,the function of Eppin and its antibody in men and its relationship with men's infertility are poorly studied.To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty cases,we developed an Escherichia coli expression system for the expression of biologically actire human Eppin.Methods The human Eppin gene was cloned into PET-28a(+)vector after induction with 0.5 mmol/L isopropy-β-D-thiogalactoside(IPTG)at 26℃ for 4 hours,and the expressed fusion protein His6-Eppin was purified by Ni2+ affinity chromatography.Afterwards,six female 8-week-old Balb/c mice were immunized with purified His6-Eppin for three weeks.Their sera were collected and polyclonal antibodies against His6-Eppin were purified,all of which were further verified by Western-blot and immunofluorescence analysis.Results About 18.33 mg His6-Eppin was obtained from 1-L flask culture.The produced polyclonal antibodies against His6-Eppin recognized the Eppin protein both in human epididymis and in HEK293T cells by over-expression of the recombinant human Eppin.Conclusion The purified His6-Eppin protein has biological activity,which might be a candidate for clinical diagnosis of infertility and development of male immuno-contraceptive agents.

  5. Inhibition of ERBB2-overexpressing Tumors by Recombinant Human Prolidase and Its Enzymatically Inactive Mutant

    Directory of Open Access Journals (Sweden)

    Lu Yang

    2015-05-01

    Full Text Available ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers. We recently found that human prolidase (PEPD, a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers. Here, we show that recombinant human PEPD (rhPEPD strongly inhibits ERBB2-overexpressing tumors in mice, whereas it does not impact tumors without ERBB2 overexpression. rhPEPD causes ERBB2 depletion, disrupts oncogenic signaling orchestrated by ERBB2 homodimers and heterodimers, and induces apoptosis. The impact of enzymatically-inactive mutant rhPEPDG278D on ERBB2 is indistinguishable from that of rhPEPD, but rhPEPDG278D is superior to rhPEPD for tumor inhibition. The enzymatic function of rhPEPD stimulates HIF-1α and other pro-survival factors in tumors, which likely attenuates its antitumor activity. rhPEPDG278D is also attractive in that it may not interfere with the physiologic function of endogenous PEPD in normal cells. Collectively, we have identified a human protein as an inhibitory ERBB2 ligand that inhibits ERBB2-overexpressing tumors in vivo. Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPDG278D may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.

  6. Refolding of recombinant human granulocyte colony stimulating factor: effect of cysteine/cystine redox system.

    Science.gov (United States)

    Tiwari, Krishnanand; Shebannavar, Sunil; Kattavarapu, Krishna; Pokalwar, Santosh; Mishra, Maheshwari K; Chauhan, Ugam Kumari

    2012-08-01

    Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine which is widely used for treating neutropenia in humans. Evaluation of alternative to expensive components of redox buffer (reduced and oxidized glutathione) is an important step in reducing the cost of production of human biotherapeutic proteins. In the present study, refolding of recombinant human G-CSF expressed as inclusion bodies (IBs) in E. coli was optimized using cysteine and cystine redox agents. The refolding to correct native form of G-CSF was assessed by reverse phase high performance liquid chromatography (RP-HPLC). The optimized concentrations of cysteine and cystine for correct refolding of G-CSF were found to be 2 mM and 1 mM, respectively. The correctly refolded G-CSF was detected as early as 4 h of incubation in renaturation buffer containing optimized concentrations of cysteine (2 mM) and cystine (1 mM) redox agents. Refolding of G-CSF in optimized redox system increased with increase in shuffling time. Overall, the results suggested the use of cysteine/cystine redox pair could be an alternative to the costlier redox pairs for successful refolding of G-CSF and possibly other human biotherapeutic proteins of importance.

  7. 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.

  8. Selective Elimination of Human Regulatory T Lymphocytes In Vitro With the Recombinant Immunotoxin LMB-2

    Science.gov (United States)

    Attia, Peter; Powell, Daniel J.; Maker, Ajay V.; Kreitman, Robert J.; Pastan, Ira; Rosenberg, Steven A.

    2006-01-01

    Summary CD4+CD25+ T-regulatory cells (Treg) can inhibit the proliferation and cytokine secretion of CD4+CD25− helper T cells in mice and humans. In murine tumor models, the presence of these Treg cells can inhibit the antitumor effectiveness of T-cell transfer and active immunization approaches. We have thus initiated efforts to eliminate Treg cells selectively from human peripheral blood mononuclear cells (PBMCs) to potentially bolster antitumor responses. LMB-2 is a recombinant immunotoxin that is a fusion of a single-chain Fv fragment of the anti-Tac anti-CD25 monoclonal antibody to a truncated form of the bacterial Pseudomonas exotoxin A. In vitro incubation of human PBMCs with LMB-2 reduced the levels of CD4+CD25+ and Foxp3-expressing cells without impairing the function of the remaining lymphocytes. The short in vivo half-life of LMB-2 makes it an attractive candidate for reducing human Treg cells in vivo before the administration of cancer vaccine or cell transfer immunotherapy approaches. PMID:16531821

  9. Calibrating the Human Mutation Rate via Ancestral Recombination Density in Diploid Genomes.

    Directory of Open Access Journals (Sweden)

    Mark Lipson

    2015-11-01

    Full Text Available The human mutation rate is an essential parameter for studying the evolution of our species, interpreting present-day genetic variation, and understanding the incidence of genetic disease. Nevertheless, our current estimates of the rate are uncertain. Most notably, recent approaches based on counting de novo mutations in family pedigrees have yielded significantly smaller values than classical methods based on sequence divergence. Here, we propose a new method that uses the fine-scale human recombination map to calibrate the rate of accumulation of mutations. By comparing local heterozygosity levels in diploid genomes to the genetic distance scale over which these levels change, we are able to estimate a long-term mutation rate averaged over hundreds or thousands of generations. We infer a rate of 1.61 ± 0.13 × 10-8 mutations per base per generation, which falls in between phylogenetic and pedigree-based estimates, and we suggest possible mechanisms to reconcile our estimate with previous studies. Our results support intermediate-age divergences among human populations and between humans and other great apes.

  10. Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency

    OpenAIRE

    Bechter, Oliver E.; Zou, Ying; Shay, Jerry W.; Woodring E. Wright

    2003-01-01

    Homologous recombination is thought to be the molecular mechanism for maintaining telomere length in alternative lengthening of telomeres (ALT) cells. We used a recombination reporter system to show that the frequency of homologous recombination is the same for ALT- and telomerase-positive cells, suggesting that if ALT cells have a recombination defect it specifically involves telomeric sequences. We compared internal and telomere-adjacent positions of our ...

  11. Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

    Institute of Scientific and Technical Information of China (English)

    Xiangli Wang; Haili Wang; Baojie Mi

    2007-01-01

    BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene.OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning.SETTING: Central Laboratory of Northern China Coal Medical College.MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University.METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus liter.MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus liter.RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified.β-galactoside staining in situ indicated that LacZ genes were

  12. Biochemical analysis of the human ENA/VASP-family proteins, MENA, VASP and EVL, in homologous recombination.

    Science.gov (United States)

    Takaku, Motoki; Ueno, Hiroyuki; Kurumizaka, Hitoshi

    2011-06-01

    MENA, VASP and EVL are members of the ENA/VASP family of proteins and are involved in cytoplasmic actin remodeling. Previously, we found that EVL directly interacts with RAD51, an essential protein in the homologous recombinational repair of double-strand breaks (DSBs) and stimulates the RAD51-mediated recombination reactions in vitro. The EVL-knockdown MCF7 cells exhibited a clear reduction in RAD51-foci formation, suggesting that EVL may function in the DSB repair pathway through RAD51-mediated homologous recombination. However, the DSB repair defects were less significant in the EVL-knockdown cells, implying that two EVL paralogues, MENA and VASP, may complement the EVL function in human cells. Therefore, in the present study, we purified human MENA, VASP and EVL as recombinant proteins, and compared their biochemical activities in vitro. We found that all three proteins commonly exhibited the RAD51 binding, DNA binding and DNA-annealing activities. Stimulation of the RAD51-mediated homologous pairing was also observed with all three proteins. In addition, surface plasmon resonance analyses revealed that MENA, VASP and EVL mutually interacted. These results support the ideas that the ENA/VASP-family proteins are functionally redundant in homologous recombination, and that all three may be involved in the DSB repair pathway in humans.

  13. 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......-56% amplification of goitre reduction at one-year post radioiodine compared to conventional (without rh-TSH) ¹³¹I therapy. Although patient satisfaction is not improved at one-year, this approach facilitates tracheal decompression and is particularly promising in large goitres. The majority of multinodular non......-toxic goitre patients may not require amplified goitre reduction. But as an alternative strategy, rh-TSH allows up to 80% reduction of the therapeutic ¹³¹I activity while still achieving goitre reduction comparable to that of conventional ¹³¹I therapy and maintaining high patient satisfaction. The dose...

  14. [Optimization of fermentation of recombinant human Endostatin (rh-Endostatin) expression in Escherichia coli].

    Science.gov (United States)

    Chang, Guo-Dong; Li, Zhuang-Lin; Qin, Jia-Yang; Ma, Cui-Qing; Luo, Yong-Zhang; Xu, Ping

    2005-07-01

    The fermentation process of recombinant human Endostatin expression in Escherichia coli BL21 (DE3) was studied. The effects of factors such as concentration of IPTG, induction time, cultivation temperature and feeding strategies were investigated. Beside that, by changing the temperature to 40 degrees C after induction, the high-density cultivation finished in a much shorter period. After 9 hours cultivation, the optical density (OD) at 600 nm reached 140 and the yield of inclusion body was 3 g/L. While E. coli system was used, protein with better activity and stability was obtained. The cost was much lower and the producing process was much steadier. It will meet the demands of the industrial production.

  15. Human, recombinant interleukin-2 induces in vitro histamine release in a dose-dependent manner

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørgen; Petersen, L J; Skov, P S

    1995-01-01

    We previously observed that human, recombinant interleukin-2 in a pharmacologic dose (200 u/ml) induced histamine release from monocyte-depleted peripheral blood mononuclear cells in vitro. Therefore, we studied the role of various pharmacologic doses of rIL-2 on in vitro histamine release......, for 1, 24 and 48 hours under standard conditions. Histamine was analysed in supernatants using the glass fiber method. Simultaneously, total cell-bound histamine was analysed in lysate from 5 x 10(6) mononuclear cells from all patients and volunteers, thus allowing determination of percent histamine...... release. Supernatant histamine concentration from unstimulated cells was 17.2 +/- 1.5 ng/ml in patients compared to 7.9 +/- 1.0 ng/ml in volunteers (#p Histamine concentration increased...

  16. 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

    = 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......]QNB (brain) and [3H]Oxo-M (brain) binding data, were shown to be predictive of pharmacologically determined intrinsic activities at m1-m5, the same rank order of intrinsic activity being observed at all five mAChRs (4a > 4d > 4b > 4c). It is concluded that within this class of high-affinity mAChR (m1-m5...

  17. Activity and stability of recombinant human superoxide dismutase in buffer solutions and hypothermic perfusates.

    Directory of Open Access Journals (Sweden)

    Senoo,Yoshimasa

    1988-06-01

    Full Text Available The stability of recombinant human superoxide dismutase (r-hSOD in buffer solutions was studied in solutions at various pH and temperatures. Additionally, we studied the effects of incubation with proteases, serum and two types of hypothermic perfusates. R-hSOD was stable in the pH range of 6-11 and at temperatures up to 80 degrees C for 30 min. R-hSOD activity was not affected by incubation with trypsin, aminopeptidase M or serum for 2 h. R-hSOD activity determined at various temperatures (4-37 degrees C did not vary remarkably. R-hSOD in hypothermic perfusates was stable at 4-37 degrees C for 24 h.

  18. Optimization of a recombinant human growth hormone purification process using quality by design.

    Science.gov (United States)

    Ortiz-Enriquez, Carolina; Romero-Díaz, Alexis de Jesús; Hernández-Moreno, Ana V; Cueto-Rojas, Hugo F; Miranda-Hernández, Mariana P; López-Morales, Carlos A; Pérez, Néstor O; Salazar-Ceballos, Rodolfo; Cruz-García, Norberto; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2016-11-16

    This work describes a strategy to optimize a downstream processing of a recombinant human growth hormone (rhGH) by incorporating a quality by design approach toward meeting higher quality specifications. The optimized process minimized the presence of impurities and degradation by-products during manufacturing by the establishment of in-process controls. Capillary zone electrophoresis, reverse phase, and size-exclusion chromatographies were used as analytical techniques to establish new critical process parameters for the solubilization, capture, and intermediate purification steps aiming to maintain rhGH quality by complying with pharmacopeial specifications. The results indicated that the implemented improvements in the process allowed the optimization of the specific recovery and purification of rhGH without compromising its quality. In addition, this optimization facilitated the stringent removal of the remaining impurities in further polishing stages, as demonstrated by the analysis of the obtained active pharmaceutical ingredient.

  19. Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.

    Science.gov (United States)

    Piano, Valentina; Nenci, Simone; Magnani, Francesca; Aliverti, Alessandro; Mattevi, Andrea

    2016-12-02

    Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Human elastin-based recombinant biopolymers improve mesenchymal stem cell differentiation.

    Science.gov (United States)

    Çelebi, Betül; Cloutier, Maxime; Rabelo, Rodrigo B; Balloni, Rodrigo; Mantovani, Diego; Bandiera, Antonella

    2012-11-01

    Elastin-based polypeptides are a class of smart biopolymers representing an important model in the design of biomaterials. The combination of biomimetic materials with cells that have great plasticity provides a promising strategy for the realization of highly engineered cell-based constructs for regenerative medicine and tissue repair applications. Two recombinant biopolymers inspired by human elastin are assessed as coating agents to prepare biomimetic surfaces for cell culture. These substrates are assayed for hBM MSC culture. The coated surfaces are also characterized with AFM to evaluate the topographical features of the deposited biopolymers. The results suggest that the elastin-derived biomimetic surfaces play a stimulatory role on osteogenic differentiation of MSCs.

  1. Review of recombinant human deoxyribonuclease (rhDNase in the management of patients with cystic fibrosis

    Directory of Open Access Journals (Sweden)

    Tacjana Pressler

    2008-09-01

    Full Text Available Tacjana PresslerCF Centre, Dept. of Pediatrics, Rigshospitalet, University of Copenhagen DenmarkAbstract: The most important problem in cystic fibrosis (CF lung disease is chronic airway inflammation and infection, which starts early in life. To prevent severe lung damage, it is important to mobilize as much sputum as possible from the lung on a daily basis. RhDNase is an enzyme that breaks down DNA strands in airway secretions, hydrolyzes the DNA present in sputum/mucus of CF patients, reducing viscosity in the lungs and promoting secretion clearance. Several well performed trials have proven its efficacy in young CF patients with mild disease as well as in older patients with more advanced lung disease. Daily inhalation of this agent slows down lung function decline and decreases the frequency of respiratory exacerbations. The drug is well tolerated by most patients independent of the severity of lung disease.Keywords: cystic fibrosis, lung disease, recombinant human deoxyribonuclease

  2. Report: Heparin-induced thrombocytopenia associated with cardiopulmonary bypass: Preliminary attempt with recombinant human thrombopoietin therapy.

    Science.gov (United States)

    Yuan, Shi-Min

    2015-09-01

    Recombinant human thrombopoietin (rhTPO) is popularly used for the treatment of chemotherapy-induced thrombocytopenia. However, rhTPO therapy for heparin-induced thrombocytopenia relating to cardiopulmonary bypass has not been previously described. A young patient developed heparin-induced thrombocytopenia during open-heart surgery. Postoperative rhTPO therapy (15000 units injection hypodermatica once daily for consecutive 3 days) made a quick platelet recovery without any side effects. Heparin-induced thrombocytopenia associated with cardiopulmonary bypass is more likely to be benign, and is curable to rhTPO therapy. The preliminary rhTPO administration of heparin-induced thrombocytopenia in association with cardiopulmonary bypass shows satisfactory pharmaceutical effects with lower dose, shorter duration treatment and shorter platelet increase time and recovery time in comparison with those for the treatment of chemotherapy-induced thrombocytopenia. rhTPO therapy does not produce any side effects and it could avoid or minimize necessary blood product infusions.

  3. 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 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.

  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 growth.

  5. 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

  6. Human, recombinant interleukin-2 induces in vitro histamine release in a dose-dependent manner

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørgen; Petersen, L J; Skov, P S

    1995-01-01

    We previously observed that human, recombinant interleukin-2 in a pharmacologic dose (200 u/ml) induced histamine release from monocyte-depleted peripheral blood mononuclear cells in vitro. Therefore, we studied the role of various pharmacologic doses of rIL-2 on in vitro histamine release....... Peripheral blood mononuclear cells (5 x 10(6) cells/ml), which also contain basophils, from 13 patients scheduled for elective colorectal cancer surgery and 10 age and sex matched healthy volunteers were stimulated with rIL-2 in concentrations of 0, 50, 100, 200, 450, 900, 1,800 and 3,600 u/ml, respectively......, for 1, 24 and 48 hours under standard conditions. Histamine was analysed in supernatants using the glass fiber method. Simultaneously, total cell-bound histamine was analysed in lysate from 5 x 10(6) mononuclear cells from all patients and volunteers, thus allowing determination of percent histamine...

  7. Effect of recombinant human basic fibroblast growth factor on angiogenesis during mandible fracture healing in rabbits

    Institute of Scientific and Technical Information of China (English)

    龚振宇; 周树夏; 顾晓明; 李涤尘; 孙明林

    2003-01-01

    Objective: To investigate the effect of recombinant human basic fibroblast growth factor (rhbFGF) on angiogenesis during mandible fracture healing in rabbit. Methods: Fifty adult white rabbits were used for animal model and randomly divided into a control group (25 rabbits) and an experimental group (25 rabbits). The membranous complex of rhbFGF and bovine type I collagen was prepared and implanted into the rabbit mandible fracture site under periosteum. The animals were sacrificed on 7, 14, 28, 56 and 84 days respectively after operation and the whole mandibles were harvested. The expression of factor VIII related antigen (F8-RA) in callus was examined with immunohistochemical staining. Results: The amounts of microvascular formation in calluses in the rhbFGF-treating group on days 7, 14, 28 and 56 were more than those of the control group (P<0.01).Conclusions: The results indicated that rhbFGF could stimulate microvascular formation during mandible fracture healing in rabbits.

  8. 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

    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...... 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...... received 5,000 IU rHuEpo every second day for 14 days, and subsequently a single dose of 5,000 IU weekly for 12 weeks. Muscle biopsies were obtained before and after 14 weeks of rHuEpo treatment. The treatment increased hematocrit (from 44.7 to 48.8%), maximal oxygen uptake by 8.1%, and submaximal...

  9. Exogenous recombinant human growth hormone effects during suboptimal energy and zinc intake

    Directory of Open Access Journals (Sweden)

    Duro Debora

    2005-04-01

    Full Text Available Abstract Background Energy and Zinc (Zn deficiencies have been associated with nutritional related growth retardation as well as growth hormone (GH resistance. In this study, the relationship between suboptimal energy and/or Zn intake and growth in rats and their response to immunoreactive exogenous recombinant human GH (GHi, was determined. Results Rats treated with GHi and fed ad-libitum energy and Zn (100/100 had increased IGFBP-3 (p Conclusion These results suggest that GHi enhances weight gain in rats with suboptimal energy and Zn intake but does not modify energy expenditure or physical activity index. Suboptimal Zn intake did not exacerbate the reduced growth or decrease in energy expenditure observed with energy restriction.

  10. Recombinant human erythropoietin and hemoglobin concentration at operation and during the postoperative period

    DEFF Research Database (Denmark)

    Qvist, N; Boesby, S; Wolff, B

    1999-01-01

    and 750 ml, respectively. The number of blood transfusions given was significantly lower in the erythropoietin group, with a mean of 0.3 (range 0-6) units compared to 1.6 (0-9) units in the control group (p concentration at the time of surgery and during the week......In a double-blind placebo-controlled study we investigated the effect of recombinant human erythropoietin (r-HuEPO), on the perioperative hemoglobin concentration and the use of blood transfusions in patients undergoing elective colorectal surgery with a preoperative hemoglobin level ... for 4 days before surgery. There were no differences between the two groups with regard to sex, height, weight, serum electrolytes, and liver function tests at study entry. The preentry hemoglobin concentration was similar in the two groups, with a median value of 7.9 (range 5.3-8.5) mmol...

  11. Mass spectrometric analysis of innovator, counterfeit, and follow-on recombinant human growth hormone.

    Science.gov (United States)

    Jiang, Haitao; Wu, Shiaw-Lin; Karger, Barry L; Hancock, William S

    2009-01-01

    We have performed a detailed characterization of recombinant human growth hormone that included the identification of the entire sequence with disulfide linkages as well as subtle modifications by a sensitive liquid chromatography coupled online with tandem mass spectrometry (LC-MS) approach using the accurate peptide mass (FTICR MS) and sequence assignment (MS/MS measurement). The extent of oxidation, deamidation, and chain cleavages were measured by the ratio of peak areas of the nonmodified peptide vs. the sum of peak area of the nonmodified and modified peptides in the same LC-MS analysis. The subtle but distinct differences were found in the recombinant human growth from the three manufacturers (the follow-on, counterfeit, and the original innovator products). In relative comparison, the follow-on product had the highest degree of oxidation at methionine residues, followed by the counterfeit product, and the original innovator product had the least amount of oxidation at all three sites with the similar oxidation order. In cases, the oxidation order was Met14 > Met125 > Met170. In contrast, the follow-on had the least amount of deamidation at aspargine (Asn149), and the counterfeit had the highest degree of deamidation at this site. For the chain cleavage, the follow-on product had the highest cleavage occurring at T 10 peptide (between Asn99 and Ser100), the counterfeit had the highest cleavage on T4 peptide, (between Glu30 and Phe31), and the original innovator product with the least amount of cleavages on both sites. These subtle but distinct differences are likely because of nonidentical manufacturing, formulation procedures, and storage conditions.

  12. Clinical experience with recombinant human thrombopoietin in chemotherapy-induced thrombocytopenia.

    Science.gov (United States)

    Vadhan-Raj, S

    2000-04-01

    Since the identification and cloning of c-Mpl ligand, two forms of recombinant human thrombopoietin have undergone clinical development. Both the full-length molecule, known as rhTPO, and the truncated version of the molecule, known as pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), have been evaluated in phase I/II clinical trials in cancer patients receiving myelosuppressive chemotherapy. Early clinical trials with PEG-rHuMGDF in cancer patients demonstrated its clinical safety and platelet-stimulating activity. However, the development of neutralizing antibodies and clinically significant thrombocytopenia in some patients and normal donors who received PEG-rHuMGDF have led to discontinuation of clinical trials with this molecule in the United States. Clinical experience with rhTPO so far indicates that this full-length glycosylated molecule is remarkably well tolerated and has a favorable safety profile. In these studies, rhTPO exhibited dose-dependent increases in circulating platelet counts and bone marrow megakaryocytes before chemotherapy. In addition, there was an increase in the frequency and proliferation of bone marrow progenitor cells and mobilization of progenitors into the peripheral blood. Early results also showed that rhTPO can attenuate chemotherapy-induced severe thrombocytopenia and reduce the need for platelet transfusions. However, in this setting, the optimal schedule of rhTPO administration may depend on the length of the regimen and anticipated timing of the platelet nadir. These initial results indicate that rhTPO is a safe and potentially useful agent in the prevention and management of chemotherapy-induced thrombocytopenia. Results of larger randomized clinical trials will determine the therapeutic potential of this novel growth factor in various clinical settings.

  13. Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15

    Science.gov (United States)

    Li, Qinglei; Rajanahally, Saneal; Edson, Mark A.; Matzuk, Martin M.

    2009-01-01

    Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin βA (Inhba) and βB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes. PMID:19651638

  14. 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.

  15. The Effect of Human Recombinant Erythropoietin on Prevention of Anemia of Prematurity

    Directory of Open Access Journals (Sweden)

    K Hajian

    2007-06-01

    Full Text Available Objective: Premature infants often develop significant anemia that requires blood transfusion, this carries significant risks. This study was carried out to determine the effect of recombinant human erythropoietin (r-HuEPO on prevention of anemia of prematurity. Material & Methods: From April 2001 to March 2002, 24 neonates in  newborn services at Amirkola childrens hospital randomly were assigned to erythropoietin group and control (no treatment group. Inclusion criteria were birth weight of ≤1750 grams and gestational age ≤34 weeks. Exclusion criteria were problems of hemolytic anemia, congenital infections, congenital malformations, severe asphyxia, intraventricular hemorrhage (grade III and IV, need for exchange transfusion and death during the first week of life. Erythropoietin group received r-HuEPO400 unit/kg/dose subcutaneously three times a week plus 4 mg/kg/day iron orally. White blood cell, hemoglobin (Hgb, hematocrit (Hct, platelet and reticulocyte count were obtained every 2 weeks until the 42nd day of life. Anemia was defined as Hgb≤8gr/dl and Hct≤24%. Student t test and Fisher exact were used to evaluate differences between the two groups.Findings: Hemoglobin and hematocrit values were significantly higher in erythropoietin group than the control group after the 14th day of the study (P<0.04 and this difference was getting higher until the end of the trial (P<0.001. Five neonates developed anemia; all of them were from control group. One of these neonates required transfusion. None of the erythropoietin group newborns developed anemia.Conclusion: The results of this study confirm the efficacy of recombinant human erythropoietin in the prevention of anemia of prematurity.

  16. Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.

    Science.gov (United States)

    Vemu, Annapurna; Atherton, Joseph; Spector, Jeffrey O; Szyk, Agnieszka; Moores, Carolyn A; Roll-Mecak, Antonina

    2016-06-17

    Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

  17. Historical review on the use of recombinant human erythropoietin in chronic renal failure.

    Science.gov (United States)

    Winearls, C G

    1995-01-01

    The success of maintenance haemodialysis in the 1960s was blighted by the problem of anaemia. Treatment with iron, folic acid, androgens and transfusions did no more than minimize its effects. The need for a renewable source of erythropoietin was appreciated very early but the hope took 25 years to realize. Cloning and expression of the human gene was achieved in 1984 and clinical trials planned even before the descriptions of the recombinant hormone were published. The Amgen material was tested in parallel studies in Seattle and England and by the end of 1986 the efficacy of recombinant human erythropoietin (r-HuEPO) given in large intravenous bolus doses in reversing the anaemia of uraemia was established. The benefits were immediately obvious: relief from transfusion dependence was the unequivocal evidence but the effect on 'wellbeing' though subjective was remarkable. Large clinical trials were completed in Europe and the USA so that r-HuEPO was licensed as a therapeutic drug less than two years later. The pilot studies flagged a number of key issues: hypertension, sometimes with encephalopathy, occurred in patients whose blood pressure was labile before treatment; vascular access failure seemed more frequent and hyperkalaemia was thought to reflect less efficient dialysis. Failure to respond focused attention on iron balance as well as on factors such as infection, aluminium, and hyperparathyroidism. A more clear understanding of the pathogenesis of the anaemia of uraemia was made possible by dissection of the specific effects of the exogenous erythropoietin on erythroid function.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Novel monoclonal antibodies broadly reactive to human recombinant sapovirus-like particles.

    Science.gov (United States)

    Kitamoto, Noritoshi; Oka, Tomoichiro; Katayama, Kazuhiko; Li, Tian-Cheng; Takeda, Naokazu; Kato, Yoji; Miyoshi, Tatsuya; Tanaka, Tomoyuki

    2012-11-01

    Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1-7], 7 GII, [GII.1-7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus-like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty-five hybrid clones producing MAbs were obtained. Twenty-four MAbs were characterized by ELISA, according to their cross-reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross-reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup-specific; and (iii) those reactive in a genotype-specific manner. Further analysis of three broadly cross-reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV. © 2012 The Societies and Wiley Publishing Asia Pty Ltd.

  19. 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.

  20. 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.

  1. 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.

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

    Science.gov (United States)

    Simon-Loriere, Etienne; Galetto, Roman; Hamoudi, Meriem; Archer, John; Lefeuvre, Pierre; Martin, Darren P; Robertson, David L; Negroni, Matteo

    2009-05-01

    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.

  3. Molecular Mechanisms of Recombination Restriction in the Envelope Gene of the Human Immunodeficiency Virus

    Science.gov (United States)

    Simon-Loriere, Etienne; Galetto, Roman; Hamoudi, Meriem; Archer, John; Lefeuvre, Pierre; Martin, Darren P.; Robertson, David L.; Negroni, Matteo

    2009-01-01

    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. PMID:19424420

  4. 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...

  5. Comparison of nonhomologous end joining and homologous recombination in human cells

    Science.gov (United States)

    Mao, Zhiyong; Bozzella, Michael; Seluanov, Andrei; Gorbunova, Vera

    2009-01-01

    The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR leads to accurate repair, while NHEJ is intrinsically mutagenic. To understand human somatic mutation it is essential to know the relationship between these pathways in human cells. Here we provide a comparison of the kinetics and relative contributions of HR and NHEJ in normal human cells. We used chromosomally integrated fluorescent reporter substrates for real-time in vivo monitoring of the NHEJ and HR. By examining multiple integrated clones we show that the efficiency of NHEJ and HR is strongly influenced by chromosomal location. Furthermore, we show that NHEJ of compatible ends (NHEJ-C) and NHEJ of incompatible ends (NHEJ-I) are fast processes, which can be completed in approximately 30 min, while HR is much slower and takes 7h or longer to complete. In actively cycling cells NHEJ-C is twice as efficient as NHEJ-I, and NHEJ-I is three times more efficient than HR. Our results suggest that NHEJ is a faster and more efficient DSB repair pathway than HR. PMID:18675941

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

    Science.gov (United States)

    Lai, Xuelei; Soler-Lopez, Montserrat; Wichers, Harry J.

    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 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. PMID:27551823

  7. Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D.

    Science.gov (United States)

    Gonzalez, Gabriel; Koyanagi, Kanako O; Aoki, Koki; Kitaichi, Nobuyoshi; Ohno, Shigeaki; Kaneko, Hisatoshi; Ishida, Susumu; Watanabe, Hidemi

    2014-08-15

    Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic "types" than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (dS). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments "universally conserved segments" (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. 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

  9. Safety and immunogenicity of recombinant human thrombin: a pooled analysis of results from 10 clinical trials.

    Science.gov (United States)

    Singla, Neil K; Foster, Kevin N; Alexander, W Allan; Pribble, John P

    2012-11-01

    To evaluate the safety and immunogenicity of recombinant human thrombin (rThrombin), an active topical stand-alone hemostatic agent. Analysis of pooled data from 10 rThrombin clinical trials. A total of 644 adult and pediatric patients treated with rThrombin; 609 patients were included in the immunogenicity analysis. In all studies, rThrombin was applied during a single surgical procedure (day 1); the procedures consisted of spinal procedures, major hepatic resection, peripheral arterial bypass, arteriovenous graft formation for hemodialysis access, and synchronous burn wound excision and skin grafting. A dosage of 1000 IU/ml of rThrombin was administered for more than 99% of patients. Adverse events and clinical laboratory values were monitored through day 29. Blood samples were obtained for immunogenicity analyses before the procedure and on day 29. Adverse events were mild or moderate in severity for the majority of patients; no patients discontinued from an rThrombin study due to adverse events. The most commonly reported adverse events in the 644 patients were incision site pain (305 patients [47.4%]), procedural pain (215 patients [33.4%]), and nausea (170 patients [26.4%]). Five patients (0.8%) died during the studies; all deaths were considered unrelated to rThrombin treatment. Antibodies to the rThrombin product developed in 5 (0.8%, 95% confidence interval 0.4-2.8%) of 609 patients by day 29, approximately 1 month after treatment; these antibodies did not neutralize the activity of native human thrombin. The development of antibodies did not appear to differ substantively by type of surgical procedure, amount of rThrombin administered, or patient age. Recombinant human thrombin was well tolerated, and adverse events were consistent with those reported in the postoperative setting in the surgical populations studied. Approximately 1 month after treatment, less than 1% of the patients had developed antibodies to the rThrombin product, and these

  10. Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

    Science.gov (United States)

    Kruzel, Marian L; Actor, Jeffrey K; Zimecki, Michał; Wise, Jasen; Płoszaj, Paulina; Mirza, Shaper; Kruzel, Mark; Hwang, Shen-An; Ba, Xueqing; Boldogh, Istvan

    2013-12-01

    Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells

    Science.gov (United States)

    Mao, Zhiyong; Bozzella, Michael; Seluanov, Andrei; Gorbunova, Vera

    2009-01-01

    DNA double-strand breaks (DSBs) are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an intrinsically error-prone pathway while HR results in accurate repair. To understand the origin of genomic instability in human cells it is important to know the contribution of each DSB repair pathway. Studies of rodent cells and human cancer cell lines have shown that the choice between NHEJ or HR pathways depends on cell cycle stage. Surprisingly, cell cycle regulation of DSB repair has not been examined in normal human cells with intact cell cycle checkpoints. Here we measured the efficiency of NHEJ and HR at different cell cycle stages in hTERT-immortalized diploid human fibroblasts. We utilized cells with chromosomally-integrated fluorescent reporter cassettes, in which a unique DSB is introduced by a rare-cutting endonuclease. We show that NHEJ is active throughout the cell cycle, and its activity increases as cells progress from G1 to G2/M (G1human somatic cells utilize error-prone NHEJ as the major DSB repair pathway at all cell cycle stages, while HR is used, primarily, in the S phase. PMID:18769152

  12. 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.

  13. Differential gene expression in human granulosa cells from recombinant FSH versus human menopausal gonadotropin ovarian stimulation protocols

    Directory of Open Access Journals (Sweden)

    Bietz Mandi G

    2010-03-01

    Full Text Available Abstract Background The study was designed to test the hypothesis that granulosa cell (GC gene expression response differs between recombinant FSH and human menopausal gonadotropin (hMG stimulation regimens. Methods Females Results After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11, bone morphogenetic protein receptor II (BMPR2, epidermal growth factor (EGF, insulin-like growth factor binding protein (IGFBP-4, IGFBP-5, and hypoxia-inducible factor (HIF-1 alpha. Conclusions Results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence.

  14. Repetitive in vivo treatment with human recombinant interleukin-1 beta modifies beta-cell function in normal rats

    DEFF Research Database (Denmark)

    Wogensen, L D; Reimers, J; Nerup, J

    1992-01-01

    It is unknown whether interleukin-1 exerts a bimodal effect on Beta-cell function in vivo, and whether interleukin-1 has a diabetogenic action in normal animals. We therefore studied: (a) acute effects 2 h after an intraperitoneal bolus injection of 4 micrograms of recombinant human interleukin-1...

  15. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF) : a randomised, placebo-controlled trial

    NARCIS (Netherlands)

    Teerlink, John R.; Cotter, Gad; Davison, Beth A.; Felker, G. Michael; Filippatos, Gerasimos; Greenberg, Barry H.; Ponikowski, Piotr; Unemori, Elaine; Voors, Adriaan A.; Adams, Kirkwood F.; Dorobantu, Maria I.; Grinfeld, Liliana R.; Jondeau, Guillaume; Marmor, Alon; Masip, Josep; Pang, Peter S.; Werdan, Karl; Teichman, Sam L.; Trapani, Angelo; Bush, Christopher A.; Saini, Rajnish; Schumacher, Christoph; Severin, Thomas M.; Metra, Marco

    2013-01-01

    Background Serelaxin, recombinant human relaxin-2, is a vasoactive peptide hormone with many biological and haemodynamic effects. In a pilot study, serelaxin was safe and well tolerated with positive clinical outcome signals in patients with acute heart failure. The RELAX-AHF trial tested the hypoth

  16. 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...

  17. 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...

  18. Recombinant human thyrotropin-stimulated radioiodine therapy of nodular goiter allows major reduction of the radiation burden with retained efficacy

    DEFF Research Database (Denmark)

    Fast, Søren; Hegedüs, Laszlo; Grupe, Peter;

    2010-01-01

    Context and Objective: Stimulation with recombinant human TSH (rhTSH) before radioiodine ((131)I) therapy augments goiter volume reduction (GVR). Observations indicate that rhTSH has a preconditioning effect beyond increasing thyroid (131)I uptake. We test the hypothesis that an equivalent GVR...

  19. Active immunotherapy of allergic asthma with a recombinant human interleukin-5 protein as vaccine in a murine model

    Institute of Scientific and Technical Information of China (English)

    TAN Guang-hong; WANG Cai-chun; HUANG Feng-ying; WANG Hua; HUANG Yong-hao; LIN Ying-ying

    2007-01-01

    Background Eosinophils are highly related to allergic asthma inflammation. Interleukin (IL)-5 is the major chemokine of eosinophils, inhibition of the activity of IL-5 thus seems to be a potential approach to asthma therapy. The current study was performed to determine whether a recombinant human IL-5 protein as a xenogeneic vaccine has the capability of inducing anti-asthma activities.Methods Recombinant human IL-5 was used as a protein vaccine. Mouse asthma model was established to observe the anti-asthma activities. Lung histology was observed; eosinophils in blood and bronchoalveolar lavage were stained and counted. Airway hyperresponsiveness was determined by whole body plethysmograph. Antibody characters and cytokines were detected with enzyme linked immunosorbent assay (ELISA) and Western blot assay.Results Vaccination with recombinant human IL-5 protein as vaccine significantly reduced airway inflammation and airway hyperresponsiveness, and shifted the cytokine production from Th2 (IL-4) to Th1 (INF-γ) in mice allergic-asthma model. Immunization with recombinant human IL-5 protein vaccine bypassed the immunological tolerance and induced production of polyclonal antibodies that were cross-reactive with murine IL-5.Conclusions Active immunization with xenogeneic homologous IL-5 may be a possible therapeutic approach to the treatment of asthma and potentially of other eosinophilic disorders.

  20. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    DEFF Research Database (Denmark)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban;

    2016-01-01

    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 str...

  1. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF) : a randomised, placebo-controlled trial

    NARCIS (Netherlands)

    Teerlink, John R.; Cotter, Gad; Davison, Beth A.; Felker, G. Michael; Filippatos, Gerasimos; Greenberg, Barry H.; Ponikowski, Piotr; Unemori, Elaine; Voors, Adriaan A.; Adams, Kirkwood F.; Dorobantu, Maria I.; Grinfeld, Liliana R.; Jondeau, Guillaume; Marmor, Alon; Masip, Josep; Pang, Peter S.; Werdan, Karl; Teichman, Sam L.; Trapani, Angelo; Bush, Christopher A.; Saini, Rajnish; Schumacher, Christoph; Severin, Thomas M.; Metra, Marco

    2013-01-01

    Background Serelaxin, recombinant human relaxin-2, is a vasoactive peptide hormone with many biological and haemodynamic effects. In a pilot study, serelaxin was safe and well tolerated with positive clinical outcome signals in patients with acute heart failure. The RELAX-AHF trial tested the hypoth

  2. Recombination analysis of intermediate human adenovirus type 53 in Japan by complete genome sequence.

    Science.gov (United States)

    Kaneko, Hisatoshi; Aoki, Koki; Ishida, Susumu; Ohno, Shigeaki; Kitaichi, Nobuyoshi; Ishiko, Hiroaki; Fujimoto, Tsuguto; Ikeda, Yoshifumi; Nakamura, Masako; Gonzalez, Gabriel; Koyanagi, Kanako O; Watanabe, Hidemi; Suzutani, Tatsuo

    2011-06-01

    Human adenovirus type 53 (HAdV-53) has commonly been detected in samples from epidemic keratoconjunctivitis (EKC) patients in Japan since 1996. HAdV-53 is an intermediate virus, containing hexon-chimeric, penton base and fiber structures similar to HAdV-22 and -37, HAdV-37 and HAdV-8, respectively. HAdV-53-like intermediate strains were first isolated from EKC samples in Japan in the 1980s. Here, the complete genome sequences of three such HAdV-53-like intermediate strains (870006C, 880249C and 890357C) and four HAdV-53 strains were determined, and their relationships were analysed. The seven HAdV strains were classified into three groups, 870006C/880249C, 890357C and the four HAdV-53 strains, on the basis of phylogenetic analyses of the partial and complete genome sequences. HAdV strains within the same group showed the highest nucleotide identities (99.87-100.00 %). Like HAdV-53, the hexon loop 1 and 2 regions of 870006C, 880249C and 890357C showed the highest identity with HAdV-22. However, these strains did not show a hexon-chimeric structure similar to HAdV-22 and -37, or a penton base similar to HAdV-37. The fiber genes of 870006C and 880249C were identical to that of HAdV-37, but not HAdV-8. Thus, the three intermediate HAdVs isolated in the 1980s were similar to each other but not to HAdV-53. The recombination breakpoints were inferred by the Recombination Detection Program (rdp) using whole-genome sequences of these seven HAdV and of 12 HAdV-D strains from GenBank. HAdV-53 may have evolved from intermediate HAdVs circulating in the 1980s, and from HAdV-8, -22 and -37, by recombination of sections cut at the putative breakpoints.

  3. Paclitaxel, ifosfamide and cisplatin with granulocyte colony-stimulating factor or recombinant human interleukin 3 and granulocyte colony-stimulating factor in ovarian cancer : A feasibility study

    NARCIS (Netherlands)

    Veldhuis, GJ; Willemse, PHB; Beijnen, JH; Piersma, H; vanderGraaf, WTA; deVries, EGE; Boonstra, J.

    1997-01-01

    The tolerability and efficacy of four courses of paclitaxel and ifosfamide plus cisplatin every 3 weeks was evaluated in patients with residual or refractory ovarian cancer. Additionally, supportive haematological effects of recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte

  4. Expression of soluble, biologically active recombinant human endostatin in Escherichia coli.

    Science.gov (United States)

    Xu, Han-Mei; Zhang, Guo-Yuan; Ji, Xiao-Dan; Cao, Lin; Shu, Luan; Hua, Zi-Chun

    2005-06-01

    Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in E. coli by employing both co-expression of the molecular chaperones and lower temperature fermentation. Two groups of chaperones Trigger factor and GroEL-GroES (GroEL/ES), DnaK-DnaJ-GrpE and GroEL/ES, were co-expressed, respectively, with rhEndostatin at different temperatures (37, 25, and 16 degrees C). It revealed that low temperature or molecular chaperones alone could enhance the production of active rhEndostatin; meanwhile, combinational employment of low temperature cultivation (16 degrees C) together with co-expression of DnaK-DnaJ-GrpE and GroEL/ES was more effective to prevent aggregation of rhEndostatin. The production of soluble rhEndostatin was about 36 mg/L, and at least 16 mg of rhEndostatin was purified from 1L flask culture. The purified rhEndostatin specifically inhibited the proliferation of endothelial cell-bovine capillary endothelial cell in a dose-dependent manner, and it showed potent anti-angiogenic capability on the chorioallantoic membrane of chick embryo in vivo. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.

  5. Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.).

    Science.gov (United States)

    Steere, Ashley N; Bobst, Cedric E; Zhang, Deshui; Pettit, Steve C; Kaltashov, Igor A; Huang, Ning; Mason, Anne B

    2012-11-01

    The Fe(3+) binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin bind Fe(3+) tightly yet reversibly. Although previously shown to be capable of delivering Fe(3+) to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin might be suitable for consideration in biopharmaceutical applications. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. A macroporous bioreactor super activated by the recombinant human transforming growth factor-beta 3

    Directory of Open Access Journals (Sweden)

    Ugo eRipamonti

    2012-06-01

    Full Text Available Macroporous single-phase hydroxyapatite (HA and biphasic HA/β-tricalcium phosphate with 33% post-sinter hydroxyapatite (HA/β-TCP were combined with 25 or 125 µg recombinant human transforming growth factor-β3 (hTGF-β3 to engineer a super activated bioreactor implanted in orthotopic calvarial and heterotopic rectus abdominis muscle sites and harvested on day 30 and 90. Coral-derived calcium carbonate fully converted (100% and partially converted to 5% and 13% hydroxyapatite/calcium carbonate (HA/CC preloaded with 125 and 250 µg hTGF-β3, and 1:5 and 5:1 binary applications of hTGF-β3: hOP-1 by weight, were implanted in the rectus abdominis and harvested on day 20 and 30, respectively, to monitor spatial/temporal morphogenesis by high doses of hTGF-β3. Bone formation was assessed on decalcified paraffin-embedded sections by measuring the fractional volume of newly-formed bone. On day 30 and 90, single phase HA implants showed greater amounts of bone when compared to biphasic specimens; 5 % and 13 % HA/CC pre-loaded with 125 and 250 µg hTGF-β3 showed substantial induction of bone formation; 250 µg hTGF-β3 induced as yet unreported massive induction of bone formation as early as 20 days prominently outside the profile of the macroporous constructs. The induction of bone formation is controlled by the implanted ratio of the recombinant morphogens, i.e. the 1:5 hTGF-β3:hOP-1 ratio by weight was greater than the inverse ratio. The unprecedented tissue induction by single doses of 250 µg hTGF-β3 resulting in rapid bone morphogenesis of vast mineralized ossicles with multiple trabeculations surfaced by contiguous secreting osteoblasts is the novel molecular and morphological frontier for the induction of bone formation in clinical contexts.

  7. Novel SLA-DQ alleles and their recombinant molecules in xenogeneic stimulation of human T cells.

    Science.gov (United States)

    Chen, Fuxiang; Xie, Jin; Li, Ningli; Zhou, Yun; Xin, Lijun; Chou, Kuang-Yen

    2005-06-01

    MHC class II antigens DR and DQ are essential for graft rejection both in allo- and xeno-transplantation. The antigens, especially the DQA and DQB gene-coencoded DQ molecules, are also involved in transplantation tolerance induced by activation of regulatory T cells. Here we report six novel DQ alleles from three properly inbred Chinese pig strains Gz, Bm and Yn. In our study, cDNA of swine leukocyte antigen (SLA)-DQA and -DQB were amplified by RT-PCR and sequenced for each strain. The ORF-containing SLA-DQA and -DQB genes are composed of 768 (or 765) and 786 nucleotides, encoding antigen molecules of 255 (or 254) and 261 amino acid residues, respectively. Sequences of both SLA-DQA and -DQB alleles showed disparities when compared either among the three pig strains or with available SLA data, which allows our novel alleles receiving their accession numbers from GenBank. The sequence analysis further revealed a phylogenic connection of our SLA-DQ alleles with SLA-DQ(c) haplotype. In addition, the homologies of MHC DQ or DQ-like molecules between Chinese pigs (SLA) and human (HLA) are higher than those between pigs and mice (H-2). By co-transfection of Bm pig DQA and DQB genes into L929 cells, the Bm-DQ heterodimer-expressed cells could effectively stimulate the human lymphoproliferation in presence of human APCs with a mean stimulation index (SI) 9.9+/-1.4. This functional assay indicated that our recombinant DQ antigens are capable of initiating human lymphoproliferation in a xeno-MLR.

  8. Cutaneous Wound Healing After Treatment with Plant-Derived Human Recombinant Collagen Flowable Gel

    Science.gov (United States)

    Roth, Sigal; Amzel, Tal; Harel-Adar, Tamar; Tamir, Eran; Grynspan, Frida; Shoseyov, Oded

    2013-01-01

    Chronic wounds, particularly diabetic ulcers, represent a main public health concern with significant costs. Ulcers often harbor an additional obstacle in the form of tunneled or undermined wounds, requiring treatments that can reach the entire wound tunnel, because bioengineered grafts are typically available only in a sheet form. While collagen is considered a suitable biodegradable scaffold material, it is usually extracted from animal and human cadaveric sources, and accompanied by potential allergic and infectious risks. The purpose of this study was to test the performance of a flowable gel made of human recombinant type I collagen (rhCollagen) produced in transgenic tobacco plants, indicated for the treatment of acute, chronic, and tunneled wounds. The performance of the rhCollagen flowable gel was tested in an acute full-thickness cutaneous wound-healing rat model and compared to saline treatment and two commercial flowable gel control products made of bovine collagen and cadaver human skin collagen. When compared to the three control groups, the rhCollagen-based gel accelerated wound closure and triggered a significant jumpstart to the healing process, accompanied by enhanced re-epithelialization. In a cutaneous full-thickness wound pig model, the rhCollagen-based flowable gel induced accelerated wound healing compared to a commercial product made of bovine tendon collagen. By day 21 post-treatment, 95% wound closure was observed with the rhCollagen product compared to 68% closure in wounds treated with the reference product. Moreover, rhCollagen treatment induced an early angiogenic response and induced a significantly lower inflammatory response than in the control group. In summary, rhCollagen flowable gel proved to be efficacious in animal wound models and is expected to be capable of reducing the healing time of human wounds. PMID:23259631

  9. Recombinant human diamine oxidase activity is not inhibited by ethanol, acetaldehyde, disulfiram, diethyldithiocarbamate or cyanamide.

    Science.gov (United States)

    Bartko, Johann; Gludovacz, Elisabeth; Petroczi, Karin; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-08-01

    Human diamine oxidase (hDAO, EC 1.4.3.22) is the key enzyme in the degradation of extracellular histamine. Consumption of alcohol is a known trigger of mast cell degranulation in patients with mast cell activation syndrome. Ethanol may also interfere with enzymatic histamine degradation, but reports on the effects on DAO activity are controversial. There are also conflicting reports whether disulfiram, an FDA-approved agent in the treatment of alcohol dependence, inhibits DAO. We therefore investigated the inhibitory potential of ethanol and disulfiram and their metabolites on recombinant human DAO (rhDAO) in three different assay systems. Relevant concentrations of ethanol, acetaldehyde, and acetate did not inhibit rhDAO activity in an in vitro assay system using horseradish peroxidase (HRP) -mediated luminol oxidation. The aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors cyanamide and its dimer dicyanamide also had no effect on DAO activity. In one assay system, the irreversible ALDH inhibitor disulfiram and its main metabolite diethyldithiocarbamate seemed to inhibit DAO activity. However, the decreased product formation was not due to a direct block of DAO activity but resulted from inhibition of peroxidase employed in the coupled system. Our in vitro data do not support a direct blocking effect of ethanol, disulfiram, and their metabolites on DAO activity in vivo.

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

    Directory of Open Access Journals (Sweden)

    Hempel Casper

    2008-01-01

    Full Text Available Abstract 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 haematopoietic properties – significant anti-inflammatory, antioxidant and anti-apoptotic effects in various brain disorders. The neurobiological responses to exogenously injected Epo during murine CM were examined. Methods Female C57BL/6j mice (4–6 weeks, infected with Plasmodium berghei ANKA, were treated 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 expression in brain tissue was measured by real time PCR. Results Treatment with rhEpo increased survival in mice with CM in a dose- and time-dependent manner and reduced apoptotic cell death of neurons as well as the expression of pro-inflammatory cytokines in the brain. This neuroprotective effect appeared to be independent of the haematopoietic effect. Conclusion These results and its excellent safety profile in humans makes rhEpo a potential candidate for adjunct treatment of CM.

  11. Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells.

    Science.gov (United States)

    Kaszubska, W; Zhang, H; Patterson, R L; Suhar, T S; Uchic, M E; Dickinson, R W; Schaefer, V G; Haasch, D; Janis, R S; DeVries, P J; Okasinski, G F; Meuth, J L

    2000-03-01

    Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.

  12. Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin.

    Science.gov (United States)

    Matsumura, I; Kanakura, Y; Kato, T; Ikeda, H; Ishikawa, J; Horikawa, Y; Hashimoto, K; Moriyama, Y; Tsujimura, T; Nishiura, T

    1995-07-15

    Thrombopoietin (TPO) is a newly identified hematopoietic growth factor that stimulates both megakaryopoiesis and thrombopoiesis through its interaction with a specific cell surface receptor encoded by the c-mpl proto-oncogene. In an effort to investigate the effect of TPO on human myeloid leukemia cells, the expression of c-mpl and the proliferative response to recombinant human (rh) TPO were investigated in a series of patients with acute myeloblastic leukemia (AML). Of 50 cases of AML, the c-mpl mRNA was detectable by means of Northern blot analysis in 26 cases, and the in vitro treatment with rhTPO led to proliferation of AML cells in 22 cases. The c-mpl expression and proliferative response to rhTPO was observed in all subtypes of AML and did not correlate with French-American-British classification, whereas all cases of M7-type AML cells expressed c-mpl and proliferated in response to rhTPO. Furthermore, rhTPO-induced proliferation of AML cells was augmented with the addition of interleukin-3 (IL-3), IL-6, stem cell factor, or granulocyte-macrophage colony-stimulating factor. These results suggested that c-mpl may be functional in terms of supporting proliferation of various types of AML cells and that TPO may contribute, at least in part, to abnormal growth of the cells, especially in combination with other hematopoietic growth factors.

  13. Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay

    Directory of Open Access Journals (Sweden)

    Zahra YADEGARI

    2015-10-01

    Full Text Available Background: Amelogenins are the major components of enamel matrix proteins. Enamel matrix derivatives (EMD can be used in periodontal diseases to regenerate periodontal tissues. The main aim of this study was to evaluate ex-pression of full-length functional recombinant human amelogenin (rhAm in Iranian lizard Leishmania (I.L.L. as an alternative eukaryotic expression system.Methods: Human cDNA encoding a 175-amino acid amelogenin expression cassette was sub cloned into a pLEXSY vector. The construct was transferred into Leishmania cells by electroporation. The protein production was surveyed in the transcription and the translation levels. The expressed protein was purified and some of its biological properties were investigated in comparison to EMD and negative control.Results: Expression of rhAm was confirmed by RT-PCR and western blot test in Leishmania cells. Purified rhAm sig-nificantly inhibited the formation of tartrate-resistant acid phosphatase positive (TRAP+ multinuclear cells in calcitriol stimulated mouse marrow cultures. Moreover, it significantly promoted proliferation and DNA synthesis in L929 mouse fibroblast cells.Conclusion: Functional rhAm was successfully expressed in I.L.L. Easy handling and post translation modification were the main advantages of this expression system. It is suggested to investigate molecular properties of this rhAm in the future.

  14. Transgenic milk containing recombinant human lactoferrin modulates the intestinal flora in piglets.

    Science.gov (United States)

    Hu, Wenping; Zhao, Jie; Wang, Jianwu; Yu, Tian; Wang, Jing; Li, Ning

    2012-06-01

    Lactoferrin (LF) is a beneficial multifunctional protein in milk. The objective of this study was to determine whether bovine transgenic milk containing recombinant human lactoferrin (rhLF) can modulate intestinal flora in the neonatal pig as an animal model for the human infant. We fed 7-day-old piglets (i) ordinary whole milk (OM), (ii) a 1:1 mixture of OM and rhLF milk (MM), or (iii) rhLF milk (LFM). LFM provided better average daily mass gain than OM (P = 0.007). PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis revealed that the LFM piglets exhibited more diversity of the intestinal flora than the OM group. Except for the colon in the LFM group, an increasing trend in microbial diversity occurred from the duodenum to the colon. Fecal flora was not different across different ages or different treatment groups, but a cluster analysis showed that the fecal flora of OM- and MM-fed piglets had a higher degree of similarity than that of LFM-fed piglets. Based on culture-based bacterial counts of intestinal content samples, concentrations of Salmonella spp. in the colon and of Escherichia coli throughout the intestine were reduced with LFM (P intestine were also increased with LFM (P ≤ 0.01). We suggest that rhLF can modulate the intestinal flora in piglets.

  15. Production of recombinant human bile salt-stimulated lipase in Pichia pastoris.

    Science.gov (United States)

    Murasugi, A; Asami, Y; Mera-Kikuchi, Y

    2001-11-01

    Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris. Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7. Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.

  16. An efficient procedure for purification of recombinant human β heat shock protein 90

    Directory of Open Access Journals (Sweden)

    M Bandehpour

    2010-03-01

    Full Text Available "nBackground and the purpose of the study: Heat Shock Protein 90 (Hsp90 is typically the most abundant chaperone in the eukaryotic cell cytoplasm, and its expression is essential for loading immunogenic peptides onto major histocompatibility complex molecules for presentation to T-cells. Therefore, it may act as a good candidate as an adjuvant molecule in vaccine technology. "n Methods: Initially the human Hsp90β gene was cloned into the heat inducible expression vector pGP1-2 and then the recombinant protein was isolated by ion exchange chromatography. After intradermal injection of confirmed purified band of protein to rabbits and isolation of the serum IgG antibody, for its affinity purification, the rabbit’s purified Hsp90 specific IgG was coupled to the cyanogen bromide-activated Sepharose 4B. Results: The recovery of the purified protein of interest by affinity chromatography was 50% . "n "nConclusion: This research enabled purification of human heat shock protein by a laboratory "n "nprepared column chromatography. "n   

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

    DEFF Research Database (Denmark)

    Wogensen, L D; Welinder, B; Hejnaes, K R

    1991-01-01

    -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......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...... the circulation with a T1/2 alpha of 2.9 min and a T1/2 beta of 41.1 min. The central and peripheral volume of distribution was 20.7 and 19.1 ml/rat, respectively, and the metabolic clearance rate was 16.9 ml/min/kg. The kidney and liver showed the highest accumulation of tracer, and autoradiography demonstrated...

  19. Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex

    Science.gov (United States)

    Willhoft, Oliver; Bythell-Douglas, Rohan; McCormack, Elizabeth A.; Wigley, Dale B.

    2016-01-01

    We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding. PMID:27257055

  20. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    OpenAIRE

    2016-01-01

    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 ...

  1. The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins.

    Science.gov (United States)

    Bosze, Zs; Hiripi, L; Carnwath, J W; Niemann, H

    2003-10-01

    Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.

  2. Patterns of Human Immunodeficiency Virus type 1 recombination ex vivo provide evidence for coadaptation of distant sites, resulting in purifying selection for intersubtype recombinants during replication

    DEFF Research Database (Denmark)

    Galli, Andrea; Kearney, Mary; Nikolaitchik, Olga A

    2010-01-01

    High-frequency recombination is a hallmark of HIV-1 replication. Recombination can occur between two members of the same subtype or between viruses from two different subtypes, generating intra- or intersubtype recombinants, respectively. Many intersubtype recombinants have been shown to circulate....../B) and between viruses with pol genes from subtype B or F (B/F). Recombination events generated during a single cycle of infection without selection pressure on pol gene function were analyzed by single-genome sequencing. We found that recombination occurred slightly ( approximately 30%) less frequently in B...... subtypes; these sites may be segregated by recombination events, causing the newly generated intersubtype recombinants to undergo purifying selection. Therefore, the ability of the recombinants to replicate is the major barrier for many of these viruses....

  3. Clinical efficacy and safety of recombinant canine erythropoietin in dogs with anemia of chronic renal failure and dogs with recombinant human erythropoietin-induced red cell aplasia.

    Science.gov (United States)

    Randolph, John E; Scarlett, Janet; Stokol, Tracy; MacLeod, James N

    2004-01-01

    The efficacy and safety of recombinant canine erythropoietin (rcEPO) therapy was evaluated in 19 dogs with anemia of chronic renal failure (group 1) and 6 dogs with chronic renal failure and recombinant human erythropoietin (rhEPO)-induced red cell aplasia (group 2). Hematocrit (Hct) and absolute reticulocyte count (ARC) were monitored weekly for the first 8 weeks, CBC (including ARC) and serum iron profiles were evaluated monthly, and serum biochemical analyses were performed every 2 months for 6 (group 2) to 12 (group 1) months. For group 1 dogs, median Hct and ARC increased significantly during the 1st week of rcEPO treatment, and median Hct was sustained at >35% after week 5. In contrast, median Hct and ARC for group 2 did not change significantly with rcEPO treatment, even with doses greater than those used in group 1. Nevertheless, 2 (33%) of the 6 dogs in group 2 developed erythroid hyperplasia, reticulocytosis, and increases in Hct with rcEPO treatment. Although median systolic blood pressure did not change significantly in either group, 5 dogs developed systolic blood pressures > or = 180 mm Hg during the study. Appetite and energy level improved in most group 1 dogs with increases in Hct. Recombinant cEPO stimulated erythrocyte production in dogs with nonregenerative anemia secondary to chronic renal failure without causing the profound erythroid hypoplasia that can occur in rhEPO-treated dogs. Unfortunately, rcEPO was not as effective in restoring erythrocyte production in dogs that had previously developed rhEPO-induced red cell aplasia.

  4. Evaluation of complications associated with off-label use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in pediatric orthopaedics.

    Science.gov (United States)

    Stiel, Norbert; Hissnauer, Tim N; Rupprecht, Martin; Babin, Kornelia; Schlickewei, Carsten W; Rueger, Johannes M; Stuecker, Ralf; Spiro, Alexander S

    2016-12-01

    The off-label use of recombinant human bone morphogenetic protein-2 to promote bone healing in adults has significantly increased in recent years, while reports of recombinant human bone morphogenetic protein-2 application in children and adolescents are very rare. The aim of this study was to evaluate the safety of single and repetitive recombinant human bone morphogenetic protein-2 use in pediatric orthoapedics. Therefore we reviewed the medical records of 39 patients who had been treated with recombinant human bone morphogenetic protein-2 at our institution. Their mean age was 10.9 years. Recombinant human bone morphogenetic protein-2 was used in 17 patients for spine fusion, in 11 patients for the treatment of congenital pseudarthrosis of the tibia or tibial nonunion, in 5 patients for the management of femoral nonunion, in 5 patients for nonunions at other locations, and in 1 case for tibial shortening. Special attention was paid to identify all adverse events that may be attributed to recombinant human bone morphogenetic protein-2 use, including local inflammatory reactions, allergic reactions, systemic toxicity, excessive wound swelling, hematoma, compartment syndrome, infection, heterotopic ossification, excessive bone growth, carcinogenicity, and the consequences of repeated applications of recombinant human bone morphogenetic protein-2. Follow-up was a mean of 39 months. Forty-six operations with application of rhBMP-2 were performed. Complications that may be due to application of recombinant human bone morphogenetic protein-2 were seen after 18 operations including swelling, increase in temperature, wound secretion, redness and hyperthermia. We consider the three cases of necessary revisions, one due to hematoma, one due to development of a compartment syndrome, and one due to deep infection, to be the only complications related to the use of recombinant human bone morphogenetic protein-2. In conclusion, we found few complications attributable to

  5. 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.

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

    Science.gov (United States)

    Wang, Hua; Liu, Jingning; Yin, Ying; Wu, Jun; Wang, Zilu; Miao, Dengshun; Sun, Wen

    2014-01-01

    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.

  7. Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

    Science.gov (United States)

    Santhanam, R; Naz, R K

    2001-09-01

    A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

  8. 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...

  9. Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

    Science.gov (United States)

    Papanagiotou, Marianthi; Dailiana, Zoe H; Karachalios, Theophilos; Varitimidis, Sokratis; Hantes, Michael; Dimakopoulos, Georgios; Vlychou, Marianna; Malizos, Konstantinos N

    2017-01-01

    AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7 (rhBMP-7) for the treatment of nonunions. METHODS Bone morphogenetic proteins (BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients (80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent joints were also carried out. Factors related to the patient (age, gender), the nonunion (location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure (graft and fixation type, amount of rhBMP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ2 test was performed. RESULTS Eighty point nine percent of the nonunions treated with rhBMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients (17.8%) and it was apparent in the routine radiological evaluation of the nonunion site, in a mean time of 5.5 mo after the rhBMP-7 application (range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhBMP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient’s gender was the only important factor for the development of heterotopic ossification (P = 0.007). CONCLUSION Heterotopic ossification after the use of rhBMP-7 in nonunions was

  10. Combination therapy with sivelestat and recombinant human soluble thrombomodulin for ARDS and DIC patients

    Directory of Open Access Journals (Sweden)

    Miyoshi S

    2014-09-01

    Full Text Available Seigo Miyoshi,1 Ryoji Ito,1 Hitoshi Katayama,1 Kentaro Dote,2 Mayuki Aibiki,3 Hironobu Hamada,1,4 Takafumi Okura,1 Jitsuo Higaki1 1Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University Graduate School of Medicine, 2Intensive Care Division, Ehime University Hospital, 3Department of Emergency and Critical Care Medicine, School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, 4Department of Physical Analysis and Therapeutic Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima, Japan Background: Neutrophil elastase, alveolar thrombin generation, and fibrin deposition play crucial roles in the development of acute respiratory distress syndrome (ARDS and disseminated intravascular coagulation (DIC. However, the usefulness of combination therapy with a selective neutrophil elastase inhibitor, sivelestat, and recombinant human soluble thrombomodulin (rhTM for patients with ARDS and DIC remains unknown. Methods: We conducted a retrospective data analysis of 142 ARDS patients with DIC to assess the effects of sivelestat combined with rhTM. Patients were divided into four groups: control (no sivelestat or rhTM treatment, sivelestat treatment alone, rhTM treatment alone, and combined treatment with sivelestat and rhTM. A Cox proportional hazard model was used to assess subject mortality rates. The efficacy of these drugs was evaluated based on survival rate, number of ventilator-free days, and change in PaO2/FIO2 (P/F ratios and DIC scores before and at 7 days after a diagnosis of ARDS with DIC. Results: Multivariate analysis showed that patient age, combination therapy, gas exchange, organ failure, cause, associated disease score, and serum C-reactive protein levels were predictors of mortality for patients with ARDS and DIC. As compared with untreated controls, combination therapy significantly improved the 60-day survival rate of patients with ARDS and DIC

  11. Effect of Recombinant Human Lecithin Cholesterol Acyltransferase Infusion on Lipoprotein Metabolism in Mice

    Science.gov (United States)

    Vaisman, Boris; Auerbach, Bruce; Krause, Brian R.; Homan, Reyn; Stonik, John; Csako, Gyorgy; Shamburek, Robert; Remaley, Alan T.

    2010-01-01

    Lecithin cholesterol acyl transferase (LCAT) deficiency is associated with low high-density lipoprotein (HDL) and the presence of an abnormal lipoprotein called lipoprotein X (Lp-X) that contributes to end-stage renal disease. We examined the possibility of using LCAT an as enzyme replacement therapy agent by testing the infusion of human recombinant (r)LCAT into several mouse models of LCAT deficiency. Infusion of plasma from human LCAT transgenic mice into LCAT-knockout (KO) mice rapidly increased HDL-cholesterol (C) and lowered cholesterol in fractions containing very-low-density lipoprotein (VLDL) and Lp-X. rLCAT was produced in a stably transfected human embryonic kidney 293f cell line and purified to homogeneity, with a specific activity of 1850 nmol/mg/h. Infusion of rLCAT intravenously, subcutaneously, or intramuscularly into human apoA-I transgenic mice showed a nearly identical effect in increasing HDL-C approximately 2-fold. When rLCAT was intravenously injected into LCAT-KO mice, it showed a similar effect as plasma from human LCAT transgenic mice in correcting the abnormal lipoprotein profile, but it had a considerably shorter half-life of approximately 1.23 ± 0.63 versus 8.29 ± 1.82 h for the plasma infusion. rLCAT intravenously injected in LCAT-KO mice crossed with human apolipoprotein (apo)A-I transgenic mice had a half-life of 7.39 ± 2.1 h and increased HDL-C more than 8-fold. rLCAT treatment of LCAT-KO mice was found to increase cholesterol efflux to HDL isolated from mice when added to cells transfected with either ATP-binding cassette (ABC) transporter A1 or ABCG1. In summary, rLCAT treatment rapidly restored the normal lipoprotein phenotype in LCAT-KO mice and increased cholesterol efflux, suggesting the possibility of using rLCAT as an enzyme replacement therapy agent for LCAT deficiency. PMID:20605907

  12. 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.

  13. 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.

  14. Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency.

    Science.gov (United States)

    Farrag, Mohamed A; Amer, Haitham M; Öhlschläger, Peter; Hamad, Maaweya E; Almajhdi, Fahad N

    2017-03-08

    The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codon-optimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M282-90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8(+) T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

  15. Effects of recombinant human growth hormone on growth of human gastric carcinoma xenograft model in nude mice

    Institute of Scientific and Technical Information of China (English)

    Dao-Ming Liang; Jia-Yong Chen; Yi Zhang; Ping Gan; Jie Lin; An-Bao Chen

    2006-01-01

    AIM: To study effects of recombinant human growth hormone (rhGH) on growth of a human gastric carcinoma cell in vivo.METHODS: Experimental mice were divided into control group, rhGH group, oxaliplatin (L-OHP) group and rhGH+L-OHP group. Cultured human gastric carcinoma cells BGC823 were inoculated into right axilla of nude mice and carcinoma xenograft model wasestablished successfully. Inhibitory rate of xenograft tumor growth was estimated by measuring tumor volume; expression of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 proteins of xenograft tumor was detected using immunohistochemical S-P method.RESULTS: Tumor growth inhibitory rate, the positive expression rate of PCNA, Bax and Bcl-2 were 49.3%,58.2%, 65.2% and 59.2% in rhGH+L-OHP group respectively; 46.6%, 62.5%, 59.7% and 64.7% in L-OHP group; 5.0%, 82.7%, 23.2% and 82.2% in rhGH group and 0, 77.8%, 23.5% and 80.3% in control group. There was significant difference between rhGH+L-OHP group (or L-OHP group ) and control group or rhGH group (P <0.05), whereas there were no significant differences (P >0.05) between L-OHP group and rhGH+L-OHP group and between rhGH group and control group.CONCLUSION: rhGH does not accelerate the proliferation of human gastric cancer cell in vivo.

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

    Science.gov (United States)

    Atsmon, Jacob; Brill-Almon, Einat; Nadri-Shay, Carmit; Chertkoff, Raul; Alon, Sari; Shaikevich, Dimitri; Volokhov, Inna; Haim, Kirsten Y; Bartfeld, Daniel; Shulman, Avidor; Ruderfer, Ilya; Ben-Moshe, Tehila; Shilovitzky, Orit; Soreq, Hermona; Shaaltiel, Yoseph

    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 50nmol/kg PRX-105, 2min before being exposed to 1.33×LD50 and 1.5×LD50 of toxin and 10min after exposure to 1.5×LD50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t½) 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 t½ in humans was substantially longer than in mice (average 26.7h). 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.

  17. EFFECTS OF TRANSFORMING GROWTH FACTOR β AND RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 ON HUMAN PERIODONTAL LIGAMENT FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    司晓辉; 刘正

    2001-01-01

    Objective To evaluate the effects of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP2) on human periodontal ligament fibroblasts ( HPDLFs ). Methods HPDLFs were done primary culture to detect the distinct concentrations of TGF-β and rhBMP2 on its proliferation, alkaline phosphatase (ALP) activity, osteocalcin ( OC) synthesis and formation of the mineralized nodules, respectively. Results TGF-β(5~100ng /ml) significantly stimulated the proliferation of HPDLFs. The ALP activity of HPDLFs was evaluated evidently by 5ng /ml TGF-β. TGF-β(0.5~100ng /ml) had no effects on OC synthesis and formation of the mineralized nodules of HPDLFs. rhBMP2 (0.25~2mg/ ml) had no rernarkable effect on the proliferation of HPDLFs. The ALP activity, OC synthesis and formation of the mineralized nodules of HPDLFs were significantly stimulated by 0.5~2mg/ml rhBMP2. Conclusion The effects of TGF-β and rhBMP2 on HPDLFs are dose-dependent. TGF-β can stimulate HPDLFs to express the early marker of osteoblastic phenotype , and it lacks the ability to promote maturation of the osteogenic phenotype. rhBMP2 can not only stimulate the expression but also promote the maturation of osteoblastic phenotype of HPDLFs.

  18. Excretory/secretory products of Fasciola hepatica but not recombinant phosphoglycerate kinase induce death of human hepatocyte cells.

    Science.gov (United States)

    Bąska, Piotr; Norbury, Luke J; Wiśniewski, Marcin; Januszkiewicz, Kamil; Wędrychowicz, Halina

    2013-06-01

    The liver fluke Fasciola hepatica infects a wide range of hosts, and has a considerable impact on the agriculture industry, mainly through infections of sheep and cattle. Further, human infection is now considered of public health importance and is hyperendemic in some regions. The fluke infection causes considerable damage to the hosts' liver. However, the mechanisms of liver destruction have not yet been completely elucidated. In the present report we incubated a human liver cell line in the presence of either F. hepatica excretory/secretory material (FhES) or recombinant phosphoglycerate kinase (FhPGK). Dosedependent cytotoxicity in the presence of FhES was observed, indicating that FhES is capable of killing human hepatocytes, supporting a role for FhES in damaging host liver cells during infection; while treatment with a recombinant intracellular protein - FhPGK, had no impact on cell survival.

  19. Comparative pharmacokinetics and pharmacodynamics of a PEGylated recombinant human growth hormone and daily recombinant human growth hormone in growth hormone-deficient children

    Directory of Open Access Journals (Sweden)

    Hou L

    2015-12-01

    Full Text Available Ling Hou,1,* Zhi-hang Chen,2,* Dong Liu,3 Yuan-guo Cheng,2 Xiao-ping Luo1 1Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 2Department of Pharmacy, Beijing Institute of Microbiology and Epidemiology, Beijing, 3Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China *These authors contributed equally to this study Objective: Recombinant human growth hormone (rhGH replacement therapy in children generally requires daily subcutaneous (sc injections, which may be inconvenient for patients. Jintrolong® is a PEGylated rhGH with the purpose of weekly sc injections. The aim of the current study was to examine the pharmacokinetics, pharmacodynamics, safety, and tolerability of multiple sc doses of Jintrolong® vs daily doses of rhGH. Design and methods: Twelve children with growth hormone deficiency participated in this single-center, open-label, crossover Phase I trial. All subjects received daily sc injections of rhGH at 0.0286 mg/kg/d for 7 days, followed by a 4-week washout period and six weekly doses of Jintrolong® at 0.2 mg/kg/w. Results: In comparison with rhGH, sc injection of Jintrolong® produced a noticeably higher Cmax, significantly longer half-life (t1/2, and slower plasma clearance, signifying a profile suitable for long-term treatment. The ratio of the area under the concentration vs time curve (AUC after the seventh and first injections (AUC(0–∞7th/AUC(0–∞1st of rhGH was 1.02, while the AUC(0–∞6th/AUC(0–∞1st of Jintrolong® was 1.03, indicating no accumulation of circulating growth hormone. There was no significant difference in the change in insulin-like growth factor-1 expression produced by 7 days of sc rhGH and weekly Jintrolong® injections. There were no severe adverse events during the trial. Conclusion: The elimination rate of Jintrolong® was

  20. Hotspots of homologous recombination in the human genome: not all homologous sequences are equal.

    Science.gov (United States)

    Lupski, James R

    2004-01-01

    Homologous recombination between alleles or non-allelic paralogous sequences does not occur uniformly but is concentrated in 'hotspots' with high recombination rates. Recent studies of these hotspots show that they do not share common sequence motifs, but they do have other features in common.

  1. Hotspots of homologous recombination in the human genome: not all homologous sequences are equal

    OpenAIRE

    Lupski, James R

    2004-01-01

    Homologous recombination between alleles or non-allelic paralogous sequences does not occur uniformly but is concentrated in 'hotspots' with high recombination rates. Recent studies of these hotspots show that they do not share common sequence motifs, but they do have other features in common.

  2. Recombinant human C1-inhibitor inhibits cytotoxicity induced by allo- and xenoantibodies.

    Science.gov (United States)

    Poirier, N; Blancho, G

    2008-03-01

    Antibody-mediated rejection (AMR) is usually poorly controlled, especially in the context of pretransplant immunization, and remains an unsolved issue in xenotransplantation. In order to study prevention and/or treatment of AMR through an early blockade of the complement classical pathway, we designed two strategies to test the effect of a new recombinant human C1-inhibitor that inhibits C1 esterase (rhC1-INH; Pharming, The Netherlands), in a complement-dependent cytotoxicity assay, in the contexts of pretransplant anti-donor alloimmunization and pig-to-primate combinations in order to compare the situations. RhC1-INH appeared to be efficient, in allo- and xenotransplantation settings to block cytotoxicity when given at the initiation of (preventive strategy) or during (curative strategy) the cytotoxicity assay. Importantly, we showed that a small amount of exogenous rhC1-INH was sufficient to prevent cytotoxicity induced by anti-donor alloantibody, thus possibly helping to prevent or treat AMR in preimmunized patients. These in vitro data lead to future in vivo studies in models of AMR in pigs and baboons in allotransplantation and xenotransplantation, in which cytotoxicity due to Gal and non-Gal antibodies is so detrimental.

  3. Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.

    Science.gov (United States)

    Mukaiyama, Hiroyuki; Giga-Hama, Yuko; Tohda, Hideki; Takegawa, Kaoru

    2009-11-01

    The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane.

  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. Pharmaceutical aspects of the recombinant human serum albumin dimer: structural characteristics, biological properties, and medical applications.

    Science.gov (United States)

    Taguchi, Kazuaki; Chuang, Victor Tuan Giam; Maruyama, Toru; Otagiri, Masaki

    2012-09-01

    Human serum albumin is the most abundant protein in the blood. It is clinically used in the treatment of severe hypoalbuminemia and as a plasma expander. The use of albumins as a carrier for drugs is currently being developed, and some are now in the preclinical and clinical trial stages. The main technologies for utilizing an albumin as a drug carrier are protein fusion, polymerization and surface modification, and so on. Among these technologies, albumin dimerization has wide clinical applications as a plasma expander as well as a drug carrier. Despite the fact that many reports have appeared on drugs using an albumin dimer as a carrier, our knowledge of the characteristics of the albumin dimer itself is incomplete. In this review, we summarize the structural characteristics of recombinant albumin dimers produced by two methods, namely, chemical linkage with 1,6-bis(maleimido)hexane and genetically linked with an amino acid linker, and the physicochemical characteristics and biological properties of these preparations. Finally, the potential for pharmaceutical applications of albumin dimers in clinical situations is discussed.

  6. 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.

  7. Pregnancy rates with recombinant versus urinary human chorionic gonadotropin in in vitro fertilization: an observational study.

    Science.gov (United States)

    Zeke, József; Kanyó, Katalin; Zeke, Helga; Cseh, Aron; Vásárhelyi, Barna; Szilágyi, András; Konc, János

    2011-01-01

    Randomized clinical trials (RCTs) demonstrated the equal efficacy of urinary human chorionic gonadotropin (uhCG) and recombinant hCG (rhCG) products in in vitro fertilisation (IVF). However, limitations inherent with RCTs necessitate the reinforcement of RCT results in real-life. We retrospectively analyzed pregnancies after treatment with rhCG and uhCG products (n = 391, and 96, resp.). We found that laboratory-verified pregnancy occurred more frequently in rhCG patients than in those on uhCG (43% versus 30%, P = 0.02). The association remains significant (P = 0.002) after its adjustment for clinical characteristics. The prevalence of laboratory-verified pregnancies was higher with GnRH agonist use (P = 0.012) and BMI under 30 kg/m(2) (P = 0.053) while decreased the age (P = 0.014) and the number of previous failed attempts (P = 0.08). Similar (but not significant) trends were observed with rates of pregnancy filled the 24th week. These results reinforce RCTs supporting the notion that rhCG is more efficient as uhCG during IVF.

  8. Multiple cycles of recombinant human thrombopoietin therapy in a patient with chronic refractory idiopathic thrombocytopenic purpura.

    Science.gov (United States)

    Hua, Baolai; Zou, Nong; Wang, Shujie; Zhu, Tienan; Zhao, Yongqiang

    2005-06-01

    We describe a 41-year-old woman with chronic idiopathic thrombocytopenic purpura who received recombinant human thrombopoietin (rhTPO) therapy. rhTPO was administrated subcutaneously at a dosage of 1.0 mug/kg daily for a maximum of 14 days until the platelet count was more than 50 x 10/l. The patient received three cycles (six, 13, and eight doses each) of rhTPO, each initiated when the platelet counts was less than 10 x 10/l. The platelet count increased to above 50 x 10/l on days 5, 11 and 8, and peaked at 456 x 10/l, 130 x 10/l and 82 x 10/l on days 9, 15 and 13 in the three respective cycles, each followed by a gradual decline. The durations of platelet counts at more than 50 x 10/l in the three cycles were 13, 7 and 10 days, respectively. rhTPO was well tolerated with no adverse event observed. Antibodies to rhTPO by enzyme-linked immunosorbent assay were not detected. Our observations suggested that rhTPO could transiently increase the peripheral platelet count in patients with chronic refractory idiopathic thrombocytopenic purpura. The reasons why the peak platelet counts decreased and the duration of response shortened after successive cycles of treatment were unclear.

  9. Recombinant Human Thrombopoietin Treatment Promotes Hematopoiesis Recovery in Patients with Severe Aplastic Anemia Receiving Immunosuppressive Therapy

    Directory of Open Access Journals (Sweden)

    Huaquan Wang

    2015-01-01

    Full Text Available Objective. To assess the effectiveness of recombinant human thrombopoietin (rhTPO in severe aplastic anemia (SAA patients receiving immunosuppressive therapy (IST. Methods. Eighty-eight SAA patients receiving IST from January 2007 to December 2012 were included in this retrospective analysis. Of these, 40 subjects received rhTPO treatment (15000 U, subcutaneously, three times a week. rhTPO treatment was discontinued when the platelet count returned to normal range. Hematologic response, bone marrow megakaryocyte recovery, and time to transfusion independence were compared. Results. Hematologic response was achieved in 42.5%, 62.5%, and 67.5% of patients receiving rhTPO and 22.9%, 41.6%, and 47.9% of patients not receiving rhTPO at 3, 6, and 9 months after treatment, respectively (P = 0.0665, P = 0.0579, and P = 0.0847, resp.. Subjects receiving rhTPO presented an elevated number of megakaryocytes at 3, 6, and 9 months when compared with those without treatment (P = 0.025, P = 0.021, and P = 0.011, resp.. The time to platelet and red blood cell transfusion independence was shorter in patients who received rhTPO than in those without rhTPO treatment. Overall survival rate presented no differences between the two groups. Conclusion. rhTPO could improve hematologic response and promote bone marrow recovery in SAA patients receiving IST.

  10. Aqueous stability of recombinant human thrombopoietin as a function of processing schemes.

    Science.gov (United States)

    Senderoff, R I; Kontor, K M; Heffernan, J K; Clarke, H J; Garrison, L K; Kreilgaard, L; Lasser, G W; Rosenberg, G B

    1996-07-01

    Preformulation studies conducted with recombinant human thrombopoietin (rhTPO), a 332 amino acid glycoprotein which stimulates platelet production, show distinctions in degradation profiles as a function of processing schemes. The stability-limiting degradation pathways change as a function of purification stage and method and are dependent upon the presence of contaminating protease. The stability-limiting degradation pathway of affinity-purified and in-process rhTPO preparations is primarily attributed to proteolysis initiated by a protease present as a fermentation contaminant. The proteolysis increases with increasing pH as a function of temperature. The degradation profiles for these preparations show that bioactivity initially increases and then decreases with increasing pH as a function of temperature. This is consistent with proteolysis to active forms which ultimately undergo degradation to less active forms. Similar studies conducted with rhTPO preparations purified by a combination of more conventional chromatographic steps show different stability-limiting degradation pathways and a different pH-stability profile when compared to affinity purified or in-process preparations. In this case, degradation is accompanied by decreases in activity under all conditions, consistent with the conversion to less active forms. These results illustrate the importance of preformulation and stability characterization of protein pharmaceuticals in support of both process and formulation development. Issues related to storage and handling of inprocess preparations differ from those with formulated product since the stability-limiting degradation pathways change as a function of purification stage.

  11. Effect of recombinant human thrombopoietin in nonhuman primates with chemotherapy-induced thrombocytopenia.

    Science.gov (United States)

    Akahori, H; Shibuya, K; Obuchi, M; Nishizawa, Y; Tsuji, A; Kabaya, K; Kusaka, M; Ohashi, H; Tsumura, H; Kato, T; Miyazaki, H

    1996-09-01

    We examined the effects of recombinant human thrombopoietin (rhTPO) on myelosuppressive chemotherapy-induced thrombocytopenia in cynomolgus monkeys. After treatment with nimustine (ACNU) on day 0, the monkeys intravenously received rhTPO at a dose of 0.04, 0.2 or 1 microgram/kg/d or monkey's serum once each day from day 1 to day 28. Administration of rhTPO reduced the severity of thrombocytopenia and accelerated the rate of platelet recovery in a dose-dependent fashion. Treatment with the highest rhTPO dose completely prevented thrombocytopenia and stimulated a marked increase in platelet counts over the normal values. Animals treated with ACNU also became neutropenic and slightly anaemic. Administration of rhTPO following ACNU treatment significantly improved neutropenia with increasing doses of rhTPO, but had no effect on anaemia. Compared to the control animals, rhTPO-treated animals exhibited no significant changes in several serum parameters. C-reactive protein concentration and some blood coagulation profiles within the study period. These results suggest a therapeutic efficacy of rhTPO in improving chemotherapy-induced thrombocytopenia.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Bagherinejad

    2016-01-01

    Full Text Available 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.

  14. Recombinant human leptin attenuates stress axis activity in common carp (Cyprinus carpio L.).

    Science.gov (United States)

    Gorissen, Marnix; Bernier, Nicholas J; Manuel, Remy; de Gelder, Stefan; Metz, Juriaan R; Huising, Mark O; Flik, Gert

    2012-08-01

    Proper functioning of the endocrine stress axis requires communication between the stress axis and other regulatory mechanisms. We here describe an intimate interplay between the stress axis and recombinant human leptin (rhLeptin) in a teleostean fish, the common carp Cyprinus carpio. Restraint stress (by netting up to 96h) increased plasma cortisol but did not affect hepatic leptin expression. Perifusion of pituitary glands or head kidneys with rhLeptin revealed direct effects of rhLeptin on both tissues. RhLeptin suppresses basal and CRF-induced ACTH-secretion in a rapid and concentration-dependent manner. The rhLeptin effect persisted for over an hour after administration had been terminated. RhLeptin decreases basal interrenal cortisol secretion in vitro, and by doing so attenuates ACTH-stimulated cortisol production; rhLeptin does not affect interrenal ACTH-sensitivity. Our findings show that the endocrine stress axis activity and leptin are inseparably linked in a teleostean fish, a notion relevant to further our insights in the evolution of leptin physiology in vertebrates.

  15. In vivo bioassay of recombinant human growth hormone synthesized in B. mori pupae.

    Science.gov (United States)

    Lan, Hanglian; Nie, Zuoming; Liu, Yue; Lv, Zhengbing; Liu, Yingshuo; Quan, Yanping; Chen, Jianqing; Zhen, Qingliang; Chen, Qin; Wang, Dan; Sheng, Qing; Yu, Wei; Chen, Jian; Wu, Xiangfu; Zhang, Yaozhou

    2010-01-01

    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 microg/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.

  16. RP-HPLC determination of recombinant human interferon omega in the Pichia pastoris fermentation broth.

    Science.gov (United States)

    Liu, Hong; Pan, Hong-Chun; Peng, Li; Cai, Shao-Xi

    2005-07-15

    A rapid and valid reversed-phase high performance liquid chromatography (RP-HPLC) method for determination of recombinant human interferon omega (rhIFNomega) in the yeast Pichia pastoris fermentation broth was developed. The method is based on the hydrophobicity of rhIFNomega followed by RP-HPLC separation with UV detection. The chromatography analysis was performed on EC 250/4 NUCLEOSIL 300-5 C18 (250 mm x 4 mm i.d., 300 A, with a particle size of 5 microm) column. The compositions of the mobile phase A and B were 999:1 (v/v) water/TFA and 999:1 (v/v) acetonitrile/TFA at a flow rate of 1.0 ml min(-1). Detection was done by spectrophotometry at 280 nm and the column temperature was 30+/-1 degrees C. Calibration curve was linear (r=0.9986, n=7) in the range of 0.074-0.555 mg ml(-1) for rhIFNomega and the regression equation was y=2.02 x 10(6)x-1.27 x 10(5). Limit of detection for rhIFNomega was 0.053 mg ml(-1). The values of R.S.D. (%) of intra-day and inter-day precision were recovery rate of recovery experiment were <1.23 (n=3) and 97.97%.

  17. 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.

  18. Quadrivalent human papillomavirus recombinant vaccine: The first vaccine for cervical cancers

    Directory of Open Access Journals (Sweden)

    Sharma Rashmi

    2007-01-01

    Full Text Available Gardasil ® is the first quadrivalent human papillomavirus (HPV- types 6, 11, 16, 18 recombinant vaccine approved by the FDA on June 8, 2006. It induces genotype-specific virus-neutralizing antibodies and prevents infection with HPV. Various clinical trials demonstrated a reduction in the incidence of vaccine-type-specific persistent infections and of associated moderate- and high-grade cervical dysplasias and carcinomas in situ after its use. Gardasil is currently approved by FDA for prevention of genital warts, cancers and precancerous conditions of cervix and vulva in 9-26 years old females. Three doses of 0.5 ml of gardasil each at 0, 2 and 6 months are given intramuscularly. It is contraindicated in individuals who are hypersensitive to the active substances or to any of the excipients of the vaccine, patients with bleeding abnormalities or patients on anticoagulant therapy and during pregnancy. However, the vaccine, at an estimated $300-500 per course, is too expensive for many women in developing countries. Moreover, question regarding the longevity of the protection by vaccine is still unsolved. Hence, longer studies are required to establish its real status in cancer prevention.

  19. Effects of recombinant human growth hormone on remnant liver after hepatectomy in hepatocellular carcinoma with cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Shi-Min Luo; Li-Jian Liang; Jia-Ming Lai

    2004-01-01

    AIM: To explore the effects of recombinant human growth hormone (rhGH) on the remnant liver after hepatectomy in hepatocellular carcinoma with liver cirrhosis.METHODS: Twenty-four patients with hepatocellular carcinoma who underwent hepatectomy were randomly divided into 2groups: parenteral nutrition (PN) group (n=12) and rhGH+PN group (n=12). Liver function, blood glucose, AFP, serum prealbumin and transferrin were detected before operation,at post-operative d 1 and d 6. Albumin (ALB) mRNA in liver biopsy specimens was detected by RT-PCR at post-operative d 6. Liver Ki67 immunohistochemical staining was studied.RESULTS: On post-operative d 6, compared with PN group,the levels of blood glucose, serum prealbumin, transferrin,the expression of hepatic ALB mRNA and liver Ki67 labeling index were higher in rhGH+PN group.CONCLUSION: rhGH can improve protein synthesis and liver regeneration after hepatectomy in hepatocellular carcinoma with liver cirrhosis.

  20. Screening of reducing agents for the PEGylation of recombinant human IL-10.

    Science.gov (United States)

    Ambrogelly, Alexandre; Cutler, Collette; Paporello, Brittany

    2013-06-01

    PEGylation is a technology commonly used to enhance the bioavailability of therapeutic proteins in patients. Reductive alkylation of a protein amino terminal alpha amine in the presence of a polyethylene glycol (PEG) chain derivatized with propionaldehyde and a reducing agent, typically sodium cyanoborohydride, is one of the technologies available to achieve quantitative and site specific PEGylation. While cyanoborohydride has proven to be a robust and efficient reagent for this type of reaction, it generates aqueous cyanide as a reaction by-product (and its corollary, the very volatile hydrogen cyanide). We report here the screening of reducing agents such as dimethylamine borane, trimethylamine borane, triethylamine borane, tert-butylamine borane, morpholine borane, pyridine borane, 2-picoline borane, and 5-ethyl-2-methyl-pyridine borane as alternatives to cyanoborohydride for the PEGylation of recombinant human IL-10. The results of our study show that pyridine borane and 2-picoline borane promote rhIL-10 PEGylation at levels comparable to those observed with cyanoborohydride.

  1. 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.

  2. Recombinant human pentraxin-2 therapy in patients with idiopathic pulmonary fibrosis: safety, pharmacokinetics and exploratory efficacy.

    Science.gov (United States)

    van den Blink, Bernt; Dillingh, Marlous R; Ginns, Leo C; Morrison, Lake D; Moerland, Matthijs; Wijsenbeek, Marlies; Trehu, Elizabeth G; Bartholmai, Brian J; Burggraaf, Jacobus

    2016-03-01

    Abnormal fibrogenic repair response upon alveolar injury is believed to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). PRM-151 (recombinant human pentraxin-2, also known as serum amyloid P), has been shown to reduce fibrosis in preclinical lung fibrosis models, and was well tolerated with a favourable pharmacokinetic profile in an earlier single-dose phase I study.A randomised, double-blind, placebo-controlled, multiple ascending dose trial was performed to assess the tolerability and pharmacokinetic and pharmacodynamic characteristics of multiple doses of PRM-151 in IPF patients. Subjects in three successive cohorts (1, 5, or 10 mg·kg(-1) versus placebo) received intravenous study drug on days 1, 3, 5, 8 and 15, and were followed-up to day 57.PRM-151 was well tolerated at all dose levels, with no serious adverse reactions. Administration of PRM-151 resulted in two- to eight-fold dose-dependent increases in circulating pentraxin-2 levels. Forced vital capacity and 6-min walk test showed trends towards improvement in the combined PRM-151 dose groups. On high-resolution computed tomography scans, stable or improved lung volume unoccupied by interstitial lung abnormality was noted in some PRM-151 subjects compared to placebo subjects on day 57.The efficacy of PRM-151 in IPF remains to be investigated in dedicated future trials. Copyright ©ERS 2016.

  3. Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses.

    Science.gov (United States)

    Hildebrand, Laura; Seemann, Petra; Kurtz, Andreas; Hecht, Jochen; Contzen, Jörg; Gossen, Manfred; Stachelscheid, Harald

    2015-12-01

    Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA(+) cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSC(TVA+) as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA(+) cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies.

  4. Value of recombinant human thyrotropin in high-dose radioiodine therapy: a case report.

    Science.gov (United States)

    Müller, Vika; Bohuslavizki, Karl H; Klutmann, Susanne; Clausen, Malte

    2002-12-01

    We report on a high-dose radioiodine therapy after injection of recombinant human thyrotropin (rhTSH) in a 61-y-old woman with compression of the spinal cord caused by metastasis of a follicular thyroid carcinoma. Fourteen years ago, the patient underwent subtotal thyroidectomy because of multinodular goiter without any histologic evidence for malignant disease, and the patient was put on thyroxine substitution (100 micro g/d). In April 2000, she developed paralysis of the right leg. Morphologic imaging revealed spinal compression caused by a space-occupying lesion within the thoracic spine. Subsequent biopsy and histology demonstrated metastasis of a follicular thyroid carcinoma. Therefore, high-dose radioiodine therapy was scheduled after 4 wk of hormone withdrawal. Within a few days of being off thyroxine, the patient's paralytic symptoms worsened rapidly. The patient was again put on thyroxine, 100 micro g/d, and high-dose radioiodine therapy under stimulation with rhTSH was performed without any side effects. The second high-dose radioiodine therapy 3 mo later, again performed under stimulation with rhTSH, showed significantly less iodine avidity, and thyroglobulin levels fell from 1,024 micro g/L to 361 micro g/L, thereby demonstrating therapeutic efficacy. Thus, rhTSH might be used as a tool not only in the diagnostic application but also in the therapeutic application of (131)I.

  5. A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE® Bone Graft)

    Science.gov (United States)

    Peckham, Steven M.; Badura, Jeffrey M.

    2007-01-01

    The combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge (ACS) carrier has been shown to induce bone formation in a number of preclinical and clinical investigations. In 2002, rhBMP-2/ACS at a 1.5-mg/cc concentration (INFUSE® Bone Graft, Medtronic Spinal and Biologics, Memphis, TN) was FDA-approved as an autograft replacement for certain interbody spinal fusion procedures. In 2004, INFUSE® Bone Graft was approved for open tibial fractures with an intermedullary (IM) nail fixation. Most recently, in March 2007, INFUSE® Bone Graft was approved as an alternative to autogenous bone grafts for sinus augmentations, and for localised alveolar ridge augmentations for defects associated with extraction sockets. The culmination of extensive preclinical and clinical research and three FDA approvals makes rhBMP-2 one of the most studied, published and significant advances in orthopaedics. This review article summarises a number of clinical findings of rhBMP-2/ACS, including the FDA-approved investigational device exemption (IDE) studies used in gaining the aforementioned approvals. PMID:17639384

  6. Expression of bioactive recombinant human fibroblast growth factor 10 in Carthamus tinctorius L. seeds.

    Science.gov (United States)

    Huang, Jian; Yang, Jing; Guan, Lili; Yi, Shanyong; Du, Linna; Tian, Haishan; Guo, Yongxin; Zhai, Feng; Lu, Zhen; Li, Haiyan; Li, Xiaokun; Jiang, Chao

    2017-10-01

    Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein. Copyright © 2016. Published by Elsevier Inc.

  7. 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.

  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. Respiratory mechanics in an infant with perinatal lethal hypophosphatasia treated with human recombinant enzyme replacement therapy.

    Science.gov (United States)

    Rodriguez, Elena; Bober, Michael B; Davey, Lauren; Zamora, Arlene; Li Puma, Annelise B; Chidekel, Aaron; Shaffer, Thomas H

    2012-09-01

    Hypophosphatasia is a rare autosomal recessive disorder caused by deficient activity of tissue nonspecific alkaline phosphatase (TNSALP) and characterized by defective bone mineralization. In the perinatal lethal form, respiratory complications due to rachitic deformities of the thoracic cage and associated hypoplastic lungs are present. ENB-0040 is a bone-targeted human recombinant TNSALP fusion protein that aims to restore skeletal mineralization. The goal of this study was to characterize pulmonary and thoracic cage mechanics in an infant with the perinatal lethal form of hypophosphatasia under enzyme replacement therapy. Pulmonary function testing was performed on a preterm, 8-week-old patient with hypophosphatasia who was mechanically ventilated since birth because of severe chest wall insufficiency. The measurements consisted of respiratory impulse oscillation measurements (resistance and reactance), ventilatory mechanics (compliance and resistance), and thoracoabdominal motion (TAM) analysis. At baseline, chest wall compliance was 50% of normal, and the TAM indicated predominantly abdominal displacement. After 12 weeks of treatment, a consistent decrease in ventilator requirements and improvement in lung function and chest wall mechanics were observed and correlated with thoracic cage radiologic findings. Measurable changes in chest wall dynamics and respiratory mechanics using noninvasive technology were useful for respiratory management and therapeutic guidance of ENB-0040 treatment in this patient.

  10. Meeting the challenges of a new millennium: optimizing the use of recombinant human erythropoietin.

    Science.gov (United States)

    Macdougall, I C

    1998-01-01

    Optimizing the use of recombinant human erythropoietin (r-HuEPO) involves choosing an appropriate dose regimen and target haemoglobin level, addressing factors that inhibit response, and considering appropriate adjuvant therapy. Subcutaneous administration of r-HuEPO two or three times weekly is optimal for most patients. Early detection and treatment of iron deficiency is mandatory. Measurement of the percentage of hypochromic red blood cells is a reliable marker of functional iron deficiency, and the treatment of choice is intravenous iron. Other factors that can affect the response to r-HuEPO include blood loss (sometimes occult), infection, inflammation, hyperparathyroidism with marrow fibrosis, aluminium toxicity, vitamin B12/folate deficiency, haemolysis, bone marrow disorders, haemoglobinopathies, under-dialysis and possibly angiotensin-converting enzyme inhibitors. These factors should be identified and corrected where possible. Ascorbic acid, vitamin D, folic acid, carnitine, other cytokines and growth factors have all been shown to augment the response to r-HuEPO in some patients. Further research is required before any of these adjuvant therapies can be incorporated into routine clinical practice. With regard to target haemoglobin value, the current practice is to aim for a level of 10-12 g/dl, but it may be argued that a higher target would achieve greater benefits in terms of physical performance, quality of life, and possibly cardiac morbidity and mortality. International multicentre trials are currently in progress to address this issue, as are studies on other substances that may be able to stimulate erythropoiesis.

  11. Recombinant human erythropoietin stimulates angiogenesis and healing of ischemic skin wounds.

    Science.gov (United States)

    Buemi, Michele; Galeano, Mariarosaria; Sturiale, Alessio; Ientile, Riccardo; Crisafulli, Costantino; Parisi, Alessandra; Catania, MariaAntonietta; Calapai, Gioacchino; Impalà, Patrizia; Aloisi, Carmela; Squadrito, Francesco; Altavilla, Domenica; Bitto, Alessandra; Tuccari, Giovanni; Frisina, Nicola

    2004-08-01

    Wound healing in ischemic tissues such as flap margins due to inadequate blood supply is still a source of considerable morbidity in surgical practice. Adequate tissue perfusion is particularly important in wound healing. We investigated the effects of recombinant human erythropoietin (rHuEPO) on wound healing in an ischemic skin wound model. Sixty-three Sprague-Dawley rats were used. Normal incisional wound and H-shaped double flaps were used as the wound models. Animals were treated with rHuEPO (400 IU/kg) or its vehicle. Rats were killed on different days (3, 5, and 10 days after skin injury) and the wounded skin tissues were used for immunohistochemistry and for analysis of vascular endothelial growth factor content and collagen content. Tissue transglutaminase immunostaining of histological specimens was used as a vascular marker to determine the level of microvessel density. The results showed a higher level of vascular endothelial growth factor protein and an increased microvessel density in ischemic wounds with rHuEPO treatment than the normal incisional wounds and ischemic control wounds. Collagen content was higher in the incisional wounds and in the ischemic wounds with rHuEPO treatment compared with the ischemic control wounds. Our results suggest that erythropoietin may be an effective therapeutic approach in improving healing in ischemic skin wounds.

  12. Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli.

    Science.gov (United States)

    Abramczyk, Olga; Tavares, Clint D J; Devkota, Ashwini K; Ryazanov, Alexey G; Turk, Benjamin E; Riggs, Austen F; Ozpolat, Bulent; Dalby, Kevin N

    2011-10-01

    The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His(6)-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ∼85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.

  13. Complex relationship between meiotic recombination frequency and autosomal synaptonemal complex length per cell in normal human males.

    Science.gov (United States)

    Pan, Zhenzhen; Yang, Qingling; Ye, Nan; Wang, Liu; Li, Jianhua; Yu, Dexin; Cooke, Howard J; Shi, Qinghua

    2012-03-01

    Although the relationship between meiotic recombination frequency and synaptonemal complex (SC) length has been of interest for a long time, how recombination frequency is related to SC length has not been carefully explored. To address this question, we have measured the meiotic recombination frequency as represented by MLH1 foci in 889 pachytene spermatocytes and measured the length of 19,558 autosomal SCs from 10 human males. A complex relationship between the number of MLH1 foci and total autosomal SC length per cell was observed. A positive correlation with significant correlation coefficients between the two variables was found in eight of the ten donors examined, with three donors showing weak correlation, and five showing moderate correlation. Two donors who did not show any correlation between the two variables were identified for the first time. Moreover, most cells with similar total autosomal SC length showed very different numbers of MLH1 foci both between individuals and even within an individual, and vice versa. Our data provide the first evidence for a complex relationship between the recombination frequency and total length of autosomal SCs per cell in human males.

  14. Mass spectrometrical analysis of recombinant human growth hormone (Genotropin®) reveals amino acid substitutions in 2% of the expressed protein

    OpenAIRE

    Roitinger Elisabeth; Cszasar Edina; Hepner Felix; Lubec Gert

    2005-01-01

    Abstract Background The structural integrity of recombinant proteins is of critical importance to their application as clinical treatments. Recombinant growth hormone preparations have been examined by several methodologies. In this study recombinant human growth hormone (rhGH; Genotropin®), expressed in E. coli K12, was structurally analyzed by two-dimensional gel electrophoresis and MALDI-TOF-TOF, LC-MS and LC-MS/ MS sequencing of the resolved peptides. Results Electrospray LC-MS analysis r...

  15. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    Science.gov (United States)

    Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

    2016-07-01

    Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

  16. Binding of levomepromazine and cyamemazine to human recombinant dopamine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Lalit K. Srivastava

    Full Text Available Background and Objectives: Clozapine (CLOZ and levomepromazine (LMP improve treatment-resistant schizophrenia. The superior efficacy of CLOZ compared with other antipsychotic agents has been attributed to an effect on D1-like and D4 receptors. We examined the binding of LMP, CLOZ and cyamemazine (CMZ, a neuroleptic analog of LMP, to human recombinant dopamine (rDA receptor subtypes. Methods: Binding studies were performed on frozen membrane suspensions of human rDA receptor subtypes expressed in Sf9 cells. Results: (i LMP has a high affinity (Ki, nM for rD2 receptor subtypes (rD2L 8.6; rD2S 4.3; rD3 8.3; rD4.2 7.9; (ii LMP and CLOZ have comparable affinities for the rD1 receptor (54.3 vs 34.6; (iii CMZ has high affinities for rD2-like and rD1-like receptors (rD2L 4.6; rD2S 3.3; rD3 6.2; rD4.2 8.5; rD1 3.9; rD5 10.7; (iv CMZ is 9 times more potent than CLOZ at the rD1 receptor and 5 times more potent than CLOZ at the rD4.2 receptor; (v CMZ has high affinities for rD1 and rD5 receptor subtypes compared with LMP and CLOZ. Conclusions: If D1 and D4 receptors are important sites for the unique action of CLOZ, the present study points to a need for clinical trials comparing CMZ with CLOZ in schizophrenia and in particular, treatment-resistant schizophrenia, especially given the risk for agranulocytosis with CLOZ.

  17. Binding of levomepromazine and cyamemazine to human recombinant dopamine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Lalit K. Srivastava

    2009-09-01

    Full Text Available Background and Objectives: Clozapine (CLOZ and levomepromazine (LMP improve treatment-resistant schizophrenia. The superior efficacy of CLOZ compared with other antipsychotic agents has been attributed to an effect on D1-like and D4 receptors. We examined the binding of LMP, CLOZ and cyamemazine (CMZ, a neuroleptic analog of LMP, to human recombinant dopamine (rDA receptor subtypes. Methods: Binding studies were performed on frozen membrane suspensions of human rDA receptor subtypes expressed in Sf9 cells. Results: (i LMP has a high affinity (Ki, nM for rD2 receptor subtypes (rD2L 8.6; rD2S 4.3; rD3 8.3; rD4.2 7.9; (ii LMP and CLOZ have comparable affinities for the rD1 receptor (54.3 vs 34.6; (iii CMZ has high affinities for rD2-like and rD1-like receptors (rD2L 4.6; rD2S 3.3; rD3 6.2; rD4.2 8.5; rD1 3.9; rD5 10.7; (iv CMZ is 9 times more potent than CLOZ at the rD1 receptor and 5 times more potent than CLOZ at the rD4.2 receptor; (v CMZ has high affinities for rD1 and rD5 receptor subtypes compared with LMP and CLOZ. Conclusions: If D1 and D4 receptors are important sites for the unique action of CLOZ, the present study points to a need for clinical trials comparing CMZ with CLOZ in schizophrenia and in particular, treatment-resistant schizophrenia, especially given the risk for agranulocytosis with CLOZ.

  18. Characterization of human platelet binding of recombinant T cell receptor ligand

    Directory of Open Access Journals (Sweden)

    Meza-Romero Roberto

    2010-11-01

    Full Text Available Abstract Background Recombinant T cell receptor ligands (RTLs are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS. RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE. The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood. Methods We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function. Results Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen. Conclusions Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

  19. A novel human recombinant antibody fragment capable of neutralizing Mexican scorpion toxins.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Gurrola, Georgina B; Possani, Lourival D; Becerril, Baltazar

    2013-12-15

    Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 (KD = 25.1 × 10(-9) M) or Cn2 (KD = 8.1 × 10(-9) M), this antibody fragment neutralized one LD50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus. Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.

  20. Recombinant human growth hormone improves cognitive capacity in a pain patient exposed to chronic opioids.

    Science.gov (United States)

    Rhodin, A; von Ehren, M; Skottheim, B; Grönbladh, A; Ortiz-Nieto, F; Raininko, R; Gordh, T; Nyberg, F

    2014-07-01

    During recent decades, the increasing use of opioids for chronic non-cancer pain has raised concerns regarding tolerance, addiction, and importantly cognitive dysfunction. Current research suggests that the somatotrophic axis could play an important role in cognitive function. Administration of growth hormone (GH) to GH-deficient humans and experimental animals has been shown to result in significant improvements in cognitive capacity. In this report, a patient with cognitive disabilities resulting from chronic treatment with opioids for neuropathic pain received recombinant human growth hormone (rhGH) replacement therapy. A 61-year-old man presented with severe cognitive dysfunction after long-term methadone treatment for intercostal neuralgia and was diagnosed with GH insufficiency by GH releasing hormone-arginine testing. The effect of rhGH replacement therapy on his cognitive capacity and quality of life was investigated. The hippocampal volume was measured using magnetic resonance imaging, and the ratios of the major metabolites were calculated using proton magnetic resonance spectroscopy. Cognitive testing revealed significant improvements in visuospatial cognitive function after rhGH. The hippocampal volume remained unchanged. In the right hippocampus, the N-acetylaspartate/creatine ratio (reflecting nerve cell function) was initially low but increased significantly during rhGH treatment, as did subjective cognitive, physical and emotional functioning. This case report indicates that rhGH replacement therapy could improve cognitive behaviour and well-being, as well as hippocampal metabolism and functioning in opioid-treated patients with chronic pain. The idea that GH could affect brain function and repair disabilities induced by long-term exposure to opioid analgesia is supported.

  1. Enhanced sialylation of recombinant erythropoietin in CHO cells by human glycosyltransferase expression.

    Science.gov (United States)

    Jeong, Yeon Tae; Choi, One; Lim, Hye Rim; Son, Young Dok; Kim, Hong Jin; Kim, Jung Hoe

    2008-12-01

    Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human alpha2,3- sialyltransferase (alpha2,3-ST) and beta1,4-galactosyltransferase (beta1,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of alpha2,3-ST and beta1,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When alpha2,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1%. When alpha2,3-ST and beta1,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of alpha2,3- ST and beta1,4-GT is more effective than the expression of alpha2,3-ST alone. Coexpression of alpha2,3-ST and beta1,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of alpha2,3-ST and beta1,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.

  2. "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.

  3. Human recombinant erythropoietin (rEpo) has no effect on tumour growth or angiogenesis.

    Science.gov (United States)

    Hardee, M E; Kirkpatrick, J P; Shan, S; Snyder, S A; Vujaskovic, Z; Rabbani, Z N; Dewhirst, M W; Blackwell, K L

    2005-12-12

    Tumour hypoxia has been shown to increase mutation rate, angiogenesis, and metastatic potential, and decrease response to conventional therapeutics. Improved tumour oxygenation should translate into increased treatment response. Exogenous recombinant erythropoietin (rEpo) has been recently shown to increase tumour oxygenation in a mammary carcinoma model. The mechanism of this action is not yet understood completely. The presence of Epo and its receptor (EpoR) have been demonstrated on several normal and neoplastic tissues, including blood vessels and various solid tumours. In addition, rEpo has been shown in two recent prospective, randomized clinical trials to negatively impact treatment outcome. In this study, we attempt to characterize the direct effects of rEpo on tumour growth and angiogenesis in two separate rodent carcinomas. The effect of rEpo on R3230 rat mammary adenocarcinomas, CT-26 mouse colon carcinomas, HCT-116 human colon carcinomas, and FaDu human head and neck tumours, all of which express EpoR, was examined. There were no differences in tumour growth or proliferation (measured by Ki-67) between placebo-treated and rEpo-treated tumours. In the mammary window chamber, vascular length density (VLD) measurements in serial images of both placebo-treated and Epo-treated rats revealed no difference in angiogenesis between the Epo-treated tumours and placebo-treated tumours at any time point. These experiments are important because they suggest that the recent clinical detriment seen with the use of Epo is not due to its tumour growth effects or angiogenesis. These studies also suggest that further preclinical studies need to examine rEpo's direct tumour effects in efforts to improve the therapeutic benefits of Epo in solid tumour patients.

  4. 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.

  5. Deprivation of arginine by recombinant human arginase in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Hsueh Eddy C

    2012-04-01

    Full Text Available Abstract Background Recombinant human arginase (rhArg has been developed for arginine deprivation therapy in cancer, and is currently under clinical investigation. During pre-clinical evaluation, rhArg has exhibited significant anti-proliferative activity in cancer cells deficient in the expression of ornithine carbamoyl transferase (OCT. Interestingly, a variety of cancer cells such as melanoma and prostate cancer deficient in argininosuccinate synthetase (ASS are sensitive to arginine deprivation by arginine deiminase. In this study, we investigated levels of gene expression of OCT and ASS, and the effects of rhArg in human prostate cancer cells: LNCaP (androgen-dependent, PC-3 and DU-145 (both androgen-independent. Results Quantitative real-time PCR showed minimal to absent gene expression of OCT, but ample expression of ASS expression in all 3 cell lines. Cell viability assay after 72-h exposure of rhArg showed all 3 lines had half maximal inhibitory concentration less than or equal to 0.02 U/ml. Addition of ornithine to cell culture media failed to rescue these cells from rhArg-mediated cytotoxicity. Decreased phosphorylation of 4E-BP1, a downstream effector of mammalian target of rapamycin (mTOR, was noted in DU-145 and PC-3 after exposure to rhArg. Moreover, there was no significant apoptosis induction after arginine deprivation by rhArg in all 3 prostate cancer cell lines. Conclusion rhArg causes significant cytotoxicity in LNCaP, DU-145 and PC-3 prostate cancer cells which all demonstrate decreased OCT expression. Inhibition of mTOR manifested by hypophosphorylation of 4E-BP1 suggests autophagy is involved as alternative cell death mechanism. rhArg demonstrates a promising novel agent for prostate cancer treatment.

  6. Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli.

    Science.gov (United States)

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Pande, Abhay H

    2015-11-01

    Human PON1 (h-PON1) is a Ca(2+)-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate.

  7. Use of recombinant human erythropoietin as an antianemic and performance enhancing drug.

    Science.gov (United States)

    Jelkmann, W

    2000-07-01

    The glycoprotein hormone erythropoietin is an essential viability and growth factor for the erythrocytic progenitors in the bone marrow. Tissue hypoxia is the main stimulus for the synthesis of the hormone in the kidneys and the liver. Endogenous erythropoietin and recombinant human erythropoietin (rHu-EPO) are similar with respect to their biological and chemical properties except for some microheterogeneities in their 4 carbohydrate chains. Generic products and alternatives to rHu-EPO are in development. Renal anemia can be corrected by rHu-EPO in a dose-dependent and predictable way without major side effects apart from a possible increase in arterial blood pressure. The optimal target hematocrit still needs to be defined. There are rare reports of antibody formation towards rHu-EPO in humans. Patients suffering from non-renal anemias may also benefit from the prescription of rHu-EPO. The drug has been approved for treatment of tumor patients with platinum-induced anemia. The cost-effectiveness and medical justification of the administration of rHu-EPO in tumor patients with respect to its positive effects on tumor oxygenation, tumor growth inhibition and support of chemo- and radiotherapy is still a matter of debate. In surgical patients, the pharmacological application of rHu-EPO can increase the yield of blood units in autologous blood donation programs and lower the severity and duration of postoperative anemia, if applicated some days prior to surgery. While rHu-EPO is a godsend in medical practice, its abuse as an performance enhancing drug by athletes in endurance sports is an unethical and potentially dangerous procedure. Unequivocal methods for detection of rHu-EPO doping still need to be established.

  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. CRYSTAL-STRUCTURE OF RECOMBINANT HUMAN TRIOSEPHOSPHATE ISOMERASE AT 2.8 ANGSTROM RESOLUTION - TRIOSEPHOSPHATE ISOMERASE-RELATED HUMAN GENETIC-DISORDERS AND COMPARISON WITH THE TRYPANOSOMAL ENZYME

    NARCIS (Netherlands)

    MANDE, SC; MAINFROID, [No Value; KALK, KH; GORAJ, K; MARTIAL, JA; HOL, WGJ

    1994-01-01

    The crystal structure of recombinant human triosephosphate isomerase (hTIM) has been determined complexed with the transition-state analogue 2-phosphoglycolate at a resolution of 2.8 Angstrom. After refinement, the R-factor is 16.7% with good geometry. The asymmetric unit contains 1 complete dimer o

  10. Methimazole-induced aplastic anemia in third exposure: successful treatment with recombinant human granulocyte colony-stimulating factor.

    Science.gov (United States)

    Mezquita, P; Luna, V; Muñoz-Torres, M; Torres-Vela, E; Lopez-Rodriguez, F; Callejas, J L; Escobar-Jimenez, F

    1998-09-01

    The major adverse reactions of antithyroid drugs are hematologic; aplastic anemia (AA) is one of the rarest and most severe complications. Use of recombinant human hemopoietic colony-stimulating factor was reported to be of benefit in patients who developed agranulocytosis, although there is still some doubt regarding the efficacy in AA. We present a case of a 58-year-old female patient with Graves' disease who developed AA in the third exposure to methimazole (MMI). The withdrawal of MMI and early treatment with 5 microg/kg per day recombinant human granulocyte colony-stimulating factor (G-CSF) for 9 days, allowed a favorable recovery of peripheral blood cell count. We conclude that the use of hemopoietic colony stimulating factors might be a suitable means to achieve the correction of severe thionamide-induced hematologic adverse reactions.

  11. Efficient mobilization of haematopoietic progenitors after a single injection of pegylated recombinant human granulocyte colony-stimulating factor in mouse strains with distinct marrow-cell pool sizes

    NARCIS (Netherlands)

    de Haan, G; Ausema, A; Wilkens, M; Molineux, G; Dontje, B

    We have compared the efficacy of a single injection of SD/01, a newly engineered, pegylated form of recombinant human granulocyte colony stimulating factor (rhG-CSF), with a single injection of glycosylated rhG-CSF (Filgrastim). SD/01 was administered to regular and recombinant inbred strains of

  12. Susceptibility of feline herpesvirus 1 and a feline calicivirus to feline interferon and recombinant human leukocyte interferons.

    OpenAIRE

    Fulton, R W; Burge, L J

    1985-01-01

    Feline lung monolayer cultures were treated with either a feline interferon (IFN) or one of two recombinant human alpha-IFNs and then challenged with feline herpesvirus 1 (FHV-1), feline calicivirus (F-9 strain), or vesicular stomatitis virus. Treatment with these IFNs reduced the viral yield for each of these three viruses as compared with that of control cultures. Vesicular stomatitis virus was more sensitive to each IFN than were FHV-1 or feline calicivirus F-9.

  13. High-level expression and purification of soluble bioactive recombinant human heparin-binding epidermal growth factor in Escherichia coli.

    Science.gov (United States)

    Khalili, Mostafa; Soleyman, Mohammad Reza; Baazm, Maryam; Beyer, Cordian

    2015-07-01

    Heparin-binding epidermal growth factor (HB-EGF) is a member of highly conserved superfamily of proteins that has potential mitogenic activity and stimulates differentiation and migration of various cell types. Since HB-EGF has three intra-molecular disulfide bonds, a high expression pattern of active HB-EGF in an E. coli expression system was not successfully established. The aim of this study was to increase production of soluble bioactive recombinant human HB-EGF in E. coli by modifying growth conditions and codon optimization. The open reading frame codons of human HB-EGF were optimized to achieve high level expression in E. coli. The optimized codon was amplified, cloned into plasmid pET-32a, and transformed into E. coli BL21 for further expression. The cultivation parameters (temperature and inducer) were optimized to produce a high yield of soluble HB-EGF. The fusion protein was purified by Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Amethylthiazole tetrazolium assay was used to evaluate the bioactivity of the produced recombinant protein. After codon optimization, the codon adaptation index (CAI) was increased from 0.255 in native gene to 0.829 using the optimized sequence. By lowering the temperature to 22°C and the inducer to 0.4 μM, we obtained 35% soluble expression of recombinant and biologically active human HB-EGF. Our data demonstrate that codon optimization increases the yield of HB-EGF in an E. coli expression system. Furthermore, the chosen modifications in cell culturing increase the solubility of recombinant human HB-EGF.

  14. Susceptibility of feline herpesvirus 1 and a feline calicivirus to feline interferon and recombinant human leukocyte interferons.

    OpenAIRE

    Fulton, R W; Burge, L J

    1985-01-01

    Feline lung monolayer cultures were treated with either a feline interferon (IFN) or one of two recombinant human alpha-IFNs and then challenged with feline herpesvirus 1 (FHV-1), feline calicivirus (F-9 strain), or vesicular stomatitis virus. Treatment with these IFNs reduced the viral yield for each of these three viruses as compared with that of control cultures. Vesicular stomatitis virus was more sensitive to each IFN than were FHV-1 or feline calicivirus F-9.

  15. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

  16. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F.; Nam, Ho-Woo

    2016-01-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  17. Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

    Science.gov (United States)

    Stratikos, E.; Alberdi, E.; Gettins, P. G.; Becerra, S. P.

    1996-01-01

    Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation. PMID:8976566

  18. Immunological characterization of recombinantWuchereria bancrofti cuticular collagen (COL-4) as putative vaccine candidate for human lymphatic filariasis

    Institute of Scientific and Technical Information of China (English)

    Chakkaravarthy Arunkumar; Pandurangan Pandiaraja; P. R. Prince; Perumal Kaliraj

    2014-01-01

    Objective:To elucidate immunoprophylactic potential of recombinantWuchereria bancrofti(W. bancrofti) cuticular collagen(COL-4) inBALB/c mice and filarial clinical samples.Methods:col-4 gene wasPCR amplified fromW. bancroftiL3 cDNA library and cloned in pRSETB vector. RecombinantCOL-4 was over expressed in salt inducible system and was purified by nickel affinity chromatography.Humoral and cellular responses were measured byELISA and peripheral blood mononuclear cells(PBMC) of various filarial clinical samples respectively using purified recombinantCOL-4 antigen.Then the protective immune responses ofCOL-4 immunizedBALB/c mice were characterized.Results:Sequence analysis ofCOL-4 with human host proteins reveals lack of homology.The recombinantCOL-4 was found to be at15 kDa fusion protein.The affinity purifiedCOL-4 showed significant reactivity with putatively immune sera and in a similar fashion it demonstrated marked proliferation inPBMC samples.Immunization studies in experimental filarial host(mice) elicited significant titers with protective antibody isotype profile(IgM and IgG).Cellular immune responses were also significant in terms of splenocytes proliferation assay on mice samples.Conclusions:Our immunological findings in experimental host suggestTh2 mediated immune response.Hence, we propose thatW. bancroftiCOL-4 could be an efficacious vaccine candidate against lymphatic filariasis.

  19. 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

  20. Improved Cardiac Contractility of Human Recombinant Growth Hormone on the Congestive Heart Failure of Pig

    Institute of Scientific and Technical Information of China (English)

    Yang Ping; He Yu-quan; Zeng Hong; Ni Jin-song; Yun Qing-jun; Huang Xiao-ping; Li Shu-mei

    2005-01-01

    The enhanced cardiac contractility effect of human recombinant growth hormone (hr-GH) on the congestive heart failure (CHF) was studied on the pig. To build a pig model of congestive heart failure, a temporary artificial cardiac pacemaker was implanted in the pig's body and paced at 220 beats to 240 beats per minute for 1 week. After the model of congestive heart failure was successfully set up, the frequency of the pacemaker was changed to 150 beats to 180 beats per minute to maintain the CHF model stable. Pigs were divided into three groups: The hr-GH group in which 0.5 mg/kg per day of hr-GH was administrated intramuscularly for 15 days, the injection control group in which an equal amount of physiological saline was injected intramuscularly, and a normal control group. The left ventricular diastolic end pressure was (10.60±2.41 ) mmHg in the hr-GH group, but (19.00±3.81) mmHg in the saline control group (P<0.01); Cardiac output was (1.86±0.13) L/min in the hr-GH group, but (1.56 ±0.18) L/min in the saline control group (P<0.05); Peripheral min) -1 in the saline control group (P<0.05); ± dp/dtmax was (2900 ±316.23) and (2280 ±286.36) in the hr-HG group and the saline control group respectively (P<0.05). The results show that hr-GH enhances myocardial contractility of CHF, and the CHF model built by a temporary artificial cardiac pacemaker at a high rate of stimulation is reasonable and applicable.

  1. 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.

  2. 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

  3. The next generation recombinant human cytomegalovirus vaccine candidates-beyond gB.

    Science.gov (United States)

    Lilja, Anders E; Mason, Peter W

    2012-11-19

    Human cytomegalovirus (HCMV) infects the majority of the global population and persists within the infected host for life; infection of healthy adults rarely leads to severe acute clinical symptoms. In contrast, HCMV is a leading infectious cause of congenital disease and a common cause of complications in transplant recipients. A vaccine to prevent HCMV disease in these populations is a widely recognized medical need. We review recent advances in our understanding of the candidate vaccine antigens and published clinical trial data for the four most recent HCMV vaccine candidates: a gB subunit adjuvanted with MF59, a DNA vaccine expressing gB and pp65, alphavirus replicon particles (VRPs) expressing gB and a pp65-IE1 fusion protein, and a pp65 peptide vaccine. The candidates are safe, although some adverse events were reported for an adjuvanted variant of the pp65 peptide vaccine. The gB/MF59 vaccine elicited strong humoral responses with limited durability. The gB/pp65 DNA vaccine elicited cellular immunity, and the pp65 peptide vaccine elicited modest cellular immunity, but only when formulated with an adjuvant. Only the VRP vaccine expressing gB and pp65-IE1 elicited both humoral and cellular immunity. The gB/MF59 vaccine showed a short-term 50% efficacy at preventing infection of seronegative women and significantly reduced viremia and need for antivirals in solid organ transplant recipients, and the gB/pp65 DNA vaccine showed signs of clinical benefit in hematopoietic stem cell transplant recipients. Importantly, the partial efficacy of the subunit and DNA vaccines is new evidence that both humoral and cellular immunity contribute to controlling HCMV-related disease. These data show the clinical feasibility of a recombinant HCMV vaccine. We discuss areas for potential improvements in the next generation of vaccine candidates.

  4. Recombinant human acid sphingomyelinase as an adjuvant to sorafenib treatment of experimental liver cancer.

    Directory of Open Access Journals (Sweden)

    Radoslav Savić

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is the most common form of liver cancer and the third leading cause of cancer death worldwide. The only approved systemic treatment for unresectable HCC is the oral kinase inhibitor, sorafenib. Recombinant human acid sphingomyelinase (rhASM, which hydrolyzes sphingomyelin to ceramide, is an orphan drug under development for the treatment of Type B Niemann-Pick disease (NPD. Due to the hepatotropic nature of rhASM and its ability to generate pro-apoptotic ceramide, this study evaluated the use of rhASM as an adjuvant treatment with sorafenib in experimental models of HCC. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, rhASM/sorafenib treatment reduced the viability of Huh7 liver cancer cells more than sorafenib. In vivo, using a subcutaneous Huh7 tumor model, mouse survival was increased and proliferation in the tumors decreased to a similar extent in both sorafenib and rhASM/sorafenib treatment groups. However, combined rhASM/sorafenib treatment significantly lowered tumor volume, increased tumor necrosis, and decreased tumor blood vessel density compared to sorafenib. These results were obtained despite poor delivery of rhASM to the tumors. A second (orthotopic model of Huh7 tumors also was established, but modest ASM activity was similarly detected in these tumors compared to healthy mouse livers. Importantly, no chronic liver toxicity or weight loss was observed from rhASM therapy in either model. CONCLUSIONS/SIGNIFICANCE: The rhASM/sorafenib combination exhibited a synergistic effect on reducing the tumor volume and blood vessel density in Huh7 xenografts, despite modest activity of rhASM in these tumors. No significant increases in survival were observed from the rhASM/sorafenib treatment. The poor delivery of rhASM to Huh7 tumors may be due, at least in part, to low expression of mannose receptors. The safety and efficacy of this approach, together with the novel findings regarding enzyme targeting

  5. Recombinant human thrombopoietin is an effective treatment for thrombocytopenia in hemophagocytic lymphohistiocytosis.

    Science.gov (United States)

    Wang, Yini; Wang, Zhao; Wu, Lin; Zhang, Jia; Wang, Jingshi; Yan, Lijuan

    2013-12-01

    The aim of this study was to investigate the effectiveness and safety in the treatment of thrombocytopenia in hemophagocytic lymphohistiocytosis (HLH) by recombinant human thrombopoietin (rhTPO). A prospective randomized study was conducted between March 2010 and December 2011 in 40 patients with adult HLH whose blood platelet counts (BPC) were lower than 40 × 10(9)/L. The 40 patients were randomly assigned into the rhTPO group or control group based on sex, age, primary disease, and BPC (20 in each group). All patients were given conventional systemic therapy for HLH. The rhTPO group was administered by subcutaneous injection of rhTPO at a dose of 300 IU/kg Qd. The BPC, platelet transfusion, bleeding, and survival rate in the two groups were monitored and compared. There was no significant difference in BPC between the two groups before the treatment. Two weeks after the treatment, the BPC of the rhTPO group was significantly higher than that of the control group at every time point (P rhTPO group and control group, however, the number of patients presented with gastrointestinal bleeding, urinary tract bleeding, and pulmonary bleeding in the control group were higher than that in the rhTPO group (P = 0.013). The frequency of platelet transfusion in the control group (7.25 per patient, 145 in 19 patients) was significantly higher than that in the rhTPO group (2.25 per patient, 45 in 14 patients) (P rhTPO groups was shorter than that in the control group (the rhTPO group vs the control group: 13.43 ± 4.62 D vs 18.00 ± 3.98 D, P = 0.013). In the early stage of HLH treatment, rhTPO combined with conventional systemic therapy can restore the BPC to normal level within a shorter period of time, reduce the frequency of platelet transfusion and severe bleeding.

  6. A multicenter randomized controlled trial of recombinant human thrombopoietin treatment in patients with primary immune thrombocytopenia.

    Science.gov (United States)

    Wang, Shujie; Yang, Renchi; Zou, Ping; Hou, Ming; Wu, Depei; Shen, Zhixiang; Lu, Xijing; Li, Yan; Chen, Xiequn; Niu, Ting; Sun, Hui; Yu, Li; Wang, Zhao; Zhang, Yin; Chang, Naibai; Zhang, Gaokui; Zhao, Yongqiang

    2012-08-01

    This multicenter, randomized trial assessed the efficacy and safety of a recombinant human thrombopoietin (rhTPO) in patients with persistent primary immune thrombocytopenia (ITP) who had failed glucocorticosteroid treatment. A total of 140 eligible patients were randomized to receive rhTPO + danazol (rhTPO group, 73 patients) or danazol (control group, 67 patients) alone. During the first phase, the increase in the mean maximal platelet counts (101.2 × 10(9)/L) and the area under curve (749.6) in the rhTPO group were significantly higher compared to control (33.3 × 10(9)/L and 316.2; P = 0.0060 and 0.0000, respectively). The major response rate (MRR) and total response rate (TRR) in the rhTPO group were 38.4 and 60.3 %, respectively, significantly higher than in control (MRR 7.9 %, P = 0.0003; TRR 36.5 %, P = 0.0104). In the control group, 45 patients with platelet counts rhTPO during the second phase and achieved MRR 31.1 % and TRR 66.7 %. The mean platelet counts in the rhTPO group were still approximately 50 × 10(9)/L on day 28 of the study. The overall incidence of rhTPO-related adverse events was 13.6 %. All the adverse events were generally mild. This study demonstrated that rhTPO was well tolerated, and it markedly increased platelet counts in chronic ITP patients. Stimulation of platelet production by rhTPO may provide a new therapeutic option for patients with ITP.

  7. New insights for identification of doping with recombinant human erythropoietin micro-doses after high hydration.

    Science.gov (United States)

    Martin, L; Ashenden, M; Bejder, J; Hoffmann, M; Nordsborg, N; Karstoft, K; Morkeberg, J; Sharpe, K; Lasne, F; Marchand, A

    2016-11-01

    To minimize the chances of being caught after doping with recombinant human erythropoietins (rhEPO), athletes have turned to new practices using micro-doses and excess fluid ingestion to accelerate elimination and decrease the probability of detection. Our objective was to test the sensitivity of detection by validated methods (IEF: isoelectric focusing; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis) when such practices are used. First, after a three-week rhEPO boost period and 10 days of wash out, detection of a single 900 IU micro-dose of Eprex® was evaluated in healthy male subjects. After an injection in the evening, urine and plasma samples were collected the following morning. Half of the subjects then drank a bolus of water and new samples were collected 80 min later. Interestingly, rhEPO was detected in 100% of the samples even after water ingestion. A second similar protocol was then performed with a single injection of a micro-dose of rhEPO (500 IU or 900 IU), without a prior rhEPO boost. In addition, urine and plasma samples were also collected 15 and 20 h post rhEPO administration. Once again drinking water did not affect the rate of detection. Urine appeared a better matrix to detect micro-doses after 10 h, enabling between 92% and 100% of identification at that time. The rate of identification decreased rapidly thereafter, in particular for the 500 IU micro-dose. However IEF analysis still resulted in 71% identification of rhEPO in urine after 20 h. These results could help to define a better strategy for controlling and identifying athletes using rhEPO micro-doses. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  8. 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

  9. Development of a sustained-release recombinant human growth hormone formulation.

    Science.gov (United States)

    Kwak, H H; Shim, W S; Choi, M K; Son, M K; Kim, Y J; Yang, H C; Kim, T H; Lee, G I; Kim, B M; Kang, S H; Shim, C K

    2009-07-20

    Recombinant human growth hormone (rhGH) therapy for short stature must be administered as a daily injection because of its poor bioavailability and short half-life. In the present study, a sustained-release formulation of rhGH (SR-rhGH), DA-3003, was prepared using double emulsion solvent evaporation with poly(D,L-lactide-co-glycolide) (PLGA), zinc oxide and hydroxypropyl-beta-cyclodextrin (HPCD) as the release modulator, stabilizer, and aggregation-prevention agent, respectively. After a single administration of DA-3003, the elevated concentration of rhGH in plasma was sustained for 14 days in rats and 28 days in monkeys. The plasma concentration of insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3), which are pharmacodynamic markers of rhGH administration, increased and remained elevated for approximately 28 days in monkeys. Monkeys administered DA-3003 did not develop antibodies to hGH, indicating safety of the SR-rhGH formulation comparable to that observed with daily rhGH injections (Growtropin II). There were no significant differences in efficacy between Growtropin II (daily dose of 5 microg/animal for 14 days) and DA-3003 (weekly dose of 35 microg/animal for 14 days with a dosing interval of a week) in hypophysectomized rats, as assessed by changes in body weight and the width of the tibial growth plate. These results show that a sustained-release rhGH formulation, DA-3003, has the potential to be used safely and efficaciously in a weekly dosing regimen.

  10. Chronic Toxicity of a Novel Recombinant Human Granulocyte Colony-stimulating Factor in Rats

    Institute of Scientific and Technical Information of China (English)

    Fei Xia; Qing-yu Zhang; Yong-ping Jiang

    2011-01-01

    Objective To assess the severity and reversibility of the chronic toxicity of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and the dose-effect relationship. Methods A total of 100 Sprague-Dawley rats (equal numbers of male and female) were randomly divided into five groups (20 rats in each group): four groups were treated with rhG-CSFa at 500, 100,10, 1 μg/kg, respectively, and one group was treated with vehicle only to serve as the control. The rats were received subcutaneous injections of rhG-CSFa or vehicle daily for 13 weeks. During the course of the chronic toxicity study, the physical status, body weight, and food consumption were monitored. Half of the rats in each group (n= 10) were sacrificed after the last rhG-CSFa administration, and the other half were sacrificed at five weeks after the last rhG-CSFa administration. Urinalyses, blood biochemistry, hematological analysis, histopathological examination, and immunological tests were performed for each of the rats. Results The hematological analyses revealed that the mean white blood cells count, neutrophils count, and neutrophils percentage were increased in male rats at the dose of 10 μg/kg or higher, and these were related with the biological activity of rhG-CSFa. Some small abnormalities were observed in the spleen of a few rats when used highest dose (500 μg/kg, a dosage of 200 folds higher than the normal clinical dosage), but these abnormalities were recovered within S-week recovery period. No other rhG-CSFa-related abnormalities were observed in this chronic toxicity study.Conclusion No significant toxicity and immunogenicity are observed with rhG-CSFa administration to rats in the chronic toxicity studies.

  11. 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.

  12. Does recombinant human erythropoietin accelerate correction of post-ulcer-bleeding anaemia? A pilot study

    Institute of Scientific and Technical Information of China (English)

    Spiros D. Ladas; Dimitrios Polymeros; Thomas Pagonis; Konstantinos Triantafyllou; Gregorios Paspatis; Maria Hatziargiriou; Sotirios A.Raptis

    2004-01-01

    AIM: Anaemia caused by acute upper gastrointestinal bleeding is treated with blood transfusion or iron, but patients usually face a two-month recovery period from posthaemorrhage anaemia. This prospective, randomised, open,pilot study was designed to investigate whether recombinant human erythropoietin (Epoetin) therapy accelerate haematocrit increase in the post-bleeding recovery period.METHODS: We studied hospitalised patients admitted because of acute ulcer bleeding or haemorrhagic gastritis,who had a haematocrit of 27-33% and did not receive blood transfusions. One day after the endoscopic confirmation of cessation of bleeding, they were randomised either to erythropoietin (20 000 IU Epoetin alfa subcutaneously, on days 0, 4 and 6) plus iron (100 mg im, on days 1- 6, (G1) or iron only (G2). Haematocrit was measured on days 0, 6, 14,30, 45, and 60, respectively.RESULTS: One patient from G1 and two from G2 were lost to follow-up. Therefore, 14 and 13 patients from G1 and G2respectively were analysed. Demographic characteristics, serum iron, ferritin, total iron binding capacity, reticulocytes, and haematocrit were not significantly different at entry to the study.Median reticulocyte counts were significantly different between groups on day six (G1: 4.0, 3.0-6.4 vsG2: 3.5, 2.1-4.4%,P=0.03) and median haematocrit on day fourteen [G1: 35.9,30.7-41.0 vsG2: 32.5, 29.5-37.0% (median, range), P=0.04].CONCLUSION: Erythropoietin administration significantly accelerates correction of anemia after acute ulcer bleeding.The haematocrit gain is equivalent to one unit of transfused blood two weeks after the bleeding episode.

  13. Effects of recombinant human growth hormone (r-hGH) on experimental osteoporotic fracture healing

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To observe the effect of recombinant human growthhormone (r-hGH) on osteoporotic fracture healing in rats, and to provide an effective therapy for osteoporotic fracture.Methods: Thirty-six female 8-month-old SD rats were randomized into two groups: therapy group and control group. After the experimental model of osteoporotic fracture was established, the therapy group was treated with r-hGH of 2.7 mg/kg body weigh/day (1 mg=3 IU) for 10 days continuously by daily subcutaneous injection; whereas the control group was treated with equivalent saline. Plasma insulin-like growth factor I concentration was detected and bone mineral density (BMD) as well as biomechanical strength of callus were measured at 2, 4, 8 weeks.Results: Plasma insulin-like growth factor I concentration in the therapy group was higher than that in the control group (P<0.005) at 2nd week and began to decline at 4th week. At 8th week, there was no significant difference between the two groups. At 4th week, callus area and BMD in therapy group were higher than those in the control group, but at 8th week, they were lower and BMD had a significant difference between the two groups (P<0.001). Biomechanical testing of callus showed that torsional strength of the therapy group was higher than that of the control group at 4th or 8th week, meanwhile maximum torsional angle had a significant difference between the two groups (P<0.005).Conclusions: The results show that exogenous r-hGH can stimulate osteoporotic fracture healing in rats.

  14. [Predictive response variables to recombinant human erythropoietin treatment in patients with anemia and cancer].

    Science.gov (United States)

    Lastiri, José M; Specterman, Sergio R; Rendo, Pablo; Pallotta, María G; Varela, Mirta S; Goldstein, Sofía

    2002-01-01

    The use of human recombinant erythropoietin (rHuEpo) has been approved by the Food and Drug Administration (FDA) in patients with anemia and cancer. Although good results have been obtained, it is too expensive to permit its use massively. For the purpose of evaluating the therapeutic effect of rHuEpo, including toxicity, predictive response variables and quality of life parameters, a prospective trial was carried out in patients with anemia and cancer. Hematimetric parameters, ferritin, Epo, cytokines, transfusions and quality of life were registered. A total of 36 patients were treated in the protocol (34 were evaluable): 16 men and 20 women, with a medium age 56.4 years; 27 patients were treated with chemotherapy (16 with cisplatinum); 15 patients presented medullar infiltration. In 73.5% patients an increase in the level of hemoglobin was registered, and in 64.7% its normalisation was attained. Transfusional requirements were reduced by 50%. The hemoglobin increase greater than 0.5 g/dl at the second week of treatment was the most significant variable of early response. Patients treated with cisplatinum, seric ferritin lower than 1,100 ng/dl and those without medullar tumoral infiltration responded best. Serum Epo, cytokines (IL-1, IL-6 and TNF) and reticulocyte count at the second week did not correlate with response. Quality of life parameters were better in patients with good response to rHuEpo. It can be concluded that good results in the treatment of patients with anemia and cancer are obtained with rHuEpo.

  15. Recombinant human interleukin-3: pharmacokinetics after intravenous and subcutaneous bolus injection and effects on granulocyte kinetics.

    Science.gov (United States)

    Hovgaard, D J; Folke, M; Mortensen, B T; Nissen, N I

    1994-08-01

    The pharmacokinetics of E. coli derived recombinant human interleukin-3 (rhIL-3) was studied following intravenous (i.v.) and subcutaneous (s.c.) bolus injection of rhIL-3. After i.v. bolus injection in eight patients, serum peak levels of 34.5-135.0 ng/ml were reached, followed by a rapid decline with a t1/2 alpha of 17 +/- 2 min and a t1/2 beta of 59 +/- 7 min. After s.c. bolus injection in five patients, the absorption was more prolonged with peak serum levels reached at 2.8 +/- 0.4 h. Elimination was also more protracted, and serum base-line levels were reached at 14-24 h. The immediate effect of rhIL-3 on peripheral white blood cells was less pronounced and more variable than previously found for G- or GM-CSF. Following i.v. administration, neutrophils showed a moderate drop to median 64% of initial values (range 42-85%) at median 30 min after injection (range 15-60 min) followed by an increase at 24 h to 69-288% of initial values. Eosinophils dropped to a median nadir of 34% and then gradually increased to maximum values in the range 135-720% at 18-24 h. The effect of rhIL-3 was further examined following i.v. injection of autologous 111Indium-labelled granulocytes in six patients. In steady state, i.v. injection of rhIL-3 caused a moderate drop in 111Indium activity of peripheral blood within 20 min without tendency to subsequent recovery. No change occurred in the activity recorded over the lungs and liver. The activity over the spleen decreased moderately in two patients. These results are strikingly different from those previously obtained after i.v. injection of rhGM-CSF.

  16. Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes.

    Science.gov (United States)

    Ettori, C; Righetti, P G; Chiesa, C; Frigerio, F; Galli, G; Grandi, G

    1992-09-01

    Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.

  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 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. Molecular Design, Expression and Evaluation of PASylated Human Recombinant Erythropoietin with Enhanced Functional Properties.

    Science.gov (United States)

    Hedayati, Mohammad Hossein; Norouzian, Dariush; Aminian, Mahdi; Teimourian, Shahram; Ahangari Cohan, Reza; Sardari, Soroush; Khorramizadeh, M Reza

    2017-02-01

    Erythropoietin (EPO) is the principal hormone which, has somewhat short half-life involved in the differentiation and regulation of circulating red blood cells. The present study was carried out to evaluate the capability of a polyethylene glycol mimetic technology as a biological alternative to improve pharmaceutical properties of human recombinant EPO. In silico models of EPO fused to 200 amino acids of proline, alanine, and serine (PAS) were initially generated and assessed by molecular dynamic (MD) simulation. The fluctuations of the modeled structure reached a plateau after 6000 ps of MD simulation. The Phi and psi analysis showed >99.2% of residues were located in the allowed regions. An expression vector consisting of EPO cDNA tagged to PAS coding sequences was synthesized and expressed in CHO-K1 Cells. The produced PASylated molecule was purified and characterized by standard analytical methods. The molecular weight of fusion protein was expanded to 70 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis method. Analytical size exclusion chromatography revealed an approximately sevenfold increase in apparent size of produced protein. Although the in vitro potency of the fusion protein was significantly reduced (1.26 ± 0.05 vs. 0.24 ± 0.03 ng/ml) but, the in vivo activity was considerably increased up to 1.58 × 10(5) IU/ml in normocythemic mice assay. Pharmacokinetic animal studies revealed strongly 15.6-fold plasma half-life extension for the PASylated EPO (83.16 ± 13.28 h) in comparison to epoetin α (8.5 ± 2.4 h) and darbepoetin α (25.3 ± 2.2h).

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

    Science.gov (United States)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian; Oturai, Peter; Meinild-Lundby, Anne-Kristine; Holstein-Rathlou, Niels-Henrik; Lundby, Carsten; Vidiendal Olsen, Niels

    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 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.

  20. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    Science.gov (United States)

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  1. [Treatment of anemia in hemodialysis patients using recombinant human erythropoietin: advantages and disadvantages].

    Science.gov (United States)

    Zehnder, C; Blumberg, A

    1989-03-04

    18 anemic patients undergoing maintenance hemodialysis were treated with recombinant human erythropoietin (EPO) 1-3 times per week for 10.7 +/- 3 months. 4 patients underwent renal transplantation whereas 14 patients could be followed up during 12 months of EPO treatment. Hemoglobin concentration rose (from 7.0 +/- 0.7 to 11.0 +/- 1.1 g/dl, p less than 0.001) with an EPO maintenance dose of 298 units/kg/week. Blood transfusions were totally eliminated. 12 patients without iron overload required iron supplements. In the course of an infectious episode and notwithstanding an increase in EPO dosage, 2 patients exhibited a fall in hemoglobin which rose again after successful treatment of the infection. The few complications observed in connection with the rise in hemoglobin were: 1. deterioration of arterial hypertension in 7/18 with hypertensive encephalopathy in 3 patients, 2. thrombotic occlusion of the vascular hemodialysis access (a-v fistula) in 3/18, 3. periarticular inflammation with calcified deposits due to an elevated calcium-phosphorus product of 6.8 mmol/l in 4/18, 4. occurrence of hyperkalemia (6.9 +/- 0.3 mmol/l) in 7/18. These complications were more frequent during the first 3 months. They were corrected with close monitoring, drug therapy for hypertension, and intensification of dialysis and of treatment with phosphate binding substances, with the result that no differences were found in 14 patients before and after 12 months of treatment with EPO (blood pressure 133 +/- 25/77 +/- 9 vs 139 +/- 26/79 +/- 13 mm Hg [ns], potassium 5.4 +/- 0.4 vs 5.6 +/- 1.0 mmol/l [ns] and calcium-phosphorus product 4.3 +/- 1.0 vs 4.6 +/- 1.3 [ns]).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Tissue eosinophilia induced by recombinant human interleukin-5 in the hamster cheek pouch membrane

    Directory of Open Access Journals (Sweden)

    M. Minnicozzi

    1995-01-01

    Full Text Available Interleukin-5 (IL-5 is a cytokine that preferentially effects the development and function of eosinophils, and is considered important in the pathophysiology of allergic inflammation. In this study, we evaluated the ability of recombinant human IL-5 (rHu IL-5 to promote tissue eosinophilia and the importance of this eosinophilia to pathological alterations in vascular function. Repetitive subcutaneous administration for 18 days of rHu IL-5 resulted in a 7-fold increase in the number of eosinophils found in the ipsilateral hamster cheek pouch membrane. The contralateral cheek pouch membrane and peritoneum of these animals showed lesser but significant elevations in the number of eosinophils. In contrast, denatured rHu IL-5 did not elevate eosinophils in these tissues. Through the use of intravital microscopy and fluorometric analysis, rHu IL-5 treated hamster cheek pouch membranes were evaluated for alterations in microvascular permeability, using plasma clearance of FITC-dextran 150 as an index. Despite promoting a prominent tissue eosinophilia, the repetitive subcutaneous injections of rHu IL-5 did not alter the clearance of FITC-dextran 150. Topical application of rHu IL-5 to the cheek pouch, also, had no effect on the clearance of FITC-dextran 150. Immunofluorescence observations using an antibody to the granule protein, eosinophil peroxidase, indicated that the recruited cells had not degranulated. Our results support the importance of IL-5 in the recruitment of tissue eosinophils, but further stimulation is probably required to cause degranulation of these cells and the ensuing tissue damage.

  3. Pharmacokinetics of His-tag recombinant human endostatin in Rhesus monkeys

    Institute of Scientific and Technical Information of China (English)

    Hai-feng SONG; Xiu-wen LIU; Hai-ning ZHANG; Bao-zhen ZHU; Shou-jun YUAN; Shang-yi LIU; Zhong-ming TANG

    2005-01-01

    Aim: To study the pharmacokinetics and accumulation of an Escherichia coliexpressed His-tag fused recombinant human endostatin (rh-endostatin) in Rhesus monkeys. Methods: Rh-endostatin was iv or sc injected in Rhesus monkeys, and the rh-endostatin concentration in serum samples was determined by an enzyme immunoassay (EIA) method. The serum drug concentration-time data were analyzed by compartmental analysis using the practical pharmacokinetic program 3p97.Results: Following iv administration at a dose rate of 1.5, 4.5, and 13.5 mg/kg in rhesus monkeys, the concentration-time curves of rh-endostatin were best fitted to a three-compartment open model. AUC(0-∞) linearly increased with dose, while Cls exhibited no significant difference among different dose groups. The terminal half-lives (λ3) were 8±8, 3.1±1.4, and 20±14 h, respectively. After sc administration at a dose rate of 1.5 mg/kg, the concentration-time curve was best fitted to a two-compartment open model, with a terminal half-life (T1/2β) of 8±3 h.Bioavailability following sc injection was approximately 70%±3%. After consecutive iv injection of rh-endostatin at a dose rate of 1.5 mg.kg-1.d-1 for 7 d, the AUC(0-24 h) substantially increased from 22± 13 mg.h.L-1 (d 1) to 50±29 mg.h.L-1 (d 7), with an accumulation factor of 2.3±0.6 (P<0.05). Conclusion: The pharmacokinetic behavior of rh-endostatin in Rhesus monkeys complies with linear kinetics within the examined dose range. It tends to be accumulated in bodies after repeated administration at a dose level of 1.5 mg.kg-1.d-1 for more than 7 consecutive days.

  4. EFFECTS OF CHINA-MADE RECOMBINANT HUMAN GROWTH HORMONE ON THE TREATMENT OF GROWTH HORMONE DEFICIENCY

    Institute of Scientific and Technical Information of China (English)

    Jing Jiang; Wei Wang; Wen-xin Sun; Xiu-min Wang; Ji-hong Ni; Feng-sheng Chen; De-fen Wang

    2004-01-01

    Objective To evaluate the therapeutic effect of China-made recombinant human growth hormone (r-hGH) in children with growth hormone deficiency (GHD) and to investigate the utilities of various biochemical parameters in GHD diagnosis and treatment.Methods Our study comprises of 30 normal children and 71 GHD children treated with China-made r-hGH substitution 3 (IGFBP-3), bone turnover markers (Ost, ICTP), and anti-growth hormone antibody (GHAb) were detected before and after r-hGH treatment.Results After the first 3 and 6 months of treatment, growth velocities of GHD children were significantly increased (13.1 + 3.7 and 12.6 ± 3.6 cm/year) compared with pretreatment values (2.9 ± 0.8 cm/year, P < 0.01). GHD Children had obviously reduced serum levels of IGF-1, IGFBP-3, and bone turnover markers (Ost, ICTP) compared with normal controls(P < 0.01), and these biochemical parameters improved significantly after treatment (P < 0.01). Growth hormone antibodies were positive in 17 of 45 cases after treatment by binding capacity detection. The binding percentage of growth hormone antibody which was increased more than 30% after the treatment showed a negative correlation with growth velocity (P < 0.01).Conclusions (1) The growth stimulating effect and safety were confirmed in using China-made r-hGH in the treatment of GHD children for 6 months. (2) The measurements of serum IGF-1 and IGFBP-3 may serve as useful parameters in the diagnosis of GHD. (3) Serum Ost and ICTP are useful laboratory criteria for evaluating the effect of r-hGH therapy in the early stage. (4) It is necessary to monitor serum levels of GHAb during r-hGH therapy.

  5. Fundamental analysis of recombinant human epidermal growth factor in solution with biophysical methods.

    Science.gov (United States)

    Kim, Nam Ah; Lim, Dae Gon; Lim, Jun Yeul; Kim, Ki Hyun; Jeong, Seong Hoon

    2015-02-01

    Correlation of thermodynamic and secondary structural stability of proteins at various buffer pHs was investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR). Recombinant human epithelial growth factor (rhEGF) was selected as a model protein at various pHs and in different buffers, including phosphate, histidine, citrate, HEPES and Tris. Particle size and zeta potential of rhEGF at each selected pH of buffer were observed by DLS. Four factors were used to characterize the biophysical stability of rhEGF in solution: temperature at maximum heat flux (Tm), intermolecular β-sheet contents, zeta size and zeta potential. It was possible to predict the apparent isoelectric point (pI) of rhEGF as 4.43 by plotting pH against zeta potential. When the pH of the rhEGF solution increased or decreased from pI, the absolute zeta potential increased indicating a reduced possibility of protein aggregation, since Tm increased and β-sheet contents decreased. The contents of induced intermolecular β-sheet in Tris and HEPES buffers were the lowest. Thermodynamic stability of rhEGF markedly increased when pH is higher than 6.2 in histidine buffer where Tm of first transition was all above 70 °C. Moreover, rhEGF in Tris buffer was more thermodynamically stable than in HEPES with higher zeta potential. Tris buffer at pH 7.2 was concluded to be the most favorable.

  6. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

    Directory of Open Access Journals (Sweden)

    Ruvinov Emil

    2008-11-01

    Full Text Available Abstract Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO would improve tissue repair in rat after myocardial infarction (MI. Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat.

  7. Recombinant human lactoferrin enhances the efficacy of triple therapy in mice infected with Helicobacter pylori.

    Science.gov (United States)

    Yuan, Yuping; Wu, Qinyi; Cheng, Guoxiang; Liu, Xuefang; Liu, Siguo; Luo, Juan; Zhang, Aimin; Bian, Li; Chen, Jianquan; Lv, Jiajun; Dong, Xiangqian; Yang, Gang; Zhu, Yunzhen; Ma, Lanqing

    2015-08-01

    Helicobacter pylori (H. pylori) is a life-threatening pathogen which causes chronic gastritis, gastric ulcers and even stomach cancer. Treatment normally involves bacterial eradication; however, this type of treatment only has a rate of effectiveness of <80%. Thus, it is a matter of some urgency to develop new therapeutic strategies. Lactoferrin, a member of the transferrin family of iron-binding proteins, has been proven to be effective in removing a vast range of pathogens, including H. pylori. In the present study, we examined the effectiveness of recombinant human lactoferrin (rhLf) isolated from transgenic goats as a treatment for H. pylori in vitro and in vivo. For the in vivo experiments, BALB/c mice received an intragastric administration of 0.1 ml of a suspension of H. pylori. The mice were then divided into 4 groups: group A, treated with saline; group B, treated with 1.5 g of rhLF; group C, treated with the standard triple therapy regimen; and group D, treated with the standard triple therapy regimen plus.5 g of rhLF. Following sacrifice, the stomach tissues of the mice were histologically examined for the presence of bacteria. For the in vitro experiments, the bacteria were cultured in BHI broth and RT-qPCR and western blot analysis were carried out to determine the mRNA and protein levels of virulence factors (CagA and VacA) in the cultures. Our results revealed that rhLf not only inhibited the growth of H. pylori, but also suppressed the expression of two major virulence factors. Moreover, rhLf markedly increased bacterial eradication and effectively reduced the inflammatory response when combined with the standard triple therapy regimen. These results provide evidence supporting the use of rhLF as an adjuvant to traditional therapeutic strategies in the treatment of H. pylori.

  8. Different enzyme kinetics of midazolam in recombinant CYP3A4 microsomes from human and insect sources.

    Science.gov (United States)

    Christensen, Hege; Mathiesen, Liv; Postvoll, Lillian W; Winther, Bjørn; Molden, Espen

    2009-01-01

    In vitro drug metabolism techniques with human CYP c-DNA expressed systems are frequently used to predict human drug metabolism in vivo. The aim of this study was to compare midazolam enzyme kinetics in recombinant expressed CYP3A4 microsomes from human and insect cells. The amounts of 1'- hydroxymidazolam and 4-hydroxymidazolam formed in CYP3A4 microsomes from transfected human liver epithelial cells (T5-3A4 microsomes) and baculovirus-infected insect cells (with and without coexpressed cytochrome b(5)) were analysed by LC-MS. Enzyme kinetic parameters were estimated by nonlinear regression. Mean K(m) for the formation of 1'-hydroxymidazolam was 3- and 4-fold higher in T5-3A4 microsomes than in insect microsomes (pmicrosomes was reflected by significantly lower Cl(int) compared to insect microsomes (pmicrosomes displayed Michaelis-Menten kinetics, while insect microsomes showed substrate inhibition kinetics. The different enzyme kinetics of midazolam observed in recombinant CYP3A4 microsomes from human and insect sources, especially the substantially higher K(m) obtained in human microsomes compared to insect microsomes, should be further evaluated since it may have implications for correlations to in vivo situation.

  9. Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

    Science.gov (United States)

    Szpakowski, Piotr; Biet, Franck; Locht, Camille; Paszkiewicz, Małgorzata; Rudnicka, Wiesława; Druszczyńska, Magdalena; Allain, Fabrice; Fol, Marek; Pestel, Joël; Kowalewicz-Kulbat, Magdalena

    2015-01-01

    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.

  10. 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.

  11. Bioactivity, pharmacokinetics, and immunogenicity assays in preclinical and clinical trials for recombinant human endostatin

    Institute of Scientific and Technical Information of China (English)

    Bi HU; Hao-wen ZHU; Li-ping ZHU; Chen LI; Zhi-gang RONG; Jia-ming XU; Zhi-wei WU; Jian-jun WANG; Gen-xing XU

    2008-01-01

    Aim: To determine the in vitro and in vivo bioactivity of recombinant human endostatin (rhEndostatin) and to analyze its pharmacokinetics and immunogenicity in rhesus monkeys and patients. Methods: The physical chemical characteristics of rhEndostatin were detected according to Pharmacopoeia of the People's Republic of China (2005 edition, part Ⅲ). Its in vitro and in vivo bioactivities were assayed via proliferation-inhibition on human umbilical vein endothelial cells and their inhibitory effect on tumor-bearing mice models. Serum concentrations of rhEndostatin in monkeys and patients were determined by an enzyme immunoassay method. Results: The corresponding specific in vitro activities of rhEndostatin obtained from the cell counting method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and lactate dehydrogenase assay, respectively, were 6.4×107, 6.7×107, and 3.8×108 U/mg, and the in vivo antitumoral potency was 4.04×107 U/mg. In rhesus monkeys, there were no gender differences in all pharmacokinetic parameters. Serum anti-rhEndostatin immunoglobulin (Ig)G antibodies were generated quickly after intravenous (iv) administration and decreased rapidly when therapy was stopped. In phase Ⅰ clinical trials, linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve and the maximum serum concentration. Serum rhEndostatin reached a steady-state level after 7 d of successive administration with the average concentration at a steady state of 272.44±91.98 ng/mL. Neither IgG nor IgM antibodies against rhEndostatin were observed in patients. Conclusion: RhEndostatin exhibited a definite proliferation-inhibition effect on HUVEC, and significant antitumoral activity in mice. The immunoreactivity of rhesus monkeys to rhEndostatin is common, and rhEn-dostatin showed no immunogenicity in patients in this trial. The results provide a basis for further clinical trials.

  12. Passive immunization against dental caries and periodontal disease: development of recombinant and human monoclonal antibodies.

    Science.gov (United States)

    Abiko, Y

    2000-01-01

    and periodontal diseases are summarized, and the biotechnological approaches for developing recombinant and human-type antibodies are introduced. Furthermore, our own attempts to construct single-chain variable fragments (ScFv) and human-type antibodies capable of neutralizing virulence factors are discussed.

  13. Detection of DNA-recombinant human epoetin-alfa as a pharmacological ergogenic aid.

    Science.gov (United States)

    Wilber, Randall L

    2002-01-01

    The use of DNA-recombinant human epoetin-alfa (rhEPO) as a pharmacological ergogenic aid for the enhancement of aerobic performance is estimated to be practised by at least 3 to 7% of elite endurance sport athletes. rhEPO is synthesised from Chinese hamster ovary cells, and is nearly identical biochemically and immunologically to endogenous epoetin-alfa (EPO). In a clinical setting, rhEPO is used to stimulate erythrocyte production in patients with end-stage renal disease and anaemia. A limited number of human studies have suggested that rhEPO provides a significant erythropoietic and ergogenic benefit in trained individuals as evidenced by increments in haemoglobin, haematocrit, maximal oxygen uptake (VO2max) and exercise endurance time. The purpose of this review is to summarise the various technologies and methodologies currently available for the detection of illicit use of rhEPO in athletes. The International Olympic Committee (IOC) banned the use of rhEPO as an ergogenic aid in 1990. Since then a number of methods have been proposed as potential techniques for detecting the illegal use of rhEPO. Most of these techniques use indirect markers to detect rhEPO in blood samples. These indirect markers include macrocytic hypochromatic erythrocytes and serum soluble transferrin receptor (sTfr) concentration. Another indirect technique uses a combination of 5 markers of enhanced erythropoiesis (haematocrit, reticulocyte haematocrit, percentage of macrocytic red blood cells, serum EPO, sTfr) to detect rhEPO. The electrophoretic mobility technique provides a direct measurement of urine and serum levels of rhEPO, and is based on the principle that the rhEPO molecule is less negatively charged versus the endogenous EPO molecule. Isoelectric patterning/focusing has emerged recently as a potential method for the direct analysis of rhEPO in urine. Among these various methodologies, the indirect technique that utilises multiple markers of enhanced erythropoiesis appears to

  14. Analysis of the kinetic mechanism of recombinant human isoprenylcysteine carboxylmethyltransferase (Icmt

    Directory of Open Access Journals (Sweden)

    Baron Rudi A

    2004-12-01

    Full Text Available Abstract Background Isoprenylcysteine carboxyl methyltransferase (Icmt is the third of three enzymes that posttranslationally modify proteins that contain C-terminal CaaX motifs. The processing of CaaX proteins through this so-called prenylation pathway via a route initiated by addition of an isoprenoid lipid is required for both membrane targeting and function of the proteins. The involvement of many CaaX proteins such as Ras GTPases in oncogenesis and other aberrant proliferative disorders has led to the targeting of the enzymes involved in their processing for therapeutic development, necessitating a detailed understanding of the mechanisms of the enzymes. Results In this study, we have investigated the kinetic mechanism of recombinant human Icmt. In the reaction catalyzed by Icmt, S-adenosyl-L-methionine (AdoMet provides the methyl group that is transferred to the second substrate, the C-terminal isoprenylated cysteine residue of a CaaX protein, thereby generating a C-terminal prenylcysteine methyl ester on the protein. To facilitate the kinetic analysis of Icmt, we synthesized a new small molecule substrate of the enzyme, biotin-S-farnesyl-L-cysteine (BFC. Initial kinetic analysis of Icmt suggested a sequential mechanism for the enzyme that was further analyzed using a dead end competitive inhibitor, S-farnesylthioacetic acid (FTA. Inhibition by FTA was competitive with respect to BFC and uncompetitive with respect to AdoMet, indicating an ordered mechanism with SAM binding first. To investigate the order of product dissociation, product inhibition studies were undertaken with S-adenosyl-L-homocysteine (AdoHcy and the N-acetyl-S-farnesyl-L-cysteine methylester (AFCME. This analysis indicated that AdoHcy is a competitive inhibitor with respect to AdoMet, while AFCME shows a noncompetitive inhibition with respect to BFC and a mixed-type inhibition with respect to AdoMet. These studies established that AdoHcy is the final product released, and

  15. Recombinant hirudin suppresses the viability, adhesion, migration and invasion of Hep-2 human laryngeal cancer cells.

    Science.gov (United States)

    Lu, Qian; Lv, Mei; Xu, Erdong; Shao, Fangyu; Feng, Ya; Yang, Jingru; Shi, Lin

    2015-03-01

    Recombinant hirudin (rH) is a highly potent and specific inhibitor of thrombin, and has been shown to inhibit the growth and metastasis of several types of cancers in experimental tumor models. The objective of this study was to evaluate the antitumor effects and explore the underlying mechanisms of rH in Hep-2 human laryngeal carcinoma (LC) cells. Hep-2 cells were treated with various concentrations of rH for 24 h. The cell viability was evaluated by a water-soluble tetrazolium salt (WST) assay. The adhesion ability of the cells was evaluated by cell adhesion to fibronectin. Cell migration and invasion were measured with the Boyden chamber assay. Cell apoptosis was detected by Hoechst 33324 fluorescence staining. A chicken chorioallantoic membrane (CAM) assay was used to assess the effects of rH on angiogenesis in vivo. Western blotting was used to detect the expression levels of vascular endothelial growth factor receptor (VEGF-R), focal adhesion kinase (FAK), Bcl-2-associated agonist of cell death (Bad) and B-cell CLL/lymphoma 2 (Bcl-2) proteins. rH significantly inhibited the cell viability and induced apoptosis in LC Hep-2 cells in a dose-dependent manner, as compared with phosphate-buffered saline (PBS) as control. These results were accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bad. Moreover, rH dose-dependently inhibited the adhesion, migration and invasion of the Hep-2 cells, compared to the vehicle PBS. In addition, rH robustly suppressed angiogenesis in the CAM assay. Importantly, the expression of adhesion and angiogenesis-associated proteins FAK and VEGF-R was significantly downregulated by rH in a dose-dependent manner. The present findings demonstrate that rH exerts antitumor effects in Hep-2 human laryngeal cancer cells via multiple mechanisms and suggests that targeting thrombin by rH is a potential strategy for the treatment of LC.

  16. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    DEFF Research Database (Denmark)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban

    2016-01-01

    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......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...

  17. Activation of coagulation by administration of recombinant factor VIIa elicits interleukin 6 (IL-6) and IL-8 release in healthy human subjects

    National Research Council Canada - National Science Library

    Jonge, de, E; Friederich, P.W; Vlasuk, G.P; Rote, W; Vroom, M.B; Levi, M.M; Poll, van der, T

    2003-01-01

    .... 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 the concentrations of interleukin 6 (IL-6) and IL-8 in plasma...

  18. 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.

  19. 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.

  20. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Science.gov (United States)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    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.

  1. The Enhancement of Osteogenesis by Scaffold Based on Mineralized Recombinant Human-like Collagen Loading with rhBMP-2

    Institute of Scientific and Technical Information of China (English)

    WU Bin; ZHENG Qixin; GUO Xiaodong; WU Yongchao; WANG Yu; CUI Fuzai

    2009-01-01

    A biomimetic scaffold based on mineralized recombinant collagen,nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid)(nHA/RHLC/PLA),was prepared with recombinant human bone morphogenic protein-2(rhBMP-2)for improving the os-teoinductive property of the scaffold.The nHA/RHLC/PLA scaffolds loaded with 10μg rhBMP-2 and the unloaded scaffolds were implanted subcutaneously in the rat model.The osteogenetic capacity of these composites was evaluated by CT scan,ALP activity test and histological observation at 4 and 8 weeks after implantation.The experimental results indicated that the osteogenic capability of the scaffolds loaded with rhBMP-2 was superior to the unloaded scaffold.It was concluded that rhBMP-2 can enhance the osteoinductive property of the nHA/RHLC/PLA scaffold and the nHA/RHLC/PLA scaffold loaded with rhBMP-2 have the good potential of being used in bone tissue engineering.

  2. A meta-analysis of controlled trials of recombinant human activated protein C therapy in patients with sepsis

    Directory of Open Access Journals (Sweden)

    Wiedermann Christian J

    2005-10-01

    Full Text Available Abstract Background Meta-analysis of two randomised controlled trials in severe sepsis performed with recombinant human activated protein C may provide further insight as to the therapeutic utility of targeting the clotting cascade in this syndrome. Methods In search for relevant studies published, two randomized clinical trials were found eligible. Results The studies, PROWESS and ADDRESS, enrolled a total of 4329 patients with risk ratio (RR and 95% confidence interval (CI data for effect on 28-day mortality relative to control treatment of 0.92 (0.83–1.02 suggesting that recombinant human activated protein C is not beneficial in severe sepsis. In PROWESS, 873 of 1690 patients presented with low risk, and 2315 of 2639 patients in ADDRESS as defined by APACHE II score Conclusion This meta-analysis, therefore, raises doubts about the clinical usefulness of recombinant activated protein C in patients with severe sepsis and an APACHE II score ≥ 25 which can only be resolved by another properly designed clinical trial.

  3. [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.

  4. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation.

    Science.gov (United States)

    Osipov, Andreyan N; Grekhova, Anna; Pustovalova, Margarita; Ozerov, Ivan V; Eremin, Petr; Vorobyeva, Natalia; Lazareva, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry; Eremin, Ilya

    2015-09-29

    Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices.

  5. Expression of human clotting factor Ⅸ mediated by recombinant lentiviral vector in cultured cells and hemophilia B mice

    Institute of Scientific and Technical Information of China (English)

    ZHU Huanzhang; CHEN Xiaoguang; LI Feng; GONG Juli; XUE Jinglun

    2003-01-01

    To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMVΔR8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.

  6. Human parvovirus B19 serology and avidity using a combination of recombinant antigens enables a differentiated picture of the current state of infection.

    Science.gov (United States)

    Pfrepper, K-I; Enders, M; Motz, M

    2005-01-01

    In order to improve serodiagnostic methods for the determination of the state of human parovirus B19 infection, a new test system, recomLine Parvovirus, based on the use of recombinant antigens, has been developed and evaluated. The test system combines the advantages of enzyme-linked immunosorbent assay (ELISA) methods with those of the Western blot technique. For the recombinant line assay, five antigens of human parvovirus B19 that were recombinantly produced in Escherichia coli were applied directly on nitrocellulose membranes: VP2, the aminoterminal and the carboxyterminal domain of VP1 (VP-N and VP-C), VP-1S another fragment of VP-N and NS1. In addition, empty virus particles isolated from eukaryotic cell cultures were also applied. The recombinant-line assay was used to detect human IgG and IgM antibodies directed against human parvovirus B19. In addition, the avidity of the IgG antibodies was investigated. The recombinant line assay was evaluated using 87 human serum samples of patients recently infected with human parvovirus B19 including 10 samples of three infection time courses and 100 serum samples of healthy blood donors. All results were compared with commercially available ELISAs. In the case of discrepancies, Western blot analysis was performed. The data revealed the recombinant line assay to be highly sensitive and specific. The individual determination of the human immune response against several recombinant antigens covering the structural proteins of human parvovirus B19 gives a deeper insight into the actual status of infection. In addition, the determination of IgG avidity against these individual recombinant antigens enables a more precise and differentiated picture of the infection event.

  7. Expression of recombinant human lysozyme in transgenic chicken promotes the growth of Bifidobacterium in the intestine and improves postnatal growth of chicken

    OpenAIRE

    Wang, Hai; Wu, Hongping; Wang, Kejun; Cao, Zhichen; Yu, Kun; Lian, Ling; Lian, Zhengxing

    2016-01-01

    Lysozyme is one kind of antimicrobial proteins and often used as feed additive which can defend against pathogenic bacteria and enhance immune function of animals. In this study, we have injected the lentiviral vector expressing recombinant human lysozyme (rhLZ) gene into the blastoderm of chicken embryo to investigate the effect of recombinant human lysozyme on postnatal intestinal microbiota distribution and growth performance of chicken. Successfully, we generated 194 transgenic chickens i...

  8. Suppression of Growth of Hela, EJ, SK-OV-3 and MDA-MB-231 Cells by Recombinant Human NK4

    Institute of Scientific and Technical Information of China (English)

    Hua Bai; Hong Chen; Chang-shan Ren

    2009-01-01

    Objective: To study the effects of recombinant human Nk4 on the growth and invasion activities of tumor cells. Methods: The inhibition of recombinant human NK4 on human oophoroma, cervical tumor, breast tumor and gallbladder tumor cells was evaluated in vitro by basement membrane invasion assay.Results: rhNK4 could markedly inhibited the growth of human oophoroma, cervical tumor and breast tumor cells. The inhibition rates of human oophoroma, cervical tumor, breast tumor and gallbladder tumor cells were 48.2%, 29.2%, 58.4% and 30.1% respectively. Conclusion: rhNK4 factor can markedly inhibit the invasion of multiple tumor cells.

  9. Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains.

    Science.gov (United States)

    Wang, Junfeng; Liu, Changting; Liu, Jinyi; Fang, Xiangqun; Xu, Chen; Guo, Yinghua; Chang, De; Su, Longxiang

    2014-03-01

    The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis.

  10. Evidence for repair of ultraviolet light-damaged herpes virus in human fibroblasts by a recombination mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Hall, J.D.; Featherston, J.D.; Almy, R.E.

    1980-09-01

    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.

  11. CONSTRUCTION AND IDENTIFICATION OF THE RECOMBINANT OF THE AAV VECTOR AND HUMAN INTERFERON-GAMMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To construct and identify further a recombinant of Adeno-associated virus and interferon-gamma for gene therapy, the full-length IFN-γcDNA containing signal peptide was amplified by PCR, and then cloned into the pUC18. After screening, the fragment from the positive clone was then subcloned into pwpl9. After the correct recombinant was identified by digestion with SacI and BamHI, it was transfected into lympho- cyte cell line H9 mediated by calcium phosphate, and the expression of IFN-γ was detected by RT-PCR and ELISA. The result showed that the IFN-y were expressed in the H9 cells transfected with pwp/IFN-y. The so constructed recombinant plasmid pwpl9/IFN-y containing the full-length IFN-y gene was expressed in mam- malian cells.

  12. N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits.

    Science.gov (United States)

    Jongen, Susanne P; Gerwig, Gerrit J; Leeflang, Bas R; Koles, Kate; Mannesse, Maurice L M; van Berkel, Patrick H C; Pieper, Frank R; Kroos, Marian A; Reuser, Arnold J J; Zhou, Qun; Jin, Xiaoying; Zhang, Kate; Edmunds, Tim; Kamerling, Johannis P

    2007-06-01

    Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the

  13. Preparation of recombinant human bone morphogenetic protein-2 loaded dextran-based microspheres and their characteristics

    Institute of Scientific and Technical Information of China (English)

    Fa-ming CHEN; Zhi-fen WU; Qin-tao WANG; Hong WU; Yong-jie ZHANG; Xin NIE; Yan JIN

    2005-01-01

    Aim: To prepare new pharmaceutical forms with sustained delivery properties of recombinant human bone morphogenetic protein-2 (rhBMP2) for tissue engineering and guided tissue regeneration (GTR) use. Methods: rhBMP2-1oaded dextranbased hydrogel microspheres (rhBMP2-MPs), which aimed to keep rhBMP2 bioactivity and to achieve long-term sustained release of rhBMP2, were prepared by double-phase emulsified condensation polymerization. The physical, chemical performances and biological characteristics of those microspheres were studied both in vitro and in vivo. Results: The microspheres' average diameter was 30.33±4.32 μm with 75.4% ranging from 20 μm to 40 μm and the drug loading and encapsulation efficiency were 7.82% and 82.25%, respectively. The rhBMP2-releasing profiles in vitro showed that rhBMP2 release could be maintained more than 10 d. The rhBMP2-MPs, with good swelling and biodegradation behavior,could be kept for 6 months at below 4 ℃ without significant characteristic change or bioactivity loss. Cytology studies showed that rhBMP2-MPs could promote the proliferation of periodontal ligament cells (PDLCs) approximately 10 d, while the bioactivity of concentrated rhBMP2 solution could keep no more than 3 d.Scanning electron microscope showed that rhBMP2-MPs could be enchased into the porous structure of calcium phosphate ceremic (CPC) and the eugonic growth of PDLCs in CPC/rhBMP2-MPs scaffolds. Animal experiments indicated that using CPC/rhBMP2-MPs scaffolds could gain more periodontal tissue regeneration than using rhBMP2 compound firsthand with CPC (CPC/rhBMP2). Conclusion:By encapsulating rhBMP2 into dextran-based microspheres, a small quantity of rhBMP2 could achieve equivalent effects to the concentrated rhBMP2 solution and at the same time, could prolong rhBMP2 retention both in vitro and in vivo.

  14. [Clinical analysis of recombinant humanized thrombopoietin for treating 25 children with severe immune thrombocytopenia].

    Science.gov (United States)

    Zheng, Jie; Ma, Jing-Yao; Su, Yan; Yang, Jing; Zhang, Rui-Dong; Zhou, Xuan; Wu, Run-Hui

    2014-04-01

    This study was aimed to evaluate the efficacy and safety of recombinant humanized thrombopoietin (rhTPO) for treating children with severe immune thrombocytopenia (ITP). A total of 25 patients with severe ITP who accepted rhTPO treatment for 14 days between December, 2009 and November, 2012 in Beijing Children's Hospital was retrospectively analyzed. The results showed that the median platelet counts of all 25 patients increased from the lowest level 4.0×10(9)/L (0×10(9)/L-10×10(9)/L) to the highest level 71×10(9)/L (14×10(9)/L-439×10(9)/L) on median 11 days (range from 3 days to 15 days). After rhTPO discontinuation, the platelet counts of patients gradually decreased. Complete response rate was 44% (11/25), response rate was 32% (8/25), non-response rate was 24% (6/25) and total response rate was 76% (19/25). The platelet count in the patients who showed complete response to rhTPO therapy reached the highest 112×10(9)/L (43×10(9)/L-439×10(9)/L) on median 12 days(range from 7 days to 15 days). The patients showed response to rhTPO treatment on median 4 days (range from 1 days to 11 days). The platelet count decreased gradually after the discontinuation of rhTPO administration but still significantly higher on 28 days than the level before the treatment (P rhTPO treatment showed response to γ-globulin after the discontinuation of rhTPO therapy. 2 patients showed mild clinical adverse reaction. It is concluded that rhTPO is an effective and safe treatment method for children with severe ITP. It will help the patient smoothly through the dangerous period of severe bleeding, but the platelet count decreases gradually after rhTPO discontinuation. Maintenance treatment is needed to consolidate the curative efficacy.

  15. Recombinant human thrombopoietin augments mobilization of peripheral blood progenitor cells for autologous transplantation.

    Science.gov (United States)

    Linker, Charles; Anderlini, Paolo; Herzig, Roger; Christiansen, Neal; Somlo, George; Bensinger, William; Fay, Joseph; Lynch, Joseph P; Goodnough, Lawrence T; Ashby, Mark; Benyunes, Mark C; Jones, Dennie V; Yang, Timothy A; Miller, Langdon L; Weaver, Charles

    2003-06-01

    This study assessed the ability of various schedules of recombinant human thrombopoietin (rhTPO) to enhance mobilization of peripheral blood progenitor cells (PBPCs) in 134 patients with cancer undergoing high-dose chemotherapy and autologous PBPC transplantation. Patients received the study drug on days 1, 3, and 5 before initiation of granulocyte colony-stimulating factor (G-CSF) 10 microg/kg/day on day 5 and pheresis starting on day 9. Randomly assigned treatments on days 1, 3, and 5 were: group 1 (n=27) placebo, placebo, rhTPO 1.5 microg/kg; group 2 (n=27) rhTPO 1.5 microg/kg, placebo, placebo; groups 3 (n=28) and 4 (n=22) rhTPO 0.5 microg/kg on all 3 treatment days; and group 5 (n=30) placebo on all 3 treatment days. After high-dose chemotherapy and PBPC transplantation, groups 1 through 4 received rhTPO 1.5 microg/kg days 0, +2, +4, and +6 with either G-CSF 5 microg/kg/day (groups 1-3) or granulocyte-macrophage colony-stimulating factor 250 microg/m(2)/day (group 4). Group 5 received placebo plus G-CSF 5 microg/kg/day. The addition of rhTPO to G-CSF increased median CD34+ cell yield/pheresis in cohorts in which rhTPO was started before day 5, with higher yields in groups 2 (2.67 x 10(6)/kg) and groups 3 and 4 (3.10 x 10(6)/kg) than in group 1 (1.86 x 10(6)/kg) or group 5 (1.65 x 10(6)/kg) (P=.006 across groups). Comparing rhTPO to placebo, higher percentages of patients achieved the minimum yield of CD34+ > or =2 x 10(6)/kg (92% v 75%; P=.050) as well as the target yield of CD34+ > or =5 x 10(6)/kg (73% v 46%; P= .041). rhTPO-treated patients required fewer phereses to achieve minimum (P= .011) and target (P= .015) CD34+ cell values. rhTPO given after transplantation did not speed platelet recovery. No neutralizing antibodies were observed. We conclude that rhTPO can safely enhance mobilization of PBPC, reduce the number of leukapheresis, and allow more patients to meet minimal cell yield requirements to receive high-dose chemotherapy with PBPC

  16. Antitumor effect of recombinant human endostatin combined with cisplatin on rats with transplanted Lewis lung cancer

    Institute of Scientific and Technical Information of China (English)

    Zhan-Wu Yu; Ying-Hua Ju; Cheng-Liang Yang; Han-Bing Yu; Quan Luo; Ye-Gang Ma; Yong-Yu Liu

    2015-01-01

    Objective:To observe the antitumor effect and mechanism of recombinant human endostatin (Endostar) injection in tumor combined with intraperitoneal injection of cisplatin on subcutaneous transplanted Lewis lung cancer in rats.Methods:A total of 30 C57 rats were selected, and the monoplast suspension of Lewis lung cancer was injected into the left axilla to prepare the subcutaneous transplanted tumor models in the axilla of right upper limb. The models were randomly divided into Groups A, B, and C. Medication was conducted when the tumor grew to 400 mm3. Group A was the control group without any interventional treatment. Group B was injected with Endostar 5 mg.kg-1.d for 10 d. Group C was given the injection of Endostar 5 mg.kg-1.d combined with intraperitoneal injection of cisplatin 5 mg.kg-1.d for 10 d. All the rats in three groups were executed the day after the 10-d medication and the tumor was taken off for measurement of volume and mass changes and calculation of antitumor rate, after which the vascular endothelial growth factor (VEGF) concentration in rats’plasma was determined by ELISA. The tumor tissues were cut for the preparation of conventional biopsies. After hematoxylin-eosin staining, the pathologic histology was examined to observe the structures of tumor tissues, VEGF score and microvessel density (MVD) in each group. Results:The volume and mass of tumor in Groups B and C were significantly lower than Group A (P< 0.05) while the tumor volume and mass in Group C were significantly lower than Group B (P < 0.05). The antitumor rate in Group C was significantly higher than Group B (P < 0.05), but the tumor VEGF score, MVD and plasma VEGF level in Group C were significantly lower than Groups A and B (P < 0.05). In Group B, the tumor VEGF score, MVD and plasma VEGF level were significantly lower than Group A (P < 0.05). The microscopic image of Group C showed that its number of active tumor cells and the blood capillary around tumor was significantly

  17. Glutamine and recombinant human growth hormone protect intestinal barrier function following portal hypertension surgery

    Institute of Scientific and Technical Information of China (English)

    Zhao-Feng Tang; Yun-Biao Ling; Nan Lin; Zheng Hao; Rui-Yun Xu

    2007-01-01

    AIM: To evaluate the effects of combined treatment of glutamine (Gln) and recombinant human growth hormone(rhGH) on intestinal barrier function following portal hypertension surgery.METHODS: This study was designed as a prospective,randomized and controlled clinical trial. Forty two patients after portal hypertension surgery were randomly assigned into 2 groups: control group (n = 20) and supplemental group (adding Gin and rhGH, n = 22). Every patient received isocaloric and isonitrogenous standard total parenteral nutrition (TPN) starting 3 d after surgery for 7 d. Blood samples were obtained before surgery and at the 3rd and 10th day postoperatively. Host immunity was evaluated by measuring levels of CD4, CD8, CD4/CD8, IgG, IgM and IgA, and the inflammatory responses were determined by assessing IL-2, TNF-α and C-reactive protein (CRP) levels. Intestinal permeability and integrity was evaluated by L/M test and histological examination, respectively.RESULTS: On postoperative d 10, CD4, CD4/CD8, IgG and IL-2 levels in supplemental group were significantly higher than those in control group (33.7 ± 5.5 vs 31.0± 5.4, P < 0.05, (1.17 ± 0.32 vs 1.05 ± 0.15, P < 0.05,13.94 ± 1.09 vs 12.33±1.33, P < 0.05, and 368.12± 59.25 vs 318.12 ± 45.65, P < 0.05, respectively),whereas the increase in serum TNF-α concentration was significantly reduced (41.02 ± 27.56 vs 160.09 ± 35.17,P < 0.05). The increase in L/M ratio was significantly lower in the supplemental group than in the control group (0.0166 ± 0.0017 vs 0.0339 ± 0.0028, P < 0.05).Moreover, mucosal integrity in the supplemental group was better than in the control group.CONCLUSION: Postoperative administration of TPN supplemented with Gin and rhGH in patients after portal hypertension surgery improves immune function,modulates inflammatory response, prevents the intestinal mucous membrane from atrophy and preserves intestinal integrity.

  18. Bioequivalence studies for three formulations of a recombinant human growth hormone: challenges and lessons learned.

    Science.gov (United States)

    Dao, Le; Jacobs, Joan; Kuebler, Peter; Bakker, Bert; Lippe, Barbara

    2010-10-01

    Two bioequivalence (BE) studies in healthy volunteers comparing new formulations of the recombinant human growth hormone (rhGH) Nutropin AQ (somatropin [rDNA origin] injection; Genentech, Inc., South San Francisco, CA) with the currently marketed formulation (5 mg/mL) were conducted to extend available dosing options. All formulations were administered by subcutaneous (SC) injection ranging in volume from 0.25 to 1.0 mL depending on the formulation concentration. Study A was a 2-period crossover design to assess the BE of 5 and 10 mg/mL. The estimate for relative bioavailability (AUC(0-24 h)) was within the prespecified BE interval (0.80-1.25). However, while the C(max) estimate (1.17) was contained within the range for BE, the 90% CI (0.986-1.38) extended beyond the prespecified BE interval. As a result, Study A failed to show BE between the 5 and 10mg/mL formulations. Review of the data showed unexpected increased variability in the observed C(max). Further review of individual data suggested that in 4 subjects, the GH concentration profile of 1 of the 2 injections closely resembled the absorption kinetics of an intramuscular injection rather than an SC injection. Because study conduct may have contributed to these results, we performed a second study, Study B. This study incorporated injection technique training, a defined injection site, and a larger sample size to accommodate variability. It also included a third formulation, creating a 3-period crossover design to assess the BE of 2.5, 5, and 10 mg/mL. Study B results demonstrated BE of the new 2.5- and 10-mg/mL formulations to the reference 5-mg/mL formulation, and BE to each other, with all 90% CIs within the BE range of 0.80 to 1.25. Thus the challenge of recognizing that design issues could affect outcomes gave us the tools to perform a second study, and the positive results taught us that demonstrating BE is an issue not only of pharmacology, but also of study methodology and execution.

  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. Effects of recombinant human growth hormone on rat septic shock with intraabdominal infection by E. coli

    Institute of Scientific and Technical Information of China (English)

    Ying Huang; Shu-Ren Wang; Cheng Yi; Ming-Ying Ying; Ying Lin; Mao-Hui Zhi

    2002-01-01

    AIM: To investigate the therapeutic effects of recombinant human growth hormone (rhGH) on rat septic shock with intraabdominal infection by E. coli and its possible mechanism. METHODS: 76 SD rats were divided into 3 groups randomly:control group (group C, n=16) without any special treatnent,of E. coli(1×1010 cfu@ L-1,15 ml@kg-1, ip), treated group (group by rhGH injection (2.25 U@kg-1@d-1, im). Group S and group T were further divided into 1d and 3d subgroups, respectively (n=15 each). Mean arterial pressure (MAP), levels of plasma TNFα and endotoxin and leukocyte count were determined on 1st day and 3rd day after E. coli injection. Another 39 SD rats were divided into groups C, S and T (n=13 each) just for observing survival rate within 1 week.RESULTS: (1) On 1st and 3rd day, MAP in group S decreased markedly, and MAP on 1st day lowered more than that of 3rd day (P<0.01), while MAP in group T just decreased slightly. The survival rate within 1 week was much higher in group T (84.6 %) than in group S (46.2 %) (P<0.01). (2)On 1st day, plasma TNFα and endotoxin elevated significantly in group S and group T (P<0.05), and endotoxin in group S had more increase (P<0.01). On 3rd day, TNFα in group S returned to the level of group C (P>0.05),while TNFα in group T went down below the level of group C(P<0.01). On 3rd day, endotoxin in group S declined, but was still higher than that of group C (P<0.01), endotoxin in group T returned to the level of group C (P>0.05). (3) On 1st day, neutrophil ratio in total leukocyte count in both group S and group T increased significantly (P<0.05 vs group C).CONCLUSION: rhGH showed beneficial effects on rat septic shock. The possible mechanisms may involve the attenuation of bacteria/endotoxin translocation and decreased systemic endotoxin level; inhibition of the production and release of TNFα; improved circulatory function; improved systemic host defenses and maintenance of intestinal mucosa barrier.

  1. Recombination Mutant Human Tumor Necrosis Factor Combined with Chemotherapy in the Treatment of Advanced Cancer

    Institute of Scientific and Technical Information of China (English)

    LIUXing; ZHANGXiangfu; ZHENGZhiweng; LUHuishan; WUXinyuan; HUANGChangmin; WANGChuan; GUANGuoxian

    2005-01-01

    Objective: Past studies showed that tumor necrosis factor (TNF) assisted anti-tumor treatment and intensified the sensitivity of chemotherapy. However its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. The objective of present study is to evaluate the therapeutic effects and adverse reactions of recombinant mutant human tumor necrosis factor (rmhTNF) combined with chemotherapy in patients with advanced malignant tumor. Methods: 105 patients with advanced malignant tumor were randomly divided into trial group, 69 patients, and control group, 36 patients.rm hTNF was injected intramuscularly to the trial group at a dose of 4×106 U/m2, from the 1st to 7th days, the llth to 17th days combined with chemotherapy course. The chemotherapy plan was as follows:CAP for patients with the NSCLC; FAM for patients with gastric cancer; FC for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. The control group was given only the same chemotherapy as the trial group. Results: In the trial group there was 1 CR case and 12 PR cases, and the response rate was 13/69 (18.84%); in the control group 1 PR case, the response rate 1/36 (2.78%). The response rate in the trial group was significantly higher than that in the control group (P=0.022). The response rate for NSCLC in the trial group was 8/17 (47.06%), and 1/6 (16.67%) in the control group. The response rates for gastric cancer and colorectal cancer in the trial groups also were higher than those in the control groups. After the treatment the KPS was 89.00+9.92 in the trial group,and 84.17±8.84 in the control group, with a significant difference between the two groups (P=0.028). The adverse reactions of rmhTNF injection included: pain in the injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia, and cold-like symptoms. All these adverse reactions were mild and bearable

  2. Repeat use of human recombinant bone morphogenetic protein-2 for second level lumbar arthrodesis.

    Science.gov (United States)

    Singh, Kern; Dumonski, Mark; Stanley, Tom; Ponnappan, Ravi; Phillips, Frank M

    2011-02-01

    Prospective randomized controlled animal model. The purpose of this study is to determine whether the readministration of human recombinant bone morphogenetic protein-2 (rhBMP-2) induces an immune response and inhibits successful fusion in repeat posterolateral spinal surgery. Little research has been performed on the effectiveness or immunoreactivity of rhBMP-2 (Infuse, Medtronic, Memphis, TN) in the context of its reuse in posterolateral fusion spinal surgery at adjacent levels. A total of 34 New Zealand White rabbits underwent posterior intertransverse process fusion with the use of rhBMP-2 delivered on an absorbable collagen sponge (rhBMP-2/ACS). Two rabbits were killed early leaving 32 total rabbits. Serologic studies (Type I bovine collagen and rhBMP-2 antibodies) were obtained at 2-week intervals throughout the experiment. At 10 weeks, posteroanterior radiographs confirmed solid fusion masses in all rabbits. The 32 rabbits were randomly separated into 2 groups of 16, and each group underwent an adjacent level, bilateral intertransverse process fusion with either rhBMP-2/ACS or iliac crest. There was no statistical difference in fusion rates with repeat use of rhBMP-2 (n = 15/16, 94%) or iliac crest (n = 11/16, 69%) (P = 0.17) at the adjacent level. Four rabbits (n = 4/32, 13%) developed rhBMP-2 antibodies. Of these 4 rabbits, 1 developed anti-rhBMP antibodies after the first exposure and 3 developed antibodies after the second surgery. Eight rabbits (n = 8/32, 25%) developed collagen antibodies with 7 rabbits developing antibodies after the first exposure and 1 rabbit developing antibodies after the second exposure. The development of antibodies did not effect fusion rates. No rabbit demonstrated evidence of a systemic or anaphylactic reaction to repeat exposure to rhBMP-2. rhBMP-2 appears to be successful in promoting intertransverse fusions when used in both primary and repeat fusion environments. The infrequent development of antibodies to rhBMP-2 after

  3. 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

  4. Properties of recombinant human N1-acetylpolyamine oxidase (hPAO): potential role in determining drug sensitivity.

    Science.gov (United States)

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Frydman, Benjamin; Valasinas, Aldonia; Fraser, Alison V; Woster, Patrick M; Casero, Robert A

    2005-07-01

    The recent cloning of the mammalian gene coding for N(1)-acetylpolyamine oxidase (PAO) provides the opportunity to directly examine the role of human PAO (hPAO) in polyamine homeostasis as well as its potential role in determining cellular response to antitumor polyamine analogues. To facilitate the study of this enzyme, the production, purification, and characterization of the recombinant hPAO is reported. hPAO oxidizes N(1)-acetylspermidine (K(m)=2.1 microM, K(cat)=15.0 s(-1)) and has very high affinity for N(1)-acetylspermine (K(m)=0.85 microM, K(cat)=31.7 s(-1)). The recombinant hPAO does not efficiently oxidize spermine, thereby demonstrating a significant difference in substrate specificity from the previously described human spermine oxidase PAOh1/SMO. Importantly, hPAO demonstrates the ability to oxidize a subset of antitumor polyamine analogues, suggesting that this oxidase activity could have a significant effect on determining tumor sensitivity to these or similar agents. Transfection of A549 human lung cancer cells with an hPAO-expressing plasmid leads to a profound decrease in sensitivity to those analogues which act as substrates, confirming its potential to alter drug response. One similarity that hPAO shares with human PAOh1/SMO, is that certain oligoamine analogues are potent inhibitors of its oxidase activity. The results of these studies demonstrate how changes in polyamine catabolism may affect drug response.

  5. Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris.

    Science.gov (United States)

    Lu, Hairong; Huang, Jinjiang; Li, Guodong; Ge, Kuikui; Wu, Hongyu; Huang, Qingshan

    2012-03-01

    Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.

  6. Genetic crossovers are predicted accurately by the computed human recombination map.

    Directory of Open Access Journals (Sweden)

    Pavel P Khil

    2010-01-01

    Full Text Available Hotspots of meiotic recombination can change rapidly over time. This instability and the reported high level of inter-individual variation in meiotic recombination puts in question the accuracy of the calculated hotspot map, which is based on the summation of past genetic crossovers. To estimate the accuracy of the computed recombination rate map, we have mapped genetic crossovers to a median resolution of 70 Kb in 10 CEPH pedigrees. We then compared the positions of crossovers with the hotspots computed from HapMap data and performed extensive computer simulations to compare the observed distributions of crossovers with the distributions expected from the calculated