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At least 415 records · Page 23Linked to original sources
PubMed2001

Induction of ovulation in World Health Organization group II anovulatory women undergoing follicular stimulation with recombinant human follicle-stimulating hormone: a comparison of recombinant human chorionic gonadotropin (rhCG) and urinary hCG.

OBJECTIVE: To compare the safety and efficacy of 250 microg recombinant hCG (rhCG) and 5,000 IU urinary hCG (uhCG), both administered s.c., for ovulation induction in anovulatory/oligo-ovulatory patients after follicular stimulation with recombinant hFSH (rhFSH). DESIGN: Phase III, double-blind, double-dummy, randomized, parallel-group, multicenter study. SETTING: Nineteen academic and private tertiary care infertility centers in Europe, Israel, Canada, and Australia. PATIENT(S): One hundred ninety-eight WHO group II anovulatory women, aged 20 to 38 years. INTERVENTION(S): Women were randomized to receive rhCG or uhCG after follicular stimulation with rhFSH in a chronic low-dose protocol. Blood samples were collected and ultrasound examinations performed during stimulation and after hCG administration. MAIN OUTCOME MEASURE(S): Ovulation (midluteal serum progesterone > or =30 nmol/L), serum progesterone, hCG levels after hCG, pregnancy, adverse events, local tolerability, and ovarian hyperstimulation syndrome (OHSS) incidence. RESULT(S): Ovulation rates did not differ between groups: 95.3% for rhCG (n = 85) and 88.0% for uhCG (n = 92). The one-sided 95% confidence interval for the observed difference fell above the predefined limit of -20%, indicating equivalence. Treatment was well tolerated, but more uhCG patients reported local reactions (particularly inflammation and pain) (P=.0001; logistic regression). CONCLUSION(S): Subcutaneous rhCG and uhCG show equivalent efficacy in ovulation induction; however, rhCG is better tolerated.

Adult↗
PubMed2002

Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells.

Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.

Animals↗
PubMed2005

Both high mobility group (HMG)-boxes and the acidic tail of HMGB1 regulate recombination-activating gene (RAG)-mediated recombination signal synapsis and cleavage in vitro.

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

Animals↗
PubMed2003

High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method.

Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins. Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage. As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs. The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility. Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date.

Animals↗
PubMed2002

Context dependence of meiotic recombination hotspots in yeast: the relationship between recombination activity of a reporter construct and base composition.

Borde and colleagues reported that a reporter plasmid inserted at different genomic locations in Saccharomyces cerevisiae had different levels of meiotic recombination activity. We show that the level of recombination activity is very significantly correlated with the GC content of DNA sequences flanking the insertion.

Base Composition↗
PubMed2004

Recombinant human LH supplementation versus recombinant human FSH (rFSH) step-up protocol during controlled ovarian stimulation in normogonadotrophic women with initial inadequate ovarian response to rFSH. A multicentre, prospective, randomized controlled trial.

BACKGROUND: In approximately 12-14% of young normogonadotrophic women treated with a depot GnRH agonist long protocol, the initial ovarian response to recombinant human FSH (rFSH) can be suboptimal. We have tested the hypothesis that these women may benefit from recombinant human LH (rLH) supplementation in a multicentre, prospective, randomized trial compared with patients treated with an rFSH step-up protocol. METHODS: A total of 260 young normogonadotrophic women undergoing controlled ovarian stimulation with a GnRH agonist long protocol for IVF/ICSI were enrolled. The starting dose of rFSH was 225 IU. One hundred and thirty patients with serum estradiol levels <180 pg/ml and with at least six follicles with a mean diameter >5 mm but none >10 mm on both day 5 and day 8 of stimulation were randomly allocated to two groups. From the eighth day of stimulation, women in group A (n=65) received 150 IU of rLH in addition to rFSH, while those in group B (n=65) had an increase of 150 IU in the daily dose of rFSH (step-up protocol). One hundred and thirty normally responding women continued monotherapy with rFSH and served as a further control population (group C). RESULTS: The mean number of cumulus-oocyte complexes retrieved in group A (9.0+/-4.3) was significantly higher (P<0.01) compared with group B (rFSH 6.1+/-2.6) but significantly lower compared with group C (10.49+/-3.7, P<0.05). Implantation and pregnancy rates were significantly lower (P<0.05) in the rFSH step-up group (10.5 and 29.3% respectively) when compared with normal responders (18.1 and 47.3% respectively). CONCLUSIONS: rLH supplementation is more effective than increasing the dose of rFSH in terms of ovarian outcome in patients with an initial inadequate ovarian response to rFSH alone.

Adolescent↗
PubMed2004

A novel recombinant dual human SCF expressed in and purified from silkworm, Bombyx mori, possesses higher bioactivity than recombinant monomeric human SCF.

A novel recombinant dual human stem cell factor (rdhSCF) gene was constructed which consisted of a full-length hSCF cDNA plus a truncated hSCF cDNA (1-145 aa), linked by a peptide (GGGGSGGGGSGG) coding region. The rdhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the polyhedrin promoter control. Silkworm larvae infected with the recombinant virus expressed rdhSCF up to 15,800 units/mL in haemolymph. The specific activity of rdhSCF purified from the haemolymph was up to 3.0 x 10(6) units/mg, about 8.6 times as high as that of monomer rhSCF from Escherichia coli, and about 9.1 times as high as that of monomer rhSCF from insect cell. The binding affinity of rdhSCF to the cell surface receptor was higher than that of monomer rhSCF.

Animals↗
PubMed1993

Recombinant mouse cytochromes P1-450 and P3-450: enzymatic characterization of the hemoprotein expressed in human cells infected with recombinant vaccinia virus.

We expressed mouse cytochrome P1-450 and P3-450 using recombinant vaccinia virus gene expression system in HeLa cells that were devoid of significant basal levels of P-450. HeLa cells were infected with the recombinant vaccinia virus containing either mouse cytochrome P1-450 or P3-450 cDNA, and the cell lysates were analyzed for the kinetics of P-450 enzyme activity and protein expression at the same time. 7-Ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities were measured as an expression of the cytochrome P-450 enzyme activities. Both cell lines began to express these enzyme activities as early as 12h after infection. The activities increased linearly up to the 24 h time point, and were kept for 36 h. Western immunoblot analysis showed that these cytochrome P-450 proteins were detected at 16 h and reached maximum quantity at 24 h after infection. These data showed a good correlation between cytochrome P-450 enzyme activity and protein concentration throughout the process of P-450 gene expression by vaccinia virus vector, suggesting a complete formation of cytochrome P-450 holoenzyme from the early stage of the protein expression.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2002

Antibodies to heterologous proteins in hemophilia A patients receiving recombinant factor VIII (Recombinate).

As a consequence of the manufacturing process, trace quantities of Chinese hamster ovary cell protein, bovine serum albumin and murine immunoglobulin G are present in Recombinate recombinant human factor VIII (rhFVIII). The development of antibodies (Abs) to these heterologous proteins was evaluated during long-term rhFVIII therapy of hemophilia A in 68 previously treated and 73 previously untreated patients. Ab prevalence was also assessed in 157 non-hemophilic subjects. Abs against heterologous proteins could be detected in varying percentages of patients and non-hemophilic subjects. Abs arose in patients sporadically, and levels were typically low. There were no adverse events associated with development or presence of anti-heterologous protein Abs. These data indicate that sustained immune responses to trace levels of heterologous proteins are very infrequent during long-term rhFVIII therapy.

Adult↗
PubMed1987

Phase II study of recombinant human interferon gamma (S-6810) on renal cell carcinoma. Summary of two collaborative studies. Recombinant Human Interferon Gamma (S-6810) Research Group on Renal Cell Carcinoma.

Seventeen institutions in Japan evaluated the antitumor activity of recombinant human interferon gamma (S-6810) as a new modality for advanced renal cell carcinoma. The response rate for 32 evaluable patients who received continuous daily administration of 8 X 10(6) U/m2 to 12 X 10(6) U/m2 of interferon for 4 weeks was 9.4%. Six of 30 patients (20%) were demonstrated responders in the case of the intermittent administration of 40 X 10(6) U/m2 of interferon on each of days 1 to 5, 15 to 19, 29, 31, 33, 43, 45, and 47 over an 8-week period. One of the responders achieved a complete response (CR). The patients tolerated this dose of interferon gamma well. Sites that responded to treatment were the lungs, lymph nodes, and brain. Major adverse effects included fever (86.8% of patients), anorexia (67.1%), fatigue (53.9%), and leukopenia (42.1%). No life-threatening side effects appeared. High doses of recombinant human interferon gamma have an antitumor activity against renal cell carcinoma.

Adult↗
PubMed1988

A phase I-II trial of the combination of recombinant leukocyte A interferon and recombinant human interferon-gamma in patients with metastatic malignant melanoma.

Twenty patients with advanced malignant melanoma received daily intramuscular recombinant leukocyte A interferon (rIFN-alpha A, Roferon-A, Hoffmann-Laroche, Nutley, NJ) concomitant with recombinant human interferon-gamma (rIFN-gamma Genentech, South San Francisco, CA). During the first week alpha dose was 2 X 10(6) U/m2 and the gamma dose was 0.01 mg/m2 with escalations, if clinically tolerable, during the second week to 5 X 10(6) U/m2 and 0.025 mg/m2, respectively. Twelve patients received the escalated doses; subsequent granulocytopenia and a flu-type illness were severe in four of the 12. We observed one partial response of MRI-documented and biopsy-confirmed osseous metastases for 7+ months. For all study participants, the median time to progression was 1 month with a median survival of 6 months. From the dose and schedule which we utilized, concurrent rIFN-alpha A and rIFN-gamma provided little impact on advanced malignant melanoma.

Adult↗
PubMed1989

Recombinant beta-serine-interferon in hairy cell leukemia compared prospectively with results with recombinant alpha-interferon.

Ten patients with hairy cell leukemia (HCL) requiring therapy were treated with recombinant beta-serine-interferon (rIFN-beta ser) (90 x 10(6) units [U] subcutaneously three times a week). Eight patients were evaluable for response and nine for toxicity. Five patients (63%) showed normalization of peripheral blood counts, and an additional two patients (25%) showed improvement in at least one hematologic variable. Persistent hairy cells were detected in the bone marrow of all patients at the completion of therapy. All patients experienced influenza-like symptoms which were not dose limiting and which resolved with continued therapy. Erythema and induration at interferon injection sites developed in five patients (56%); one required dose reduction and another was removed from the study for this reason. Data from matched historical controls treated with recombinant alpha-interferon are presented for comparison. We conclude that rIFN-beta ser has activity in HCL.

Female↗
PubMed2002

Continued expression of recombination-activating genes and TCR gene recombination in human peripheral T cells.

It has been reported that a small population of peripheral T lymphocytes are capable of expressing V(D)J recombinase and initiating secondary V(D)J rearrangements. To determine whether RAG-expression and secondary TCR gene recombination in peripheral T cells are an antigen-driven process in secondary lymphoid tissues, we examined naive CD4(+) T cells, activated/memory CD4(+) T cells, and germinal center T cells from human tonsils. Our results showed that low levels of RAG-1 and RAG-2 mRNA were present in all T cell subpopulations except CD3(+)/CD4(-) T cells. LM-PCR analyses for double-strand DNA breaks showed that all the T cell subsets expressing RAG genes contain double-strand signal break ends, indicating ongoing TCR gene recombination. Continued RAG gene expression, introducing and repairing of double-strand DNA breaks at the TCR loci in the periphery may have significant implications in the development of some T cell neoplasms.

Cell Differentiation↗
PubMed1993

Recombinant human granulocyte-macrophage colony-stimulating factor plus recombinant human erythropoietin may improve anemia in selected patients with myelodysplastic syndromes.

The purpose of this study was to improve erythropoiesis in patients with anemia due to myelodysplastic syndromes (MDS). We treated 13 patients first with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 6 weeks, then with recombinant human erythropoietin (rhEpo) and rhGM-CSF for the next 12 weeks. Five patients had refractory anemia (RA), 3 refractory anemia with ringed sideroblasts (RAS), and 5 refractory anemia with excess of blasts (RAEB). Ten patients were transfusion-dependent at the time of inclusion. Eleven patients completed this phase II study. Five responded with an increase in hemoglobin level (3 patients) or a reduction in transfusion requirement (2 patients). We registered no response in the remaining 6 patients during treatment. Patients responding to combined treatment had relatively low concentrations of plasma Epo and plasma ferritin before treatment with rhEpo and a normal karyotype throughout the study. Long-term bone marrow cultures did not predict the response. Still, responders seemed to have a higher number of colony-forming progenitors than nonresponders. In conclusion, combined therapy with rhGM-CSF and rhEpo may stimulate hematopoiesis and correct or improve anemia in some patients with MDS.

Aged↗
PubMed2007

Enhanced intra-switch region recombination during immunoglobulin class switch recombination in 53BP1-/- B cells.

Immunoglobulin class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. U:G mismatches created by AID are processed to produce lesions that recruit and activate DNA damage response proteins including Ataxia-telangiectasia mutated (ATM), histone H2AX, Nijmegen breakage syndrome 1 (Nbs1), and p53 binding protein 1 (53BP1). Among these proteins, absence of 53BP1 produces the most severe impairment of class switching. Here, we demonstrate that AID is targeted normally to switch region DNA and that intra-switch region recombination is enhanced in 53BP1-/- B cells. In addition, Smicro-Sgamma1 switch region junctions cloned from 53BP1-/- B cells show unusual insertions suggestive of failed class switching. Our data are consistent with a role for 53BP1 in stabilizing the synapsis of switch regions during CSR.

Animals↗
PubMed1987

Combination tumor-immunotherapy with recombinant tumor necrosis factor and recombinant interleukin 2 in mice.

Recombinant human tumor necrosis factor (r-TNF) inhibits growth of various mouse tumor cell lines both in vitro and in vivo. Treatment of established tumor nodules with intratumoral (i.t.) injection of r-TNF caused hemorrhagic necrosis of tumor and temporary disappearance of tumor mass. However, a small number of tumor cells remained and later formed fresh nodules. In striking contrast, combination therapy with r-TNF and recombinant human interleukin-2 (r-IL-2) resulted in a marked inhibition of regrowth of tumor cells. More than 60% of MBL-2-bearing mice were completely cured of tumor by treatment with r-TNF and r-IL-2. Cured mice could also reject rechallenged MBL-2 lymphoma cells, indicating the generation of anti-tumor effector cells in vivo. However, lymphocytes obtained from mice cured of MBL-2 showed no significant in vitro cytotoxic activity against MBL-2 lymphoma cells. In contrast, in vitro sensitization of spleen cells from cured mice with mitomycin-C-treated MBL-2 lymphoma cells resulted in the generation of cytotoxic cells against MBL-2 lymphoma cells. Moreover, spleen cells from mice cured of MBL-2 by treatment with r-TNF and r-IL-2 revealed a strong anti-tumor activity upon in vivo neutralization tests. These results strongly suggest that tumor-bearing mice can acquire systemic immunological memory after combination therapy with r-TNF and r-IL-2.

Animals↗
PubMed1993

Recombinant soluble IgA Fc receptor: generation, biochemical characterization, and functional analysis of the recombinant protein.

We previously described the cloning of a human myeloid cell surface receptor for the Fc region of immunoglobulin A (Fc alpha R). In the present study, a soluble version of the Fc alpha R (solFc alpha R) was generated by removing the transmembrane and cytoplasmic coding regions from full-length Fc alpha R cDNA and ligating into a mammalian expression vector. COS-7 cells transfected with the solFc alpha R plasmid secreted a protein that inhibited both immunoglobulin A (IgA) and anti-Fc alpha R monoclonal antibody (mAb) binding to Fc alpha R+ U937 cells. Furthermore, the solFc alpha R bound specifically to and could be eluted from an anti-Fc alpha R mAb-immunoaffinity column, retaining biological activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the recombinant full-length Fc alpha R migrates over a molecular mass range of approximately 40-60 kd, consistent with the reported size and heterogeneity of the naturally occurring myeloid cell surface Fc alpha R. The solFc alpha R ran on SDS-PAGE as a smaller band (37-55 kd) that reduced to two bands of 23 and 25 kd following N-glycanase treatment, indicating that the Fc alpha R is a heavily glycosylated protein. The biochemical data, coupled with flow cytometry studies showing that the recombinant Fc alpha Rs bind to five different anti-Fc alpha R mAbs, clearly demonstrate that the cloned Fc alpha R corresponds directly to the major Fc alpha R species expressed on human monocytes, neutrophils, and myeloid cell lines. The generation of soluble receptor protein will permit investigations of the role of Fc alpha R in IgA-mediated immunoregulation, effector functions, and disease.

Antigens, CD↗
PubMed1993

Recombinant bovine GM-CSF primes superoxide production but not degranulation induced by recombinant bovine interleukin-1 beta in bovine neutrophils.

Bovine neutrophil activation, superoxide production, and beta-glucosaminadase release induced by various biological stimuli were examined. Platelet-activating factor (PAF) and recombinant bovine interleukin-1 beta (r-BoIL-1 beta) induced superoxide production and beta-glucosaminadase release in bovine neutrophils. When these two responses were compared, the dose requirement for maximum activation was similar for PAF (1 x 10(-6) M). However, the concentration of r-BoIL-1 beta required for the maximum degranulation (2.5 x 10(-7) M) was 100-fold higher than that for the maximum superoxide production (2.5 x 10(-9) M). Furthermore, pretreatment of cells with recombinant bovine granulocyte-macrophage colony-stimulating factor (r-BoGM-CSF) enhanced both superoxide production and beta-glucosaminidase release induced by PAF. In contrast, whereas superoxide production induced by r-BoIL-1 beta was enhanced by r-BoGM-CSF priming, beta-glucosaminidase release induced by r-BoIL-1 beta was significantly reduced by pretreatment with r-BoGM-CSF. CL 184,005, a PAF antagonist, inhibited PAF-induced glucosaminidase release and superoxide production but did not inhibit r-BoIL-1 beta-induced superoxide production and degranulation. In addition, it did not inhibit the priming effect of r-BoGM-CSF on r-BoIL-1 beta-induced superoxide production. These results suggest that (1) PAF and r-BoIL-1 beta activate bovine neutrophils by different mechanisms, (2) r-BoGM-CSF primes superoxide production and degranulation induced by PAF, (3) r-BoGM-CSF primes superoxide production but not degranulation induced by r-BoIL-1 beta, and (4) the priming effect of r-BoGM-CSF is not mediated by PAF.

Animals↗