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PubMed1995

Treatment of chronic hepatitis C with cirrhosis with recombinant human granulocyte colony-stimulating factor plus recombinant interferon-alpha.

Interferon therapy in cirrhotic patients with hepatitis C virus infection is not efficient. In an attempt to improve the response rate, a pilot study using recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone, and in combination with recombinant interferon-alpha (rIFN), was carried out. Fifteen cirrhotic patients with hepatitis C virus infection were randomly allocated into 3 groups to receive treatment: 0.5, 1, or 1.5 micrograms/kg of rhG-CSF daily for 4 weeks, followed by a 4 week resting period, and by the same dose of rhG-CSF daily, plus 6 MU of rIFN 3 times weekly for 4 weeks. They then continued to receive 6 MU of rIFN alone for 4 weeks, followed by 3 MU of rIFN for 16 weeks. After the 4 weeks of treatment with rhG-CSF alone, no changes in alanine aminotransferase (ALT) levels were observed. No changes occurred during the resting period. Three of 10 patients who ended the rhG-CSF plus rIFN treatment period had normal ALT values. During the treatment with rIFN alone, two responders suffered a relapse. The other responder had normal ALT until the first month of follow-up. In summary, the combination of rhG-CSF and rIFN seems promising for the treatment of patients with chronic hepatitis C and liver cirrhosis.

Adult↗

PubMed2001

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains.

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.

Amino Acid Sequence↗

PubMed1997

Comparison of ectopic osteoinduction in vivo by recombinant human BMP-2 and recombinant Xenopus BMP-4/7 heterodimer.

Two micrograms recombinant human bone morphogenetic protein-2 (rhBMP-2) and 2 microg recombinant Xenopus bone morphogenetic protein-4/7 heterodimer (rxBMP-4/7ht) were implanted into a calf muscle pouch in rats using atelopeptide type I collagen (CL) solution as the carrier. Three weeks later induced bone in the rhBMP-2 + CL group (group A; n=5) and the rxBMP-4/7ht + CL group (group B; n=5) was investigated radiologically and histologically. Bone trabeculum and marrow were induced in all cases of both groups. In group A the area of radioopaque oval shadows was 5.78 mm2, wider than 4.64 mm2 in group B. The radoopacity in group A was 74.6, higher than 57.6 in group B. Histometry of the microscopic views showed that the trabecular area was 0.92 mm2 in group A and 0.22 mm2 in group B, while the trabecular percentage occupied in the overall lump area was 18.07% in group A and 5.51% in group B. The trabecular form in group A was more massively coral than in group B. This study indicated that rhBMP-2 has a higher ectopic osteoinduction ability in vivo than rxBMP-4/7ht.

Animals↗

PubMed1998

Neuropeptide specificity and inhibition of recombinant isoforms of the endopeptidase 3.4.24.16 family: comparison with the related recombinant endopeptidase 3.4.24.15.

Endopeptidase EC 3.4.24.16 (EP24.16c, neurolysin) and thimet oligopeptidase EC 3.4.24.15 are close related members of a large family of metalloproteases. Besides their cytosolic and membrane bound form, endopeptidase EC 3.4.24.16 appears to be present in the inner membrane of the mitochondria (EP24.16m). We have overexpressed two porcine EP24.16 isoforms in E. coli and purified the recombinant proteins to homogeneity. We show here that these peptidases hydrolyse a series of neuropeptides with similar rates and at sites reminiscent of those elicited by classically purified human brain EP24.16c. All neuropeptides, except neurotensin, were similarly cleaved by recombinant endopeptidase 3.4.24.15 (EP24.15, thimet oligopeptidase), another zinc-containing metalloenzyme structurally related to EP24.16. These two EP24.16 isoforms were drastically inhibited by Pro-Ile and dithiothreitol and remained unaffected by a specific carboalkyl inhibitor (CFP-AAY-pAb) directed toward the related EP24.15. The present purification procedure of EP24.16 should allow to establish, by mutagenesis analysis, the mechanistic properties of the enzyme.

Amino Acid Sequence↗

PubMed1996

Specific in vitro interaction between recombinant Strongylocentrotus purpuratus bindin and a recombinant 45A fragment of the putative bindin receptor.

The interaction between recombinant Strongylocentrotus purpuratus sperm bindin and a recombinant fragment of the putative egg bindin receptor of the same species was measured in vitro. In solution these molecules interact with simple bimolecular kinetics, displaying an equilibrium dissociation constant of about 0.1 microM. Thus, as implied by many observations in vivo and in vitro, bindin and the putative egg receptor display a specific affinity for one another.

Animals↗

PubMed1988

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21.

A new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containing the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30,126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3' ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3' ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.

Amino Acid Sequence↗

PubMed1989

Recombinant human interleukin-1 alpha and recombinant human interleukin-1 beta stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis.

Recombinant human interleukin-1 alpha (rhIL-1 alpha) and recombinant human interleukin 1 beta (rhIL-1 beta) stimulated the time- and concentration-dependent release of glycosaminoglycan (GAG) from bovine nasal cartilage explants. Maximum GAG release occurred during six to eight days of cartilage exposure to either species of rhIL-1; and rhIL-1 alpha was consistently more potent than rhIL-1 beta. In addition to inducing cartilage matrix resorption, rhIL-1 alpha and rhIL-1 beta also inhibited the incorporation of [35SO4]sulfate into cartilage, which is a reflection of the suppression of GAG synthesis. IL-1 had no capacity to stimulate GAG relase from or inhibit GAG synthesis by dead cartilage. Cycloheximide, an inhibitor of protein synthesis, and 1, 10-phenanthroline, a metalloproteinase inhibitor, suppressed rhIL-1-stimulated cartilage matrix resorption. Polyclonal antisera to rhIL-1 alpha and rhIL-1 beta specifically neutralized the respective cytokines.

Animals↗

PubMed1992

Serum antibodies to HIV-1 in recombinant vaccinia virus recipients boosted with purified recombinant gp160. NIAID AIDS Vaccine Clinical Trials Network.

Serum antibody responses were studied in detail in four vaccinia-naive volunteers in a phase I trial evaluating primary vaccination with a recombinant vaccinia virus expressing the HIV-1 gp160 envelope glycoprotein (HIVAC-1e, Oncogen/Bristol-Myers Squibb), followed by booster immunization with baculovirus-derived rgp160 (VaxSyn, MicroGeneSys). Prior to boosting, low-titer Fc receptor (FcR)-mediated, antibody-dependent enhancing (ADE) activity was detected in two of four volunteers but no IgM, IgG, IgA, neutralizing activity, or complement-mediated ADE activity was detected. Two weeks after boosting, all four volunteers developed HIV-1-specific IgG with titers of 1:160 to 1:640 by immunofluorescence assay. IgG1 was present in sera from each individual, while IgG2 and IgG3 were present in sera from two individuals, and IgG4 was present in serum from one individual. IgM and IgA were undetectable in all sera. Only one volunteer had IgG to the heterologous HIV-1 isolates, RF, MN, and SF2, after boosting. Serum from this volunteer neutralized the vaccine strain, LAV/IIIB, but not the heterologous strains, RF, MN, and SF2. Antibodies from the remaining volunteers had no neutralizing activity. The neutralizing serum had a positive reaction in a peptide-based ELISA utilizing a peptide corresponding to the principal neutralizing domain of the third hypervariable region (i.e., V3 loop) of the envelope glycoprotein. Neutralizing activity was partially removed by adsorption to this peptide, suggesting that it contained a type-specific neutralizing vaccine epitope. A low titer (1:40 to 1:80) of complement-mediated ADE activity to HIV-1 IIIB was present in sera from three vaccinees after boosting. FcR-ADE activity for HIV-1 SF2 and SF-128A were present in sera from two of these three vaccinees. None of the volunteers developed antisyncytial antibodies. These results indicate that inoculation with recombinant vaccinia followed by rgp160 boosting is the most effective strategy to date for inducing serum antibodies to the envelope glycoproteins of HIV-1, but further study is needed to optimize the functionality and cross-reactivity of these responses.

AIDS Vaccines↗

PubMed1988

The effects of recombinant interleukin-1 and recombinant tumor necrosis factor on non-specific resistance to infection.

Natural and synthetic immunomodulators that increase non-specific resistance to infection induce the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Therefore, we investigated the effect of IL-1 and of TNF on the survival of lethally-infected mice. Mice were injected with 1 x 10(6) Klebsiella pneumoniae in the thigh muscle. When recombinant human IL-1 beta was given as a single i.p. injection 24 h before the infection, survival was increased. Using 80 ng IL-1 beta per mouse, survival compared to control animals was 80% versus 20% 48 h after the infection (p less than 0.001). No effect of IL-1 was observed when it was given 1/2 h before or 6 h after the infection. IL-1 alpha proved to be at least as potent as IL-1 beta. Numbers of bacteria cultured from the blood, thigh muscle, liver, spleen, and kidney were similar in IL-1-treated and control animals. Protection against death by IL-1 was also investigated in granulocytopenic mice with a Pseudomonas aeruginosa infection. Administration of the cyclooxygenase-inhibitor, ibuprofen, did not affect the beneficial effect of IL-1. In this model human recombinant TNF was at least tenfold less active than IL-1 beta. Pretreatment with IL-1 also had a significant effect on survival of mice that received a high dose of bacterial lipopolysaccharide.

Adjuvants, Immunologic↗

PubMed1996

Combination immunotherapy with OK-432, recombinant granulocyte-colony-stimulating factor and recombinant interleukin-2 for human hepatocellular carcinoma.

The antitumor effects of immunotherapy using streptococcal preparations (OK-432), recombinant granulocyte-colony-stimulating factor (rG-CSF) and recombinant interleukin-2 (rIL-2) were examined for human hepatocellular carcinoma (HCC). Following subcutaneous injection of OK-432 (2 KE) and rG-CSF (50-60 microg), low-dose intratumoral administration of OK-432 (3-12 KE) was performed. Thereafter, 2 x 10(5) JRU of rIL-2 was subcutaneously injected. This therapeutic regimen was repeated twice. Serum alpha-fetoprotein levels were markedly decreased in three of seven patients with HCC by this treatment. Post-therapeutic histological examination revealed that trabecular cords or pseudoglandular arrangements of tumor cells were completely disordered in all cases and that extensive infiltration of lymphocytes into the tumor stroma was present in five cases. The number of CD4- and CD57-positive cells among tumor-infiltrating lymphocytes after immunotherapy was significantly higher than that in patients without immunotherapy (P <0.01). These findings suggest that even a small intratumoral injection of OK-432 can induce extensive infiltration of helper/inducer and natural killer cells into the tumor stroma when combined with subcutaneous injection of OK-432, rG-CSF and rIL-2 and that these cells might play important roles in tumor cytotoxicity.

Aged↗

PubMed1998

Detection of anti-recombinant beta 2-glycoprotein 1 and anti-recombinant beta 2-glycoprotein 1 fifth-domain antibodies in sera from patients with systemic lupus erythematosus.

Two kinds of plasmid expression vectors which expressed beta 2-glycoprotein 1 (beta 2GP1) and the fifth domain of beta 2-glycoprotein 1 (beta 2GP1-D5) were constructed respectively in this study. The antigenicity of recombinant beta 2GP1 (r beta 2GP1) and beta 2GP1-D5 (r beta 2GP1-D5) was identified by immunoblots using rabbit anti-beta 2GP1 antibodies, and the recombinant proteins were purified. Both anti-r beta 2GP1 and anti-beta 2GP1-D5 antibodies in 112 patients were detected by ELISA using r beta 2GP1 and r beta 2GP1-D5 as coating antigens. A significant statistical correlation (r = 0.667, P < 0.01) between the levels of anti-beta 2GP1 and anticardiolipin (ACL) antibodies was found. The presence of anti-r beta 2GP1 antibodies was associated with an increased frequency of history of thrombosis and/or recurrent abortion; hence anti-r beta 2GP1 assay provided better specificity than conventional ACL assay. Detection of anti-r beta 2GP1 antibodies may be of potential value in evaluating the risk of thrombosis and/or symptoms associated with other antiphospholipid syndromes (APS). The binding of anti-r beta 2GP1 from the sera of patients with APS to r beta 2GP1 was inhibited by r beta 2GP1-D5. Meanwhile, of 28 patients who had positive anti-r beta 2GP1 antibodies in sera, 27 (96.4%) had positive anti-r beta 2GP1-D1 antibodies. This indicated that the antigenic epitope of beta 2GP1 may be located in its fifth domain.

Abortion, Habitual↗

PubMed1997

A de novo recombination in the ABO blood group gene and evidence for the occurence of recombination products.

We have encountered a paternity case where exclusion of the putative father was only observed in the ABO blood group (mother, B; child, A1; putative father, O), among the many polymorphic markers tested, including DNA fingerprints and microsatellite markers. Cloning a part of the ABO gene, PCR-amplified from the trio's genomes, followed by sequencing the cloned fragments, showed that one allele of the child had a hybrid nature, comprising exon 6 of the B allele and exon 7 of the O1 allele. Based on the evidence that exon 7 is crucial for the sugar-nucleotide specificity of A1 and B transferases and that the O1 allele is only specified by the 261G deletion in exon 6 of the consensus sequence of the A1 allele, we concluded that the hybrid allele encodes a transferase with A1 specificity, resulting, presumably, from de novo recombination between the B and O1 alleles of the mother during meiosis. Screening of random populations demonstrated the occurrence of four other hybrid alleles. Sequencing of intron VI from the five hybrid alleles showed that the junctions of the hybrid alleles were located within intron VI, the intron VI-exon 7 boundaries, or exon 7. Recombinational events seem to be partly involved in the genesis of sequence diversities of the ABO gene.

ABO Blood-Group System↗

PubMed2006

Recombinant luteinizing hormone supplementation to recombinant follicle-stimulation hormone during induced ovarian stimulation in the GnRH-agonist protocol: a meta-analysis.

PURPOSE: to compare the efficacy of recombinant LH supplementation for controlled ovarian stimulation in recombinant FSH and GnRH-agonist protocol. METHODS: Search strategies included on-line surveys of databases. The fixed effects model was used for odds ratio and effect size (weighted mean difference). Four trials fulfilled the inclusion criteria. RESULTS: a fewer days of stimulation (p<0.0001), a fewer total amount of r-FSH administered (p<0.0001) and a higher serum estradiol levels on the day of hCG administration (p<0.0001) were observed for the r-LH supplementation protocol. However, differences were not observed in number of oocyte retrieved, number of mature oocytes, clinical pregnancy per oocyte retrieval, implantation and miscarriage rates. CONCLUSIONS: more randomized controlled trials are necessary before evidence-based recommendations regarding exogenous LH supplementation in ovarian stimulation protocols with FSH and GnRH-agonist for assisted reproduction treatment can be provided.

Female↗

PubMed1987

Characterization of recombinant glycosylated human interleukin 2 produced by a recombinant plasmid transformed CHO cell line.

A recombinant plasmid containing expression units for human pre-interleukin 2 (pre-IL-2) and the selectable marker mouse DHFR, was constructed and used to transform DHFR- CHO cells to the DHFR+ phenotype. Selected colonies were isolated and tested for IL-2 production. Twelve highly IL-2-producing clones were amplified in stepwise increasing concentrations of methotrexate. The IL-2 secreted into the culture medium by one of these clones was purified to homogeneity and partially characterized. N-terminal sequence analysis showed that pre-IL-2 was correctly processed during secretion. SDS gel electrophoresis and chromatofocusing experiments in conjunction with neuraminidase treatment indicated a posttranslational glycosylation of the secreted mature protein similar to that described for the tetrasaccharide structure of the N2 form of natural IL-2. This recombinant IL-2 has a specific activity of 2.5 x 10(7) U/mg.

Amino Acid Sequence↗

PubMed1994

Recombinant human insulin. III. High-performance liquid chromatography and high-performance capillary electrophoresis control in the analysis of step-by-step production of recombinant human insulin.

The production of recombinant human insulin consists of five main stages, accompanied by considerable transformation of molecules, concerning size, secondary structure and the presence of charged groups. The application of different methods, i.e., size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) (capillary zone electrophoresis and micellar electrokinetic capillary chromatography), to the analysis of insulin, insulin-related and non-insulin-related substances was studied. A combined HPLC-HPCE system for the step-by-step control of recombinant human insulin production technology is suggested. The advantages and shortcomings of these methods are discussed.

Amino Acid Sequence↗

PubMed1989

Additive fibrinolysis by recombinant tissue-type plasminogen activator (r-t-PA) and recombinant single-chain urokinase type plasminogen activator (r-scu-PA) in rabbit pulmonary thrombosis.

The mode of interaction between recombinant tissue-type plasminogen activator (r-t-PA) and recombinant single-chain urokinase-type plasminogen activator (r-scu-PA) has been investigated in in vivo experiments. 125J-fibrin-labeled clots were embolized via a jugular vein into the lungs of anesthetized rabbits. In saline-treated control rabbits the spontaneous lysis, shown as decrease in radioactivity of the retrieved clots, amounted to 10.4 +/- 1.4% at 255 min after the pulmonary embolization. r-t-PA (0.464 - 4.64 micrograms/kg.min) and r-scu-PA (4.64 - 46.4 microns/kg.min), infused for 60 min, produced dose-dependent lytic effects to a similar extent (maximum lysis rate 53.9 +/- 5.8 and 55.4 +/- 7.2%, resp.). When various ratios of submaximal doses of r-t-PA and r-scu-PA were combined the lytic effects of these combinations were not higher than the calculated summation of the lysis rates by the single components. The fractional dose-response curves of r-t-PA and r-scu-PA and the combination of them, fitted by linear regression analysis, are overlaying each other. The results indicate that r-t-PA and r-scu-PA produce in vivo lysis in rabbits with pulmonary embolized clots in a purely additive manner.

Animals↗

PubMed1993

Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.

15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa 6.0-6.5. The fluorescein-conjugated aequorin exhibited fluorescence in addition to the Ca(2+)-triggered luminescence, thus it can be used to visualize the diffusion and distribution of aequorin in cells. The data obtained, particularly the Ca(2+)-sensitivity curves, are useful in selecting a suitable semi-synthetic aequorin for an experiment.

Aequorin↗

PubMed1989

Cooperative effects of human recombinant granulocyte-macrophage colony stimulating factor and human recombinant erythropoietin in inducing erythroid differentiation of the human erythroleukaemia cell line K 562 clonogenic cells.

Human acute erythroleukaemia arises from the inability of the haemopoietic stem cell to differentiate. K 562 cell line provides a homogeneous population of primitive erythroleukaemic cells that are at the same point of differentiation. The effect of human recombinant granulocyte-macrophage colony-stimulating factor and human recombinant erythropoietin on the differentiation of K 562 clonogenic cells was studied. Cells were cultured in methylcellulose culture for 5 days at 37 degrees C in humidified atmosphere containing 5% CO2 in air and scored for erythroid differentiation by benzidine staining. A combination of both growth factors induced erythroid differentiation in more than 80% of K 562 clonogenic cells. This combination may be useful in the treatment of patients with erythroleukaemia.

Cell Differentiation↗