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PubMed1983

Effects of recombinant and hybrid recombinant human leukocyte interferons on cytotoxic activity of natural killer cells.

Two recombinant human leukocyte interferons (A and D), five hybrid interferons containing varying portions of A and D, and one fibroblast recombinant interferon were tested over a wide range of concentrations for their ability to modulate cytolytic activity of human natural killer (NK) cells. All of the interferons tested were purified to homogeneity. Although all the interferons were active, there were significant quantitative differences in their ability to augment cytolysis and the rank order of potency was reproducible among donors. The various recombinant interferons were also tested for their ability to augment mouse NK activity and the parental D and the A/D hybrids exhibited significant augmentation of cytolysis, which was consistent with their interspecies reactivity in viral neutralization assays. There generally was a direct correlation between antiviral activity and the ability of interferon to augment mouse NK activity; however, this correlation was not evident when tested on human cells. The study of these hybrids led to the identification of two molecules (A/D Bgl and A/D Pvu) which are very active in augmenting mouse NK activity. In addition, considerable insight has been obtained regarding the structure-function relationship of these leukocyte interferons and their ability to boost murine NK. This biological activity was associated with the COOH-terminal portion of the D interferon.

Animals↗
PubMed1980

[Formal genetic study of the enzymology of T4 phage recombination. I. Effect of the antimutational T4 DNA-polymerase on noncorrection-type recombination].

Effect of temperature sensitive antimutator L42 allele of gene 43 of T4 phage on the parameters of non-correction-type mechanism of genetic recombination have been studied under permissive conditions. By comparison of a point mutant with a deletion one, the critical frequency R (xi) corresponds to recombination between two markers separated by a distance equal to mean genetic length of the hybrid region was measured to be 4.15-10(-2). This value exceeds the frequency R (xi) determined with the parallel data in normal (43+) crosses by a factor of 1.6. The length of the hybrid region was shown not to change in the presence of L42 allele. The frequency of formation of the hybrid regions with recombinant flanks and of those with parental flanks increases in the presence of L42 by a factor of 1.3 and 1.8 respectively.

Alleles↗
PubMedDate not supplied

Identification of antioxidants for prevention of peroxide-mediated oxidation of recombinant human ciliary neurotrophic factor and recombinant human nerve growth factor.

Peroxides present in non-ionic surfactants used to stabilize certain recombinant protein formulations (e.g. polysorbate 80) can result in the oxidative degradation of proteins. In this study, the ability of various pharmaceutically acceptable antioxidants to prevent the oxidative degradation of two therapeutic proteins, recombinant human Ciliary Neurotrophic Factor (rhCNTF) and recombinant human Nerve Growth Factor (rhNGF), caused by alkyl hydroperoxides and hydrogen peroxide was studied. For rhCNTF, the rank order of effectiveness of the antioxidants tested was: thiols (cysteine, glutathione, thioglycerol) >> thioethers (methionine). Other parenterally acceptable antioxidants (ascorbic acid, propyl gallate and sodium bisulfite) destabilized the protein. The thiol antioxidants (cysteine and glutathione) were also the most effective antioxidants for rhNGF; however, in contrast to rhCNTF, ascorbic acid did not destabilize rhNGF. The rank order of effective antioxidants for rhNGF was: thiols (cysteine, glutathione) > > thioethers (methionine) > ascorbic acid.

Antioxidants↗
PubMed1996

[The characteristics of homologous DNA recombination in somatic cells. III. Intrachromosomal recombinations of the aminoglycoside phosphoribosyltransferase gene in transgenic mice].

Intrachromosomal homologous recombination has been revealed in the DNA from transgenic mice of three pedigrees. The recombination DNA of aminoglycosid phosphoribosiltransferase (neo) gene was observed in liver, kidney, spleen, heart, skeletal muscle, germinal glands and tail. It is concluded that the constructed model can be used for studying recombinations in cells of various organs and tissues both in the course of embryogenesis and during their malignant transformation and tumor progression.

Animals↗
PubMed1997

Effect of recombinant human IL-4 on tryptase, chymase, and Fc epsilon receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells.

The effect of recombinant human IL-4 (rhIL-4) on the development of recombinant human stem cell factor-dependent fetal liver-derived mast cells was examined. RhIL-4 attenuates the number of mast cells that develop, preferentially affecting the MC(T) type of mast cell. Cellular levels of tryptase and chymase mRNA normalized to that of glyceraldehyde-3-phosphate dehydrogenase were not appreciably affected. Tryptase mRNA levels peaked at least 2 wk before tryptase protein and before chymase mRNA and protein, indicating that tryptase mRNA expression is an early marker of commitment to a mast cell lineage. In contrast, alpha-tryptase and beta-tryptase mRNA levels increased and decreased in parallel. The most dramatic effect of rhIL-4 was to induce expression of functional surface Fc epsilonRI. Expression was maximal by 21 days with 20 ng/ml of rhIL-4 and reached a plateau by 2 ng/ml of rhIL-4 at 4 wk. Fc epsilonRI+ cells increased modestly when myeloma IgE was added to the developing mast cells, but increased synergistically when both myeloma IgE and rhIL-4 were present together. Delayed addition of rhIL-4 progressively diminished Fc epsilonRI expression, as did withdrawal of rhIL-4 during the first 2 wk of culture. RhIL-4 selectively increased Fc epsilonRI alpha mRNA levels at least 10-fold. Mast cells developed in the presence of rhIL-4 released tryptase when exposed to anti-Fc epsilonRI alpha. In conclusion, induction of functional Fc epislonRI on recombinant human stem cell factor-dependent human fetal liver-derived mast cells by rhIL-4 harmonizes with the well-accepted ability of this cytokine to enhance IgE production by B cells.

Cells, Cultured↗
PubMed1998

Recombinant His-tagged DNA polymerase. I. Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase.

The Tth DNA polymerase gene from the thermophilic Thermus thermophilus (strain HB8) was amplified, cloned and expressed in Escherichia coli. The recombinant DNA polymerase containing a polyhistidine tag at the N-terminus was isolated in a single step by Ni2+ affinity chromatography. The purified recombinant enzyme, showing high polymerase activity contained 43 additional amino-acid residues (including a cluster of six histidine residues inserted for purification of the recombinant protein by metal-affinity chromatography) at N-terminus. The applied overexpression system was very efficient giving 700,000 u of DNA polymerase activity from 1 liter of induced culture. The enzyme was characterized and displayed high DNA polymerase and reverse transcriptase activities and high thermostability as compared to the native Tth DNA polymerase.

Amino Acid Sequence↗
PubMed2001

Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (ALVAC) cytokine gene delivery induces destruction of established prostate tumors.

Prostate-specific antigen (PSA) is expressed by prostate epithelial cells and has a highly restricted tissue distribution. Prostatic malignancies in 95% of patients continue to express PSA, making this antigen a good candidate for targeted immunotherapy. The goals of our studies are to generate a recombinant PSA adenovirus type 5 (Ad5-PSA) that is safe and effectively activates a PSA-specific T-cell response capable of eliminating prostate cancer cells, and to characterize the immunologic basis for this rejection. Here we show that immunization of mice with Ad5-PSA induced PSA-specific cellular and humoral immunity that was protective against a subcutaneous challenge with RM11 prostate cancer cells expressing PSA (RM11psa), but not mock-transfected RM11 tumor cells (RM11neo). Mice immunized with recombinant adenovirus type 5 encoding beta-galactosidase (Ad5-lacZ) did not generate protective immunity. Antitumor activity was predominantly mediated by CD8(+) T lymphocytes. Although Ad5-PSA immunization prior to RM11psa challenge was protective, Ad5-PSA immunization alone was not able to control the growth of existing RM11psa tumors. In contrast, established RM11psa tumors ranging in size from 500 to 1,000 mm(3) were efficiently eliminated if Ad5-PSA priming was followed 7 days later by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin-12 (IL-12), IL-2, and tumor necrosis factor-alpha. In this case, antitumor immunity was still dominated by CD8(+) T lymphocytes, but natural killer cells became necessary for a maximal response. These data provide information on the effector cell populations in a protective immune response to prostate cancer and demonstrate the utility of an Ad5-PSA vaccine combined with cytokine gene delivery to eliminate large established tumors that are refractory to other interventional methods.

Adenoviridae↗
PubMed1994

Analysis of meiotic recombination events near a recombination hotspot in the yeast Saccharomyces cerevisiae.

The region of yeast chromosome III between the HIS4 and LEU2 genes has an unusually high frequency of meiotic recombination. In order to determine the pattern of cross-over and gene conversion events, we constructed a strain with a number of heterozygous markers in this 25-kb interval. We found that very high levels of recombination are localized to regions of DNA near HIS4. In addition, analysis of the patterns of co-conversion of adjacent markers suggests that there is more than one initiation site contributing to recombination of HIS4.

Chromosomes, Fungal↗
PubMed2004

Live vaccinia-rabies virus recombinants, but not an inactivated rabies virus cell culture vaccine, protect B-lymphocyte-deficient A/WySnJ mice against rabies: considerations of recombinant defective poxviruses for rabies immunization of immunocompromised individuals.

Presently, commercially available cell culture rabies vaccines for humans and animals consist of the five inactivated rabies virus proteins. The vaccines elicit a CD4+ helper T-cell response and a humoral B-cell response against the viral glycoprotein (G) resulting in the production of virus neutralizing antibody. Antibody against the viral nucleoprotein (N) is also present, but the mechanism(s) of its protection is unclear. HIV-infected individuals with low CD4+ T-lymphocyte counts and individuals undergoing treatment with immunosuppressive drugs have an impaired neutralizing antibody response after pre- and post-exposure immunization with rabies cell culture vaccines. Here we show the efficacy of live vaccinia-rabies virus recombinants, but not a cell culture vaccine consisting of inactivated rabies virus, to elicit elevated levels of neutralizing antibody in B-lymphocyte deficient A/WySnJ mice. The cell culture vaccine also failed to protect the mice, whereas a single immunization of a vaccinia recombinant expressing the rabies virus G or co-expressing G and N equally protected the mice up to 18 months after vaccination. The data suggest that recombinant poxviruses expressing the rabies virus G, in particular replication defective poxviruses such as canarypox or MVA vaccinia virus that undergo abortive replication in non-avian cells, or the attenuated vaccinia virus NYVAC, should be evaluated as rabies vaccines in immunocompromised individuals.

Animals↗
PubMed2000

The use of oligonucleotide directed cleavage of DNA and homologous recombination in the production of large recombinant adenoviral vectors.

The genetic manipulation of large plasmid DNA often requires the fortuitous presence of convenient restriction enzyme sites. For large plasmids, such as those containing full length recombinant adenovirus, it is desirable to direct the cloning or sequence alterations without having to depend on such convenient restriction sites. We report a general and efficient method to modify or clone large covalently closed circular DNA molecules at any predetermined sequence. This procedure involves two main steps. First, supercoiled DNA is hybridized to a short pre-selected synthetic oligonucleotide to form a D-loop. This hybrid is then linearized in vitro at that target site by digestion with S1 nuclease. Second, D-loop/S1 linearized DNA is transformed into E. coli with a second linear DNA fragment carrying a foreign gene flanked by sequences homologous to the target site. In vivo recombination results in the desired recombinant construct. We demonstrate the use of this method by cloning the SV40 origin of replication into the E3 transcription unit of human adenovirus type 5.

Adenoviruses, Human↗
PubMed2000

Delivery of recombinant product from subcutaneous implants of encapsulated recombinant cells in canines.

Delivering recombinant therapeutic proteins from a universal microencapsulated cell line is an alternate method for gene therapy. It has proved effective in the treatment of several murine models of human genetic diseases. However, in scaling up to large animal models, intraperitoneal Implantations of these microcapsules in canines were associated with excessive Inflammatory response and rapid degradation. We now show that subcutaneous implantation of microencapsulated cells in canines is effective in delivering recombinant product systemically for extended periods, provides a surgically benign site, leads to less inflammatory response, and permits longer-term survival of microcapsules. Allogeneic MDCK cells engineered to secrete human growth hormone (hGH) were microencapsulated in alginate-poly-L-lysine-alginate and implanted subcutaneously. Systemic delivery of hGH was evident within 4 hours and peaked by day 1 after implantation in all dogs. The gradual decline of hGH in the circulation in the first 2 weeks coincided with the development of anti-hGH antibodies by day 11. The high titer persisted for more than 1 month, demonstrating indirectly the persistent delivery of hGH. Microcapsules retrieved from the subcutaneous implant maintained their structure throughout the experiment and were free of host cellular adhesions. The mechanical integrity of the subcutaneously implanted microcapsules also appeared superior to that of the intraperitoneal implant. Hence the subcutaneously implanted microcapsules required minimal surgical intervention and led to a low level of inflammatory response, and the implant survived for at least 1 month, thus demonstrating the feasibility of systemic delivery of recombinant products via subcutaneous implantation in large animals.

Animals↗
PubMed1999

HIV-1MN recombinant glycoprotein 160 vaccine-induced cellular and humoral immunity boosted by HIV-1MN recombinant glycoprotein 120 vaccine. National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

We evaluated prime-boost immunization with two recombinant envelope glycoprotein subunit vaccines (HIV-1MN recombinant gp160 vaccine in alum adjuvant [MN rgp160] and HIV-1MN recombinant gp120 vaccine in alum adjuvant [MN rgp120]) for safety and immunogenicity in healthy, HIV-1-uninfected adults. The rationale was to combine the helper T cell memory and binding antibody responses typically induced by rgp160 vaccines with the superior neutralizing antibody responses induced by rgp120 vaccines. In a double-blinded, controlled trial, volunteers were randomly assigned to receive MN rgp160 or adjuvant placebo, and a subset later received MN rgp120. The two vaccines were safe, but reactions to MN rgp160 and its adjuvant placebo exceeded those to MN rgp120. MN rgp160 induced IgG binding antibodies, including all IgG subclasses, to MN rgp160 in all vaccine recipients. HIV-1MN-neutralizing and anti-V3 MN peptide-binding antibodies were observed in a majority of volunteers after the fourth MN rgp160 immunization, but at lower levels compared with immunization with MN rgp120 in historical controls. HIV-1-binding, neutralizing, and fusion inhibition antibodies were boosted to the highest levels among MN rgp160 recipients after MN rgp120 booster injections. MN rgp120 boosting appeared to alter the distribution of MN rgp160 vaccine-induced, anti-MN rgp160 IgG subclass antibodies. MN rgp160 induced helper T cell memory, measured by lymphocyte proliferation, Thl and Th2 cytokine production, and skin testing. Strategies including both subunit vaccines may help maximize antibody and helper T cell memory responses to HIV-1 envelope glycoprotein.

AIDS Vaccines↗
PubMed1989

Mechanism of immunity to influenza: maternal and passive neonatal protection following immunization of adult ferrets with a live vaccinia-influenza virus haemagglutinin recombinant but not with recombinants containing other influenza virus proteins.

Neonatal ferrets are protected against infection with influenza virus by milk-derived anti-influenza virus IgG after suckling on an immune mother. Live vaccines protect better than killed vaccines despite their stimulation of lower maternal haemagglutination-inhibiting antibody levels. This suggests that antibody to virus proteins other than the haemagglutinin may also be involved. To investigate this, adult ferrets were immunized intradermally with live vaccinia-influenza virus recombinants each expressing one of the 10 influenza virus polypeptides. Adult ferrets immunized with a recombinant expressing the H3 haemagglutinin were completely protected, and also passively protected their offspring, against a live challenge with clone 7a of the reassortant influenza virus A/Puerto Rico/8/34-A/England/939/69 (H3N2), immunity being mediated by IgG antibody. However, ferrets immunized similarly with recombinants expressing the H1 haemagglutinin, neuraminidase (N1 or N2), polymerases (PB1, PB2 or PAC), matrix protein (M1 or M2), nucleoprotein (NP) or non-structural proteins (NS1 or NS2) were completely susceptible to the influenza virus.

Animals↗
PubMed1989

Expression of glycoprotein D of herpes simplex virus type 1 in a recombinant baculovirus: protective responses and T cell recognition of the recombinant-infected cell extracts.

Recombinant baculoviruses expressing glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) have been generated. The proteins expressed from the recombinants have been characterized using monoclonal antibodies on Western blots and by immunoprecipitation. Partially glycosylated 48K polypeptides have been identified as products of the gD gene. Polyclonal sera from H-2k mice infected with HSV-1 recognized the same polypeptides. Furthermore, draining lymph node cells from H-2k mice infected with HSV-1 proliferated in vitro in response to recombinant-infected cell extracts. Immunization with such extracts generated high titre complement-dependent and -independent neutralizing antibody and the mice were protected against a challenge with HSV-1.

Animals↗
PubMed2003

DNA immunization with hepatitis C virus (HCV) polycistronic genes or immunization by HCV DNA priming-recombinant canarypox virus boosting induces immune responses and protection from recombinant HCV-vaccinia virus infection in HLA-A2.1-transgenic mice.

We studied immune responses to hepatitis C virus (HCV) genes delivered as DNA encoding the entire HCV protein coding genome in two polycistronic plasmids encoding HCV capsid-E1-E2-NS2-NS3 and HCV NS3-NS4-NS5 in HLA-A2.1-transgenic mice. Immune responses to HCV DNA prime and recombinant canarypox virus boost were also studied with the above constructs. At 8 weeks after a canarypox virus boost, the DNA prime/canarypox virus boosting regimen induced potent cellular immune responses to HCV structural and nonstructural proteins on target cells expressing the HLA-A2.1 allele. High frequencies of gamma interferon-secreting cells, as detected by enzyme-linked immunospot assay, were obtained in response to several endogenously expressed HCV proteins. We also observed cytotoxic-T-lymphocyte reactivity in response to endogenously expressed HCV proteins in fresh spleen cells without in vitro expansion. Upon challenge with a recombinant vaccinia virus expressing HCV proteins at 2 months postimmunization, the HCV DNA prime/canarypox virus-immunized mice showed a complete reduction in vaccinia virus titers compared to HCV DNA prime/boost- and mock-immunized controls. Immune responses were still detectable 4 months after canarypox virus boost in immunized mice. Interestingly, at 10 months postimmunization (8 months after canarypox virus boost), the protection in HCV DNA prime/boost-immunized mice against recombinant HCV-vaccinia virus challenge was higher than that observed in HCV DNA prime/canarypox virus boost-immunized mice.

Animals↗
PubMed1992

Recombinant circle PCR and recombination PCR for site-specific mutagenesis without PCR product purification.

Two simple methods for site-specific mutagenesis are described and compared. In each method, the PCR is used in two separate amplifications to mutate the site of interest and to add ends to one PCR product that are homologous to the ends of the other PCR product. In the first method, the two products are combined, denatured and reannealed prior to transformation of E. coli in order to form recombinant circles in vitro, while in the second method, the two linear products are co-transfected directly into E. coli without prior manipulation, resulting in transformation of E. coli with the recombinant of interest by recombination in vivo. Each PCR amplification uses a plasmid template that has been linearized by restriction enzyme digestion outside the region to be amplified. This permits use of unpurified PCR products in these two protocols and generation of the mutant of interest with no other enzymatic manipulation in vitro apart from PCR amplification. In each protocol greater than or equal to 50% of the resulting clones contained the mutation of interest without detected errors.

Base Sequence↗
PubMed1995

Mouse VH7183 recombination signal sequences mediate recombination more frequently than those of VHJ558.

Mouse VH gene segments are conventionally classified into 13 families on the basis of sequence similarity. The 7183 family lies close to the 3' end of the locus and is preferentially used in BALB/c mice; J558, the largest family, lies close to the 5' end of the VH stretch and is preferentially used in C57BL/6 mice. To investigate whether differential effectiveness of the RSSs in the two families might contribute to the overusage of 7183 in the primary repertoire of BALB/c, we constructed recombination substrates in which the recombination signal sequences (RSSs) of VH segments 7183 and J558 compete with each other for a single RSS, DFL16.1, after transfection into two transformed cell lines derived from C57BL/6 and two cell lines from BALB/c mice. In both strains, the 7183 RSS was found to be preferentially used (83%). Thus, the 7183 RSS mediates recombination more frequently than does that of J558, and this preference must thereby influence the primary repertoire, but the strain difference cannot be accounted for by a difference in the RSSs.

Animals↗
PubMed1993

The effector roles of kringle 1 and kringle 2 in the enzymatic properties of recombinant tissue-type plasminogen activator as revealed by generation of recombinant molecules containing each kringle linked to the protease domain.

Recombinant DNA technology has been employed to construct and express in human kidney 293 cells cDNAs encoding deletion-mutant recombinant (r) tissue-type plasminogen activators (tPA) retaining only the kringle 1 ([K1tPA]) and serine protease (P) functional domains (r-[K1tPA]P), and the kringle 2 ([K2tPA]) and P domains (r-[K2tPA]P), along with a variant of r-tPA containing a W253S mutation (r-tPA/W253S). Of these mutants, only r-[K2tPA]P retained its ability to interact with omega-amino acid effector molecules. The Km for single-chain wild-type (wt) r-tPA toward the synthetic substrate H-D-Ile-L-Pro-L-Arg-p-nitroanilide (S2288) was decreased by approximately 3-fold in the presence of a saturating concentration of human fibrinogen (Fg), along with a small (1.14-fold) increase in the kcat for this same reaction. The kinetic activation (dissociation) constant (KA) for Fg was 2.4 microM. Fg did not influence the steady-state amidolytic properties of two-chain wtr-tPA. Similar effects of Fg on the Km for hydrolysis of S2288 were displayed for single-chain forms of r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S, with additional small effects of Fg on the kcat of this reaction. The KA for Fg toward these proteins ranged from 2.4 microM for wtr-tPA to 5.2 microM for r-[K1tPA]P. The amidolytic properties of the two-chain forms of these variants were also unaffected by Fg. The activation rate of [Glu1]-plasminogen ([Glu1]Pg) by wtr-tPA was stimulated approximately 7-fold by Fg and approximately 139-fold by the same concentration (in Fg equivalents) of human fibrin (Fn) (Fn/Fg stimulatory ratio = 19.9). The Fn/Fg ratio was 10.6, 20.9, and 18.0 for r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S, respectively. Quantitative [Glu1]Pg-enriched clot lysis assays revealed that r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S were approximately 18, 72, and 54%, respectively, as effective as wtr-tPA in catalyzing the plasminogen activation event leading to lysis. The antifibrinolytic agent epsilon-aminocaproic acid, inhibited clot lysis with approximately equal effectiveness in [Glu1]Pg-enriched clots when wtr-tPA, r-[K1tPA]P, r-[K2tPA]P, or r-tPA/W253S were employed as the activators. These studies demonstrated that Fg- and Fn-based stimulatory effects on the enzymatic properties of r-tPA and its variants were generally not proportional to the macroscopic binding abilities of these proteins with omega-amino acids or with Fg and Fn.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗