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PubMed2001

Genetic study of patients with dexamethasone-suppressible aldosteronism without the chimeric CYP11B1/CYP11B2 gene.

Glucocorticoid-remediable aldosteronism is an inherited disorder caused by a chimeric gene duplication between the CYP11B1 (11beta-hydroxylase) and CYP11B2 (aldosterone synthase) genes. The disorder is characterized by hyperaldosteronism and high levels of 18-hydroxycortisol and 18-oxocortisol, which are under ACTH control. The diagnosis of glucocorticoid-remediable aldosteronism had been traditionally made using the dexamethasone suppression test; however, recent studies have shown that several patients with primary aldosteronism and a positive dexamethasone suppression test do not have the chimeric CYP11B1/CYP11B2 gene. The aim of this work was to evaluate whether other genetic alterations exist in CYP11B genes (gene conversion in the coding region of CYP11B1 or in the promoter of CYP11B2) that could explain a positive dexamethasone suppression test and to determine another genetic cause of glucocorticoid-remediable aldosteronism. We also evaluated the role of 18-hydroxycortisol as a specific biochemical marker of glucocorticoid-remediable aldosteronism. We studied eight patients with idiopathic hyperaldosteronism, a positive dexamethasone suppression test, and a negative genetic test for the chimeric gene. In all patients we amplified the CYP11B1 gene by PCR and sequenced exons 3-9 of CYP11B1 and a specific region (-138 to -284) of CYP11B2 promoter. We also measured the levels of 18-hydroxycortisol, and we compared the results with those found in four subjects with the chimeric gene. None of eight cases showed abnormalities in exons 3-9 of CYP11B1, disproving a gene conversion phenomenon. In all patients a fragment of 393 bp corresponding to a specific region of the promoter of CYP11B2 gene was amplified. The sequence of the fragment did not differ from that of the wild-type promoter of the CYP11B2 gene. The 18-hydroxycortisol levels in the eight idiopathic hyperaldosteronism patients and four controls with chimeric gene were 3.9 +/- 2.3 and 21.9 +/- 3.5 nmol/liter, respectively (P < 0.01). In summary, we did not find other genetic alterations or high levels of 18-hydroxycortisol that could explain a positive dexamethasone suppression test in idiopathic hyperaldosteronism. We suggest that the dexamethasone suppression test could lead to an incorrect diagnosis of glucocorticoid-remediable aldosteronism.

Adult↗
PubMed1989

Chimeric bovine-human growth hormone prepared by recombinant DNA technology: binding properties and biological activity.

A chimeric bovine GH (amino acids Met-Asp-Gln-greater than 1-23) and human GH (hGH) (amino acids 24-191) plasmid was constructed and expressed in Escherichia coli. The purified protein (chimeric GH) exhibited a 2-3 order of magnitude lower affinity toward lactogenic receptors in Nb2 lymphoma cells, microsomal fractions from bovine mammary gland and male rat liver. The affinity towards somatogenic receptors in IM-9 human lymphocytes and male rat liver was decreased to a much lesser degree. This diminished affinity towards lactogenic receptors was accompanied by a parallel decrease in the ability of the chimeric GH to stimulate proliferation of Nb2-11C lymphoma cells and the lipogenesis in bovine mammary gland. This implies that occupation of the respective receptors by either chimeric GH or hGH leads to identical postreceptoral effects. The chimeric GH was also capable of down-regulating the lactogenic receptors in Nb2 lymphoma cells and was recognized by three anti-hGH monoclonal antibodies. These and previously published results indicate that the N-terminal part of hGH is essential for the high affinity binding to lactogenic receptors and subsequent biological effect. Removal or replacement by a corresponding part of bovine GH converts the hormone, respectively to weak antagonist or agonists. Analysis of our data, based on hydropathy index leads us to suggest that the high affinity binding site of the hGH towards lactogenic receptors is mainly confined to amino acids nos. 8-18.

Animals↗
PubMed1999

Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules.

Genetic analysis through construction of chimeric genes and their transfection in mammalian cells could provide a better understanding of biological functions of native or modified proteins, and would allow the design of new gene constructs encoding peptides that mimic or block ligand interaction with target tissues. To identify the hGH domains responsible for induction of adipose differentiation we constructed hGH/hPL chimeric molecules using homologous DNA mutagenesis, since hGH, but not human placental lactogen (hPL), promotes adipose differentiation in mouse 3T3-F442A cells. We assayed their adipogenic activity in an autocrine/paracrine biological model consisting of transiently transfected 3T3-F442A cells with the chimeric constructs. Plasmid DNAs carrying these constructs were transfected into growing 3T3-F442A cells, and cultures were further maintained for 7 days to differentiate into adipocytes. Secretion of transfected hGH/hPL chimeric proteins into the medium was in the range of 5-25 ng/ml. Adipogenic activity was a property only of those chimeric proteins that contained hGH exon III together with either hGH exon II or hGH IV. Our results also suggest that hGH binding site-2 is composed of two structural subdomains: subsite 2A encoded by exon II of hGH and subsite-2B encoded by exon IV. We also suggest that full adipogenic activity requires the presence of binding site-1 and any of the subsites of binding site-2. This simple autocrine/paracrine biological model of gene transfection allows the analysis of specific biological activity of products encoded by modified genes.

3T3 Cells↗
PubMed2004

Construction of chimeric glucansucrases for analyzing substrate-binding regions that affect the structure of glucan products.

A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making alpha-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.

Bacterial Proteins↗
PubMed2003

Pre-exposure treatment of cats with anti-FHV-1 and anti-FCV mouse-cat chimeric antibodies.

Prior to pre-exposure treatment of cats with two mouse-cat chimeric antibodies, FJH2 and F1D7, having neutralizing activity to feline herpesvirus-1 (FHV-1) and cat calicivirus (FCV), respectively, these chimeric antibodies were labeled with (125)I and administered to cats to examine their blood kinetics. Concentrations of the both administered chimeric antibodies in the blood reached maximum at the 48th hour post-administration, and the level was 34% for FJH2 and 54% for F1D7. Then the concentration levels declined gently, and decreased afterwards to 8.2% for FJH2 and 25% for F1D7 on the 20th day post-administration. The blood half-lives of FJH2 and F1D7 were 8.3 days and 10.7 days, respectively. In order to examine effectiveness in pre-exposure treatment of cats with these chimeric antibodies, cats were administered on the 15th day prior to the challenge infections with FHV-1 and FCV by subcutaneous route with 0.5 ml/kg of an FJH-F1D7 mixture being adjusted to contain each chimeric antibody of 10 mg/ml. The cats that received the pre-exposure treatment with the cocktail, showed obvious reductions in manifestations of symptoms caused by those viral infections. The protective effectiveness of the pre-exposure treatment against these viral challenge infections was almost equal to that of the treatment given at right after these challenge infections.

Animals↗
PubMed2004

Targeted cancer therapy with gonadotropin-releasing hormone chimeric proteins.

Tumor-associated antigens (TAAs) have been identified mainly to determine cancer prognosis. In the past few years, TAAs have been used in the development of treatment modalities such as tumor vaccination. This review describes an additional application of TAAs: as a target for specific antitumor treatment. Since TAAs are overexpressed on the tumor cell surface, they can be targeted to deliver drugs directly to cancer cells. One such delivery system exploits chimeric proteins. Chimeric proteins are a class of targeted molecules designed to recognize and specifically destroy cells that overexpress specific receptors. These molecules, designed and constructed by gene fusion techniques, comprise both cell-targeting and cell-killing moieties. The authors' laboratory has developed a number of chimeric proteins using gonadotropin-releasing hormone (GnRH) as the targeting moiety. These chimeras recognize a GnRH binding site that is expressed on adenocarcinoma cells. GnRH was fused to a large number of killing moieties, including bacterial and human proapoptotic proteins. All GnRH-based chimeric proteins selectively killed adenocarcinoma cells both in vitro and in vivo. Utilizing chimeric proteins for targeted therapy represents a new and exciting therapeutic modality for the treatment of cancer in humans.

Adenocarcinoma↗
PubMed2005

Induction of human CYP1A2 and CYP3A4 in primary culture of hepatocytes from chimeric mice with humanized liver.

Chimeric mice with near-completely humanized liver were constructed by transplantating hepatocytes from a Japanese and Caucasian donor. In the present study, we investigated the induction of human CYP1A2 and CYP3A4 mRNA in a primary culture of the cryopreserved chimeric mouse hepatocytes. beta-naphthoflavone (beta-NF) and rifampicin (Rif) were used as typical cytochrome P450 (CYP) inducers for CYP1A2 and CYP3A4, respectively. Analysis was performed by the real-time reverse-transcription polymerase chain reaction method. CYP1A2 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 3.8-, 6.3-, and 3.3-fold by 5 microM beta-NF exposure, respectively, compared with the 0.1% DMSO treated control (p<0.01). CYP3A4 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 8.4-fold (p<0.001), 2.2-fold (p<0.01), and 2.3-fold (p<0.05) by 50 microM Rif exposure, respectively, compared with the 0.1% DMSO treated control. The present study demonstrated that a primary culture of cryopreserved hepatocytes from chimeric mice with humanized liver could be used for evaluating the induction of drug metabolizing enzymes in human. This in vitro method may be a useful method for screening the induction potency of new drug candidates on drug metabolizing enzymes.

Adolescent↗
PubMed1996

Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells.

We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.

Amino Acid Sequence↗
PubMed2004

Diagnostic chimerism analysis after allogeneic stem cell transplantation: new methods and markers.

Analysis of chimerism after allogeneic hematopoietic cell transplantation is important for assessing engraftment and the early detection of graft failure. In addition, the monitoring of minimal residual disease and early detection of imminent relapse has also become an important issue. Novel transplant procedures, for example dose-reduced conditioning protocols, rely on chimerism analysis to guide intervention, i.e. the reduction of immunosuppression or infusion of donor lymphocytes. During the last 30 years, several methods for the analysis of chimerism after hematopoietic cell transplantation have been published. Currently, fluorescent in situ hybridization (XY-FISH) analysis of sex chromosomes after transplantation from a sex-mismatched donor or analysis of polymorphic DNA sequences, i.e. short tandem repeats (STR) or variable number of tandem repeats (VNTR), are the most widely used procedures used in the assessment of chimerism. Two major diagnostic fields can be defined for chimerism analysis: the period of engraftment and the detection of minimal residual disease. Although STR-PCR and FISH analysis are very useful in the diagnosis of engraftment and graft failure, they are only of limited use in the monitoring of minimal residual disease, largely because of its limited level of sensitivity (1-5% for the minor population). Several novel procedures to improve this level of detection have been reported in recent years. One focus has been the use of real-time PCR techniques based on analysis of the Y-chromosome or, more recently, single nucleotide polymorphism (SNPs). These procedures combine quantitative analysis with high sensitivity (10(-4) to 10(-6)), and hold great potential for the future. In addition, the combination of cell sorting based on leukemia-specific immunophenotype and STR-PCR has been successfully used for minimal residual disease detection. First clinical data using these procedures indicate that intervention (e.g. the reduction of immunosuppression or donor lymphocyte infusion) may be effective in the minimal residual disease situation, even in high risk diseases like acute myeloid leukemia and acute lymphoblastic leukemia. The optimal timing of these diagnostic interventions is a critical issue and has to be further optimized. Whether this will ultimately improve the survival of patients with leukemia after transplantation has to be shown in prospective studies.

Animals↗
PubMed1998

Importance of mixed chimerism to predict relapse in persistently BCR/ABL positive long survivors after allogeneic bone marrow transplantation for chronic myeloid leukemia.

Determination of hematological chimerism could be helpful in understanding the biology of leukemic relapse after allogeneic bone marrow transplant (BMT) for chronic myeloid leukemia (CML), because the detection of malignant residual cells carrying the bcr/abl message by qualitative RT-PCR is of limited value in predicting disease progression for individual patients. We have studied the chimerism pattern and the bcr/abl status by Southern-blot in 15 CML patients (M/F:6/9) transplanted with unmanipulated BM from HLA identical sibling donors, persistently bcr/abl positive by RT-PCR. The median age of the series was 31 years (18-49) and disease status at BMT was: chronic phase: 11, accelerated phase: 3 and blast crisis: 1 patient. Of the 15 patients, 9 are alive and in complete remission (CR), 4 have died in CR and 2 are alive but suffered relapse at + 19 and +26 months post-BMT. The median follow-up is 81 months (13,7-168). Rearrangement of the BCR gene was performed by Southern-blot using P32-labeled transprobe-1. PCR analysis of chimerism was assessed using primers for the following VNTR loci: D1S80, D1S111, 33.1, APO-B, YNZ-22, lambdag3 and DXS52. Seventy-nine samples were analyzed (median per patient 5 (range 2-9)). Thirteen patients showed complete chimerism and lacked BCR rearrangement over time by Southern-blot. The 2 patients who relapsed showed mixed chimera status from +9 and +5 months respectively until the end of the study. Persistent BCR rearrangement was observed in these 2 patients from +12 and +11 months respectively. Our data suggest that mixed chimerism may predict hematologic or cytogenetic relapse by several months in those patients who are persistently PCR-positive post-BMT.

Adult↗
PubMed1996

Regulated expression of artificial chimeric genes contained in retroviral vectors: implications for virus-directed enzyme prodrug therapy (VDEPT) and other gene therapy applications.

Replication-defective retroviral vectors were created that contained chimeric genes composed of either the albumin (ALB) or the alpha-fetoprotein (AFP) transcriptional regulatory sequences linked to the coding domain of the thymidine kinase gene from Varicella zoster virus (VZV TK). These viruses were used to infect the human hepatoblastoma cell line, HepG2. Subsequent to infection, the infected cells were single-cell cloned. The level of expression of VZV TK from the chimeric genes correlated with the level of endogenous expression of ALB or AFP in most clones, indicating that the transcription of the chimeric VZV TK gene is controlled in a similar manner to the endogenous ALB or AFP genes, and that sites of viral integration are less important to overall gene expression. Most importantly, as the expression of the endogenous ALB gene was modified, so was expression of VZV TK from the ALB/VZV TK chimeric gene. This demonstrates that retroviruses can deliver a chimeric gene containing tissue-specific transcriptional regulatory sequences that can respond to endogenous cell regulatory signals resulting in regulated gene expression.

Albumins↗
PubMed2005

Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus.

AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier. METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR. RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preS1 (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro. CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

Animals↗
PubMed2001

Human dendritic cells are activated by chimeric human papillomavirus type-16 virus-like particles and induce epitope-specific human T cell responses in vitro.

Human papillomavirus (HPV)-derived chimeric virus-like particles (VLPs) are the leading candidate vaccine for the treatment or prevention of cervical cancer in humans. Dendritic cells (DCs) are the most potent inducers of immune responses and here we show for the first time evidence for binding of chimeric HPV-16 VLPs to human peripheral blood-derived DCS: Incubation of immature human DCs with VLPs for 48 h induced a significant up-regulation of the CD80 and CD83 molecules as well as secretion of IL-12. Confocal microscopy analysis revealed that cell surface-bound chimeric VLPs were taken up by DCS: Moreover, DCs loaded with chimeric HPV-16 L1L2-E7 VLPs induced an HLA-*0201-restricted human T cell response in vitro specific for E7-derived peptides. These results clearly demonstrate that immature human DCs are fully activated by chimeric HPV-16 VLPs and subsequently are capable of inducing endogenously processed epitope-specific human T cell responses in vitro. Overall, these findings could explain the high immunogenicity and efficiency of VLPs as vaccines.

Antigen Presentation↗
PubMed2004

Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain.

Chimeric receptors that include CD28 signaling in series with TCRzeta in the same receptor have been demonstrated to activate prestimulated human primary T cells more efficiently than a receptor providing TCRzeta signaling alone. We examined whether this type of receptor can also activate resting human primary T cells, and whether molecules other than CD28 could be included in a single chimeric receptor in series with TCRzeta to mediate the activation of resting human primary T cells. Human CD33-specific chimeric receptors were generated with CD28, inducible costimulator, CD134, or CD137 signaling regions in series with TCRzeta signaling region and transfected by electroporation into resting human primary T cells. Their ability to mediate Ag-specific activation was analyzed in comparison with a receptor providing TCRzeta signaling alone. Inclusion of any of the costimulatory signaling regions in series with TCRzeta enhanced the level of specific Ag-induced IL-2, IFN-gamma, TNF-alpha, and GM-CSF cytokine production and enabled resting primary T cells to survive and proliferate in response to Ag in the absence of any exogenous factors. Inclusion of CD28, inducible costimulator, or CD134 enhanced TCRzeta-mediated, Ag-specific target cell lysis. Chimeric receptors providing B7 and TNFR family costimulatory signals in series with TCRzeta in the same receptor can confer self-sufficient clonal expansion and enhanced effector function to resting human T cells. This type of chimeric receptor may now be used to discover the most potent combination of costimulatory signals that will improve current immunotherapeutic strategies.

Animals↗
PubMed2005

Expression vectors for human-mouse chimeric antibodies.

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded 30 pg cell(-1) day(-1) at 10(-6 )M MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

Animals↗
PubMed1975

Canine marrow transplantation: are serum blocking factors necessary to maintain the stable chimeric state?

Nine long-term canine radiation chimeras and their canine histocompatibility locus (DL-A) compatible, mixed leukocyte culture negative littermate marrow donors were studied between 545 and 1282 days after 1,200 R total body irradiation and marrow grafting. Before the time of testing, marrow donors were immunized against their chimeras by repeated skin grafts which they rejected. Skin fibroblasts from chimeras and their donors were tested for cell inhibition (CI) in the microcytotoxicity assay be exposure to lymphocytes from chimeras, donors and normal dogs and the effects of various sera on CI were evaluated. Lymphocytes from sensitized marrow donors consistently inhibited fibroblasts from the chimeras (eight of nine dogs); CI was abrogated by chimeric serum in only three of eight cases. Only two chimeras showed consistent CI of their "own" fibroblasts; CI was blocked by chimeric serum in one of the two. The remaining seven chimeras did not show consistent CI. Sequential studies in 16 additional recipients of DL-A compatible littermate marrow were carried out from 45 to 439 days after marrow grafting. Seven of the 16 did not show CI of chimeric fibroblasts by chimeric lymphocytes at any time. Nine showed CI on one or several occasions. Serum blocking factors were seen on one occasion in each of two chimeras. In conclusion, the CI assay is able to detect immunity against "minor" histocompatibility systems in dogs. Both long- and short-term chimeras occasionally demonstrated CI of chimeric fibroblasts but serum blocking factors did not appear to be necessary for maintaining stable graft-host "tolerance."

Animals↗
PubMed2001

Major differences in stability and dimerization properties of two chimeric mutants of human stefins.

Stefins A and B are cysteine proteinase inhibitors that have considerable sequence similarity but marked differences in their stability and folding properties. Two chimeric proteins were designed to shed light on these differences. The chimeric mutants have been expressed in Escherichia coli and have been isolated. The first, A37B, consists of 37 residues of stefin A, comprising the N-terminal and the alpha-helix, joined to 61 residues of stefin B; the second, A61B, consists of 61 N-terminal residues of stefin A, followed by 37 residues of stefin B. Spectroscopic properties of the chimeric proteins (absorption, CD, and NMR spectra), together with activity measurements, have confirmed that both have well-defined tertiary structure and are active as cysteine proteinase inhibitors. Characterization consisted of GuHCl denaturation, ANS binding as a function of pH, and monitoring of dimerization under partially denaturing conditions. The c(m) values are 1.3 M GuHCl for A61B as compared with 2.7 M GuHCl for stefin A, and 2.1 M GuHCl for A37B as compared with 1.4 M GuHCl for stefin B (all at pH 7.5, 25 degrees C). However (G degrees (N-U) is lower for both chimeric proteins (18 +/- 3 kJ/mol) than for the parent stefins (28 +/- 3 kJ/mol). In pH denaturation, unlike stefin B, neither chimeric mutant unfolds to I(N) below pH 5.4. At pH 3, where stefin B forms a molten globule and stefin A is native, both A37B and A61B show increased ANS fluorescence and aggregate visibly. Dimers at pre-denaturation conditions are observed in all the proteins under study, but they remain "trapped" only in stefin A.

Circular Dichroism↗
PubMedDate not supplied

In vivo events of retroviral long terminal repeat integration into Marek's disease virus in commercial poultry: detection of chimeric molecules as a marker.

The present study demonstrated, for the first time, that not only in vitro, but also in vivo, coinfections with Marek's disease virus (MDV) and each of the three avian retroviruses (reticuloendotheliosis virus [REV], avian lymphoid leukosis virus [ALV], and ALV-J) lead to retroviral long terminal repeat (LTR) integration into MDV. A total of 306 chicken and 59 turkey commercial flocks, submitted for differential avian oncogenic virus diagnosis, served to evaluate the flock mixed virus infection rate, the rate of birds with a multiple virus infection, and the issue of retroviral LTR integration into MDV in vivo. About a quarter of the tumor-bearing commercial flocks carried a mixed MDV and retrovirus infection. A total of 2926 DNA samples were analyzed, including 2428 chicken and 498 turkey DNA samples. Of these, 991 DNAs originated from flocks with a multiple virus infection. In 103 DNA preparations from that group (103/991, 10.4%), including 38 and 56 from chicken blood and tumor tissues, respectively, and nine samples from turkey blood, multiple virus sequences were detected by polymerase chain reaction (PCR). Fifty-six of the 103 samples were further analyzed by the previously developed hot spot-combined (HS-cPCR assay, of which 48% (27/56) contained chimeric MDV and retroviral LTR molecules. When extrapolated to the total samples derived from the flocks with multiple virus infection, that rate implies that about 5% of the DNA samples would carry MDV-retrovirus integration events. Several birds held a variety of chimeric molecules, indicating that several recombination events occurred simultaneously. The validation of the MDV and retroviral LTR chimeric constitution of these molecules was derived by the MDV and retroviral heterologous primers used for their creation by the HS-cPCR assay, Southern blotting and their detection by retroviral LTR probes, and LTR amplification from the gel-purified chimeric molecules. From several molecules, the LTR was sequenced, and a 161-bp retroviral LTR sequence was demonstrated. Our biochemical data imply that a recent integration occurred in the birds. The viability of recombinant viruses represented by the chimeric molecules will be further approached.

Animals↗