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Biomedical subjects

T A Stewart

Publications and source records attributed to T A Stewart.

At least 55 records · Page 3Linked to original sources
PubMed1989

Transgenic mice as a model to test the immunogenicity of proteins altered by site-specific mutagenesis.

Second generation therapeutic proteins are now being produced by in vitro mutagenesis of the relevant genes. Of some concern, however, is the possibility that these altered proteins will be immunogenic and the antibodies raised will also recognize the endogenous protein with undesirable consequences. We have designed a biological system to test these possibilities. In this model, transgenic mice produce and secrete human tissue plasminogen activator (h.tPA) to which these mice are immunologically tolerant. However, when challenged with a form in which a single amino acid has been substituted these mice will produce antibodies capable of recognizing h.tPA. These results indicate that there are immunological consequences connected with the design and administration of second generation therapeutic proteins.

Animals↗
PubMed1989

Animal models of human disease. Pathology and molecular biology of spontaneous neoplasms occurring in transgenic mice carrying and expressing activated cellular oncogenes.

This present review focuses on spontaneous neoplasms occurring in transgenic mice carrying and expressing activated cellular oncogenes. The historical development of transgenic mice as in vivo disease models is briefly traced, followed by a brief description of the actual technology in such systems. Additional emphasis is placed on the concept of targeting activated cellular oncogenes to specific tissues in transgenic mice. Cumulative experience with activated (Vmyc, ras, and neu (erb-B2] oncogenes in transgenic mice is considered in detail, with particular attention paid to the observed pathology, as well as to the kinetics of disease occurrence. It is concluded that transgenic mice offer the interested investigator(s) an excellent prospective, in vivo model of oncogenesis.

Animals↗
PubMed1988

Insulin-dependent diabetes mellitus induced in transgenic mice by ectopic expression of class II MHC and interferon-gamma.

We have produced transgenic mouse strains harboring class II major histocompatibility complex or interferon-gamma genes linked to the human insulin promoter. These experiments were designed to investigate the consequences of the expression of immunological effector molecules by nonimmunological cells. In both of these studies we observed the disappearance from the pancreas of the insulin-producing beta cells coinciding with the development of insulin-dependent diabetes mellitus. Transgenic mice expressing both chains of the I-A gene showed progressive atrophy of the islets of Langerhans, whereas mice expressing interferon-gamma suffered an inflammatory destruction of the islets.

Animals↗
PubMed1988

Multiple regulatory domains in the mouse mammary tumor virus long terminal repeat revealed by analysis of fusion genes in transgenic mice.

Transcription initiated within the mouse mammary tumor virus (MTV) long terminal repeat (LTR) is regulated by glucocorticoids, androgens, and estrogen. However, expression of the virus in vivo and transcription of MTV LTR fusion genes in transgenic mice are not readily interpretable solely in terms of the influence of these hormones. To investigate whether there is a regulatory role for sequences within the LTR but outside the region known to be responsible for glucocorticoid induction, we have produced transgenic mice carrying genes in which various regions of the LTR have been linked to the human growth hormone gene. Analysis of expression of the fusion genes in these transgenic mice has demonstrated that the 5' end of the LTR can profoundly influence transcription initiated within the MTV LTR.

Animals↗
PubMed1988

Haploid-specific transcription of protamine-myc and protamine-T-antigen fusion genes in transgenic mice.

The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.

Animals↗
PubMed1987

Parental legacy determines methylation and expression of an autosomal transgene: a molecular mechanism for parental imprinting.

We have created a transgenic mouse strain in which an autosomal transgene bearing elements of the RSV LTR and a translocated c-myc gene obeys very unusual rules. If the transgene is inherited from the male parent, it is expressed in the heart and no other tissue. If it is inherited from the female parent, it is not expressed at all. This pattern of expression correlates precisely with a parentally imprinted methylation state evident in all tissues. Methylation of the transgene is acquired by its passage through the female parent and eliminated during gametogenesis in the male. These observations provide direct molecular evidence that autosomal gene expression can depend upon the sex of the parent from which the gene is inherited. They also provide a plausible mechanism for understanding parental imprinting that may be relevant to the failure of parthenogenesis in mammals, the apparent non-Mendelian behavior of some autosomal genes, and the role of methylation in gene regulation.

Animals↗
PubMed1987

A comparison of monoclonal antibodies against distinct colon tumor-associated antigens in immunohistochemistry and in tumor localization.

Monoclonal antibodies to colon/ovary tumor antigen (COTA), carcinoembryonic antigen (CEA), colon-specific antigen (CSA), and colon-specific antigen "protein" (CSAp) were evaluated for specificity, reactivity with normal tissues, and tumor localizations using athymic rats bearing xenografted human colon tumors. Radioiodine labeled anti-CSA and anti-COTA retained immunoreactivity and effectively localized the tumors; anti-CSAp retained immunoreactivity, but localized less effectively; and anti-CEA lost most of its immunoreactivity and localized poorly. Of the antibodies tested, anti-COTA showed potential for human colorectal tumor radiolocalization.

Animals↗
PubMed1987

Gonadal development and gametogenesis in the hypogonadal mouse are restored by gene transfer.

These results describe controlled regulation of a mammalian neural gene in transgenic mice. Analysis of truncated GnRH-GAP genes in transgenic mice will enable us to define the DNA sequences responsible for this control. Furthermore, by separate mutation of the GnRH and GAP coding sequences we will be able to determine the relative importance of these two peptides in the development and maintenance of reproductive function.

Animals↗
PubMed1986

The hypogonadal mouse: reproductive functions restored by gene therapy.

The hypogonadal (hpg) mouse lacks a complete gonadotropin-releasing hormone (GnRH) gene and consequently cannot reproduce. Introduction of an intact GnRH gene into the genome of these mutant mice resulted in complete reversal of the hypogonadal phenotype. Transgenic hpg/hpg homozygotes of both sexes were capable of mating and producing offspring. Pituitary and serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin were restored to those of normal animals. Immunocytochemistry and in situ hybridization showed that GnRH expression was restored in the appropriate hypothalamic neurons of the transgenic hpg animals, an indication of neural-specific expression of the introduced gene.

Animals↗
PubMed1986

Consequences of widespread deregulation of the c-myc gene in transgenic mice: multiple neoplasms and normal development.

We have constructed a transgenic mouse strain in which a mammary tumor virus LTR/c-myc fusion gene is anomalously expressed in a wide variety of tissues. The deregulated c-myc transgene, now glucocorticoid inducible, contributes to an increased incidence of a variety of tumors, including those of testicular, breast, lymphocytic (B cell and T cell), and mast cell origin. The deregulated gene does not, however, otherwise disturb cell proliferation, nor does it interfere with normal development in these animals. Moreover, since not all tissues that express the transgene develop neoplasms, these results begin to define the transforming spectrum of the c-myc oncogene. They also extend to several organ systems the notion that elements in addition to an activated c-myc gene are required to induce malignancy in the living organism.

Adenocarcinoma↗
PubMed1986

Production of monoclonal antibody SP-21 to colon-ovarian tumor antigen, COTA.

An IgG monoclonal antibody, SP-21, directed against colon-ovarian tumor antigen, COTA, is reported. The antibody had no reactivity with CEA, normal colonic mucin, CSAp, ABO blood group antigens, or with normal human lung, liver, spleen, kidney, plasma and saliva in studies using the enzyme-linked immunoassay method (ELISA). Immunoperoxidase staining of colon, lung, kidney, and prostate cancer tissues and benign and inflammatory colon disease tissues revealed a specificity identical to that of the polyclonal (goat) anti-COTA antibodies.

Animals↗
PubMed1984

Transcription and promoter usage of the myc gene in normal somatic and spermatogenic cells.

In somatic cells the level of myc transcription is not restricted to particular cell types but correlates closely with the rate of cell division. Such transcription involves the use of two active myc promoters and produces two messenger RNA species that are differentially represented among the transcripts of different tissues. In apparent contrast to somatic cells, mitotically and meiotically dividing germ cells have very few myc transcripts and appear to proliferate, at least for a few divisions, in the absence of myc transcription. These results raise interesting questions regarding the role of the myc gene product in terminally differentiating cells, particularly of the germ line series.

Animals↗
PubMed1984

Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTV/myc fusion genes.

We have produced 13 strains of transgenic mice that carry an otherwise normal mouse myc gene in which increasingly larger portions of the myc promoter region have been replaced by a hormonally inducible mouse mammary tumor virus promoter. Although expression of the fusion genes varies among these animals, the female founders of two of these transgenic strains spontaneously developed mammary adenocarcinomas during one of their early pregnancies. Both the tumors and the breast tissue of these founder animals expressed RNA transcripts corresponding to the fusion gene. Furthermore, in the best studied strain, all the available F1 female progeny that inherited the MTV/myc gene also developed mammary adenocarcinomas during their second or third pregnancies. Thus, although it has no obvious effect on the early development of these mice, the constitutionally deregulated myc gene appears to act as a heritable, predisposing factor favoring the accelerated development of a tissue-specific adenocarcinoma.

Adenocarcinoma↗
PubMed1983

Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line.

The mutagenic potential of recombinant DNA in the germ line was investigated in the descendants of mice obtained from eggs injected with the human growth-hormone gene in a pBR322 vector. Six positive animals (including one mosaic) were produced and had 1-20 copies of the donor sequences in unique and often complex integration patterns indicative of a single or an interrupted insertion. All were heterozygotes and transmitted the foreign insert to their progeny, forming six new HUGH strains. Matings between heterozygotes yielded viable healthy homozygotes in four of the strains. However, in the HUGH/3 and HUGH/4 strains, no postnatal homozygotes were found and litter sizes at birth were small. These two independent cases of mutation, both homozygous recessive prenatal lethals, are attributable to disruption of native sequences by alien ones. They constitute the first instances of insertional mutagenesis due to integration of recombinant DNA in the germ line of the mouse. The mutants provide new possibilities for molecular identification of gene functions necessary for normal development.

Animals↗
PubMed1981

Purification and characterization of human converting enzyme (kininase II).

We purified peptidyl-dipeptidase (converting enzyme, EC 3.4.15.1) to homogeneity from the membrane fraction of human lung and for comparison, from human and hog kidney. The membrane-bound lung enzyme was purified 1800-fold with 19% yield, and the kidney enzyme 640-fold with 10% yield. The specific activities with Bz-Gly-His-Leu were 81 mumol/min/mg for the lung and 65 for the kidney enzyme. The lung enzyme was homogeneous in gel electrophoresis with Mr = 155,000 and Sw,20 = 8.0 in ultracentrifugation. Antibodies elicited against lung or kidney enzyme cross-reacted with enzyme from other organ, but not with the hog enzyme. In isoelectric focusing both human enzymes had a major form with a pI of 5.2. The lung preparation also contained more acidic forms (pI = 4--5), which were eliminated by treatment with neuraminidase. Lung and kidney converting enzyme hydrolyzed bradykinin, angiotensin I, and enkephalins and had similar kinetic constants. Bradykinin was the best substrate, as indicated by its kcat/Km, but Met5-enkephalin had the highest turnover number. The hydrolysis of Bz-Gly-His-Leu was inhibited by captopril (SQ 14225) competitively, and by Keto-ACE, a non-peptide derivative of Bz-Phe-Gly-Pro, non-competitively.

Animals↗