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Biomedical subjects

T A Stewart

Publications and source records attributed to T A Stewart.

68 records · Page 4Linked to original sources
PubMed1981

Successive generations of mice produced from an established culture line of euploid teratocarcinoma cells.

The possibility of utilizing mouse teratocarcinoma stem cells as intermediaries for production of new strains of mice with preselected mutant or foreign genes requires that, after propagation in culture (to allow for genetic manipulation and selection), the cells be capable of normalization and orderly development in carrier embryos and, ultimately, of germ-cell formation. Heretofore, no in vitro cell line has fulfilled all these requirements. A karyotypically normal teratocarcinoma culture line was recently established in this laboratory and now has been investigated as a candidate. The line, designated METT-1, is chromosomally female (X/X) and was obtained from the 129 (agouti-colored) inbred strain [Mintz, B. & Cronmiller, C. (1981) Somat Cell Genet 7, 489-505]. The developmental potential of these cells was tested, after prolonged culture and freezing and thawing, by microinjecting them into early (blastocyst stage) embryos of the C57BL/6 (black) strain. Among 312 experimental animals examined at 1 week of age, there were 41 mice (21 females and 20 males) that displayed the coat colors of both strains. This frequency (13%), as well as the extent of the coat areas derived from the cell line, greatly surpasses the contributions observed in all previous experiments, whether with other in vitro teratocarcinoma cell lines or with in vivo transplant lines. The developmental totipotency of METT-1 cells became evident from the presence of substantial amounts of 129-strain cells (bearing an isozyme marker) in all internal tissues of an individual whose coat was largely agouti. The culture-cell lineage also proved to be capable of giving rise to reproductively functional oocytes. Of nine mosaic-coat females testmated to C57BL/6 males, one produced progeny of the diagnostic agouti color in two litters; these heterozygous F(1) offspring in turn transmitted their marker genes to F(2) homozygous segregants. Thus, the METT-1 teratocarcinoma line bridges the gap between in vitro cell propagation and in vivo development and between the soma and the germ line. This creates the option of producing new mouse strains with predetermined genetic changes designed as probes of developmental regulation or as models of human genetic diseases.

Alleles↗
PubMed1981

The human beta-globin gene and a functional viral thymidine kinase gene in developing mice.

Two foreign cloned genes--one encoding a tissue-specific protein and one encoding a constitutive enzyme--were introduced into mouse eggs by microinjection into a pronucleus shortly after fertilization. They were the adult human genomic beta-globin gene and the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of herpes simplex virus (HSV), ligated in the pBR322 plasmid. Thirty-three developing mice were autopsied in late fetal life; all appeared normal. Blot hybridization tests revealed that the DNA of as many as five (15%) of the fetuses (from separate litters), and of their corresponding placentas, contained copies of the human beta-globin gene and of the HSV tk gene that had been retained and replicated without significant loss or rearrangement. The estimated total numbers of copies per cell were 3-50 for the donor globin gene and 3-20 for the donor tk. In some of the fetuses, these totals included some copies of molecular weight higher than that of the intact sequence; the additional segments may have arisen through changes such as deletions or duplications. The foreign genes in the five positive fetuses appear to be present in high molecular weight DNA. Assays capable of distinguishing between foreign and native TK indicated that at least one of the fetuses with the HSV tk gene had some TK enzyme of the HSV type and, therefore, at least one gene copy that was being accurately transcribed and translated to produce a functional protein, despite the absence of selective pressure. Thus, pure recombinant genes introduced into mice at the onset of their development can remain intact and be stably incorporated and even expressed. These experiments provide a practical basis for novel investigations of the developmental control of normal gene expression in vivo of the causes and possible cures of genetic diseases.

Animals↗
PubMed1981

Prolylcarboxypeptidase (angiotensinase C) in human lung and cultured cells.

The activity of prolylcarboxypeptidase (PCP), or angiotensinase C, was measured in lung tissues, leukocytes, and cultured human cells using Cbz-Pro-[14C]Ala as a substrate. A lysosomal fraction of homogenized rat or human lung contained most of the PCP activity in that tissue. Polymorphonuclear neutrophils, macrophages, and lymphocytes isolated from human blood had PCP activity. Fibroblasts cultured from human tissues had the highest activity (0.56-1.15 mumol/h per 10(6) cells), more than endothelial cells cultured from human pulmonary arteries. PCP of cultured human fibroblasts was similar to the human renal enzyme because it was resistant to moderate heating and was not inhibited by p-chloromercuriphenyl sulfonic acid. These properties and the substrate specificity distinguish PCP from cathepsin A, which is also in fibroblasts. Antibody to human renal PCP reacted with fibroblast PCP in immunofluorescence, indicating common antigenic determinants. Hydrocortisone changed PCP activity in fibroblasts in parallel with changes in beta-glucuronidase activity and cell-protein concentration; the activity was depressed at low concentration of the hormone. PCP activity was also found in synovial fluid from arthritic joints and in fibroblasts from the synovium. That PCP is found in both inflammatory exudates and in cells that appear at sites of inflammation indicates that, in addition to inactivating angiotensins, this enzyme may have a role in inflammation.

Angiotensin II↗
PubMed1978

Purification and properties of prolylcarboxypeptidase (angiotensinase C) from human kidney.

Prolylcarboxypeptidase was purified from human kidney 1200-fold with 18% yield. The enzyme had no cathepsin A activity and appeared to be homogeneous in gel electrophoresis. The molecular weight of prolylcarboxypeptidase was estimated to be 115,000 by gel filtration. Under denaturing conditions the enzyme dissociated into subunits of 45,000 and 66,500 molecular weight. The enzyme cleaved benzyloxycarbonyl (Cbz)-Pro-Phe, representing the COOH-terminal end of angiotensin II and des-Asp1-angiotensin II (angiotensin III), at a rate of 31 micronmol/h/mg of protein. The rate of hydrolysis increased when phenylalanine in the N-protected dipeptide was replaced with alanine, valine, or leucine or when the octapeptide angiotensin II or the heptapeptide angiotensin III were the substrates. The enzyme also cleaved the angiotensin II antagonist saralasin (Sar1-Ala8-angiotensin II). The Km values were 1 mM, 2mM, and 0.77 mM with Cbz-Pro-Phe, angiotensin II, and angiotensin III, respectively. The enzyme had an acid pH optimum (4.5 to 5.5), but hydrolyzed angiotensin III at pH 7 at 50% of the optimal rate. Prolylcarboxypeptidase was inhibited by diisopropyl phosphorofluoridate and pepstatin, but not by sequestering agents or -SH reagents.

Angiotensin II↗
PubMed1976

Regulation of nitrogen catabolic enzymes in chick liver -- effects of amino acids.

It is well-known that in the chick, dietary protein increases the levels of several hepatic enzymes that are involved in nitrogen metabolism and excretion. However, the biochemical mechanism of this response is essentially unknown. The experiments presented in this paper show that the chick is responding to alpha-amino nitrogen and not to any specific amino acid. Furthermore, it is shown that this system responds to endogenous sources of nitrogen as well as dietary protein and that the xanthine dehydrogenase response involves regulation of enzyme synthesis without changing the rate of degradation.

Amino Acids↗
PubMedDate not supplied

An inherited limb deformity created by insertional mutagenesis in a transgenic mouse.

We have created an insertional mutation that leads to a severe defect in the pattern of limb formation in the developing mouse. The novel recessive mutation is phenotypically identical and non-complementary to two previously encountered limb deformity mutations, and is closely linked to a dominant mutation that gives rise to a related limb dysmorphism. The inserted element thus provides a molecular genetic link with the control of pattern formation in the mammalian embryo.

Animals↗
PubMedDate not supplied

Carboxyhemoglobin trend in Chicago blood donors, 1970-1974.

An 18% reduction in the carboxyhemoglobin (HbCO) saturation in nonsmoking Chicago blood donors occurred between 1970 and 1974, indicating that current donors are being exposed to a lower average carbon monoxide (CO) concentration than had been experienced by 1970 donors. In contrast to the situation in 1970, when it was discovered that 74% of the nonsmokers in Chicago were being exposed to CO in excess of the amount permitted by the federal air quality standards, in 1974 only 41% of the nonsmokers were being overexposed. The observed reduction in HbCO correlates well with both the ambient CO levels recorded at the air monitoring stations and the reduction in CO emission from automobiles. If the current trend continues, Chicago should reach compliance with air quality standards for CO by 1985. The measurement of HbCO in a representative urban population is an accurate index of actual CO exposure and supplements the air pollution data provided by air monitoring stations.

Air Pollutants↗
PubMedDate not supplied

Inhibition of human converting enzyme in vitro by a novel tripeptide analog.

We have studied inhibition of homogeneous human converting enzyme by a new inhibitor, a ketomethylene derivative of the blocked tripeptide substrate, Bz-Phe-Gly-Pro (ketoACE). KetoACE inhibited the hydrolysis of Hip-His-Leu and Hip-Phe-Arg at different concentrations (I50 values were 4 X 10(-8) M and 2 X 10(-7) M, respectively). Kinetic studies indicated that ketoACE inhibits the hydrolysis of both substrates by a similar, non-competitive mechanism. At the lowest enzyme concentration tested, using 3H-Hip-Gly-Gly as substrate, the I50 of ketoACE was 6 X 10(-9) M. KetoACE protected a functional tyrosine residue in the active site of human converting enzyme from modification with N-acetylimidazole. It is proposed that there are alternate (hydrophobic) binding sites for both inhibitors and substrates in the active site of human converting enzyme. It should be possible to develop other high-affinity inhibitors of this class that bind to hydrophobic sites and do not require metal binding via a sulfhydryl group.

Angiotensin-Converting Enzyme Inhibitors↗