Isolation and characterization of a spontaneously transformed malignant mouse mammary epithelial cell line in culture

A method is described that permits the selection of spontaneously transformed mammary epithelial colonies from an untransformed mouse mammary epithelial cell line, NMuMG, and utilizes a long-term anchorage-independent growth of the transformants on soft agarose. These transformed cells (NMuMG-ST) are shown to be distinguishable from the untransformed cells by morphology, growth characteristics, induced carcinomas when transplanted into nude mice and ability to metastasize. This transformed phenotype displayed focal, multilayer growth and higher saturation density in comparison with the untransformed phenotype. Transplanted tumors as well as metastatic lung tumors in nude mice were adenocarcinomas morphologically similar to typical mammary tumors in humans. This selection procedure of mutant mammary cells from an immortalized cell line derived from normal mammary glands could be very useful to identify the genomic biomarkers in the growth regulation and malignant progression of breast cancer.     

Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma

A canine lymphoma cell line (CL-1) was established in culture from tumor cells found in the pleural fluid of a 7-year old female Japanese terrier with thymic form lymphoma. The CL-1 cells were positive for CD45 and MHC class II and negative for CD4, CD5, CD8, Thy-1 and B-cell specific antigen and surface immunoglobulin. The CL-1 cells had a rearranged T-cell receptor beta-chain gene and a germ-line form immunoglobulin gene, indicating that the CL-1 cells represented a monoclonally expanded population of canine alpha beta T-cell lineage.     

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.     

Isolation and partial characterization of a kallikrein from mouse submaxillary glands

A simple procedure to obtain relatively large amounts of purified kallikrein from male mouse submaxillary gland is described. Some chemical and biological properties of this kallikrein have been investigated. The enzyme has a m.w. of 32,000 and shows strong BAEE-esterase activity, as well as kininogenase activity. It is partially inhibited by Aprotinin.     

Radio-sensitive murine thymoma cell line 3SB: characterization of its apoptosis-resistant variants induced by repeated X-irradiation

This paper describes public health nurses' perceptions of changes in their practice. The participants were 28 public health staff nurses from six Alberta, Canada health units serving urban and rural populations. Data were collected in 1993-94 using individual and focus group interviews. Content analysis was used to identify the following themes: "pulling back", "from hands on to arms length", "handing over responsibility", "developing working partnerships", and "doing less surveillance". These themes are discussed in terms of their implications for population health and for public heath nursing, using as a point of reference the principles of Primary Health Care. Continuing research is needed to chronicle further changes in public health nursing practice that will result from health care restructuring and health system reform.     

Isolation, cloning and characterization of a putative type-1 astrocyte cell line

The high prevalence of cholesterol gallstone disease in hypertriglyceridemic patients may be associated with frequent metabolic defects in cholesterol and bile acid syntheses and in the concomitant formation of bile supersaturated with cholesterol. This study had the two aims: 1) to assess whether the defects as well as the degree of biliary cholesterol supersaturation in patients with hyperlipoproteinemia (HLP) can be estimated by the simultaneous determination of plasma mevalonate (MVL) and 7alpha-hydroxy-4-cholesten-3-one (C4); and 2) to assess the possible application of an estimated cholesterol saturation index ([CSI]E) as a means of evaluating the clinical effects of simvastatin on biliary lipid composition. Biliary cholesterol supersaturation was observed in patients with both IIa and IV HLP types. Consistent with the high activity and steady-state messenger RNA level of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase, plasma MVL was significantly higher in 86 patients with HLP (38 type IIa, 44.1 +/- 2.4 nmol/L and 48 type IV, 56.7 +/- 2.3; P < .01) than in 41 normolipidemic subjects (34.2 +/- 1.5), closely correlating with the molar percentage of cholesterol in bile (r = .61, P = .0001; n = 86). On the other hand, consistent with the high activity and messenger RNA level of cholesterol 7alpha-hydroxylase, plasma C4 was significantly higher in patients with HLP (type IIa, 28.8 +/- 2.3 nmol/L and type IV, 38.3 +/- 2.7; P < .01) than in normolipidemic subjects (17.4 +/- 1.5). Plasma C4 was closely correlated with plasma MVL (r = .40, P = .0001; n = 86), but was inversely correlated with the molar percentage of bile acids in bile (r = .49, P = .0001; n = 86). Assuming that cholesterol supersaturation in patients with HLP may be governed by both an enhanced cholesterol secretion (closely reflected by plasma MVL) and a decreased secretion of bile acids (closely reflected by plasma C4), the multivariate linear regression-analyses revealed that an index defined as estimated CSI ([CSI]E) (%) in patients with HLP was given by the following equation using plasma MVL and C4 (nmol/L): [CSI]E = 1[MVL] + 0.7[C4] + 44.4. Biliary cholesterol supersaturation in patients treated with simvastatin improved in a manner parallel to the time course of decreases in plasma MVL and C4. The [CSI]E before and at the end of treatment were correlated with biliary CSI. These results indicate that defects of hepatic cholesterogenesis, and bile acid synthesis, and the degree of biliary cholesterol supersaturation in patients with HLP can be estimated exactly by the simultaneous determination of plasma MVL and C4; furthermore [CSI]E may be adopted for clinical use as a convenient index of biliary CSI.     

Isolation and characterization of high-osmolarity-sensitive mutants of fission yeast

For the fission yeast Schizosaccharomyces pombe, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (gpd1(+)) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive S. pombe mutants, including wis1, sty1, and gpd1. In this study, we attempted to further isolate novel osmolarity-sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated hos1, hos2, and hos3 (high osmolarity sensitive) mutants. One of them, the hos1 mutant, was characterized in detail. The hos1 mutant was demonstrated to have a mutational lesion in the known ryh1(+) gene, which encodes a small GTP-binding protein. Disruption of the ryh1(+) gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the delta ryh1 mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of S. pombe.     

Isolation and characterization of dendritic cells from mouse heart and kidney

Dendritic cells (DC) are thought to be distributed throughout lymphoid and most nonlymphoid tissues. Single cell suspensions were prepared from mouse hearts and kidneys. Subsets of MHC class II-positive (Ia+) leukocytes from both sources expressed markers such as CDw32 Fc receptors, F4/80, and complement receptor type 3 (CD11b/CD18). The capacity of these cells to initiate primary in vitro immune responses was assessed using oxidative mitogenesis and allogeneic mixed leukocyte responses. After fractionation by density centrifugation, cell sorting, immunomagnetic bead separation, or cell panning, the stimulatory activity of kidney cell suspensions was found to reside in the low density, Ia+ leukocyte fractions after overnight culture (day 1). In contrast, freshly isolated (day 0) cells had considerably less or no activity in these assays. However, depletion of Ia+ or CD45+ cells on day 0 followed by overnight culture removed the stimulatory activity on day 1. Therefore, day 0 kidney cells contain Ia+ leukocytes that can acquire or up-regulate their stimulatory activity during overnight culture. Similar observations were made for cells isolated from hearts, except that a population of uncharacterized nonleukocytes with stimulatory activity was detected on day 0 but not day 1. The phagocytic capacity of the leukocytes was then examined. Subsets of Ia+ cells phagocytosed zymosan, as shown by two-color flow cytometry and other immunofluorescence studies, and the zymosan-positive cells from kidney were able to initiate primary responses. Overall, these data demonstrate the existence of DC in kidneys and hearts, and suggest that in situ these cells resemble immature rather than mature DC.     

Isolation and characterization of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9

Norepinephrine (NE) (2.5 micrograms/kg/min) was administered to 5-week-old male Sprague Dawley rats by subcutaneous osmotic mini pumps for 14 days to generate an in vivo cardiac hypertrophy model and the responses with respect to aging examined. In the model, ventricles were significantly hypertrophied without myocardial necrosis and without significant increases in heart rate or blood pressure; the beta adrenergic system was down-regulated. In 37-week-old rats receiving 1.0 microgram/kg/min NE, there was a tendency towards heart failure, and myocardial necrosis and interstitial fibrosis were revealed by histological examinations. The density of beta adrenergic receptors and adenylyl cyclase activity was lower in the older rats. The excess stimulation of adrenergic receptors caused severe cardiac injury in old rats regardless of down regulation of beta adrenergic receptors.     

Isolation and characterization of a novel bladder cancer cell line: inhibition by epidermal growth factor

A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells' proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.     

Isolation and genetic characterization of pGKL killer-insensitive mutants (iki) from Saccharomyces cerevisiae

The linear double stranded DNA plasmid pGKL1 encodes the yeast killer toxin complex (Gunge et al., 1981) of which the killing mechanism is not understood. We isolated and characterized eight mutants in Saccharomyces cerevisiae that were insensitive to both the intracellularly expressed 28-kDa killer subunit and the native killer toxin complex. These mutations (iki1 through iki5) were all recessive, and classified into five complementation groups. The iki2 mutation was mapped to a position near the centromere on chromosome XIII. We developed a novel screening system to isolate the DNA fragments complementing the iki mutations from a Saccharomyces gene library, and isolated three DNA fragments that complement the iki1, iki3, and iki4 mutations, respectively.     

Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10(-6)). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.     

Isolation and characterization of mouse nasal-associated lymphoid tissue

SETTING: Mycobacterial galactofuran is essential to the linking of the peptidoglycan and mycolic acid cell wall layers. Galactofuran biosynthesis should thus be essential for viability. OBJECTIVE: The objective was to determine the pathway of galactofuranosyl biosynthesis and to clone a gene encoding an essential enzyme necessary for its formation. DESIGN: Specific enzymatic conversions involved in formation of galactopyranose and galactofuranose residues in other bacteria were tested for in Mycobacterium smegmatis. M. tuberculosis deoxyribonucleic acid (DNA) was identified by homology. RESULTS: It was shown that the de novo synthesis of the galactose carbon skeleton occurred in M. smegmatis by the transformation of UDP-glucopyranose to UDP-galactopyranose via the enzyme UDP-glucose 4-epimerase (E.C. 5.1.3.2). The N-terminal sequence of this enzyme was obtained after purification. The galactose salvage pathway enzyme, UDP-glucose-galactose-1-phosphate uridylyltransferase (E.C. 2.7.7.12), was also shown to be present. The critical biosynthetic transformation of the galactopyranose to galactofuranose ring form was shown to occur at the sugar nucleotide level via the enzyme UDP-galactopyranose mutase (E.C. 5.4.99.9). The M. tuberculosis DNA encoding this enzyme was sequenced, the gene expressed in Escherichia coli, and the expected enzymatic activity demonstrated. CONCLUSION: Galactofuranose biosynthesis can now be pursued as a potential drug target in M. tuberculosis.     

Isolation and characterization of a rheumatoid arthritis-specific antigen (RA-A47) from a human chondrocytic cell line (HCS-2/8)

Two types of 47 kDa antigen specifically recognized by sera from rheumatoid arthritis (RA) patients were isolated from the membrane fraction of a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by a 2-step procedure: preparative SDS-PAGE and reverse-phase HPLC. An N-terminal amino acid sequence in one of the 47 kDa antigens, named RA-A47, had 81% homology to that deduced from the DNA sequence of the colligin gene which is reported as human hsp47 gene, and 100% homology to that deduced from the DNA sequence of colligin-2 gene, a homologue of colligin. The RA-A47 cross-reacted with a monoclonal antibody raised against chick heat shock protein (Hsp) 47 and bound to gelatin. The expression of the ra-a47 gene was enhanced by heat shock treatment and TGF-beta stimulation. These findings suggest that RA-A47 is a Hsp47-like protein, presumably the product of the colligin-2 gene, and that a collagen-specific molecular chaperone(s) such as Hsp47 and/or RA-A47 is involved in cartilage destruction in RA.     

Isolation and characterization of a novel inositol hexakisphosphate binding protein from mammalian cell cytosol

Inositol hexakisphosphate (IP6) is present in most mammalian cells, although its intracellular function is as yet undefined. We find that the total protein fraction from bovine brain cytosol contains a significant level of specific binding for IP6 precipitable with 40% saturated ammonium sulfate. A protein complex has been isolated from this fraction that specifically binds IP6 and is purified about 500-fold over the cytosol. The IP6 binding protein (IP6BP) chromatographs as a single peak of binding activity on a gel exclusion column, with a Stokes radius equivalent to 266 +/- 14 kDa. The IP6BP is a heterooligomeric complex composed of a number of subunits with molecular weights varying from 23,000 to 60,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Scatchard analyses of IP6 binding of both the crude ammonium sulfate fraction and the purified complex show the presence of a similar high-affinity binding site (Kd approximately 6.0 nM). Bmax for the purified fraction is 1.8 nmol of IP6/mg of protein or 0.48 mol of IP6 bound/mol of complex. Other inositol polyphosphates, such as inositol 1,3,4,5,6-pentakisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate, are poor competitors for IP6 binding to the purified complex. The purification scheme, when applied to a rat liver cytosol fraction, yields a similar IP6BP. This complex has an apparent size of 512,000 using gel exclusion chromatography and contains an additional protein band with M(r) = 97,000 by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment and characterization of a novel human myoepithelial cell line and matrix-producing xenograft from a parotid basal cell adenocarcinoma

Myoepithelial cells exert important paracrine effects on epithelial morphogenesis and mitogenesis through direct cell-cell interactions and through synthesis of a basement membrane extracellular matrix. To study these effects further, this study established the first immortalized human myoepithelial cell line, HMS-1, and transplantable xenograft, HMS-X, from the rare parotid basal cell adenocarcinoma. The cell line exhibited a fully differentiated myoepithelial phenotype and the xenograft exhibited the rare property of accumulating an abundant extracellular matrix composed of both basement membrane and nonbasement membrane components with the latter predominating. With HMS-1 as a feeder layer, dramatic and specific induction of epithelial morphogenesis (spheroid formation) occurred with selected normal epithelial and primary carcinoma target cells. HMS-1 and HMS-X provide distinct advantages over the conventional murine matrices in existence. They will be invaluable in future studies of human tumor-myoepithelial and matrix interactions important for tumor cell growth, invasion, and metastasis.