On somatic recombination in the central nervous system of transgenic mice.
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Animals↗
Biomedical subjects
M Katsuki
Publications and source records attributed to M Katsuki.
Inhibition of chemical carcinogenesis in vivo by azatyrosine.
A single painting of 7,12-dimethylbenz[a]anthracene on the skin of transgenic mice harboring the human protooncogene c-Ha-ras induced papillomas at 100% incidence after 20 weeks (M. Izawa et al., unpublished data). Application of L-beta-(5-hydroxy-2-pyridyl)alanine (azatyrosine) to the skin at a dose of 2 mg/mouse once every 3 days after initiation with 7,12-dimethylbenz[a]anthracene greatly reduced the percentage incidence, number per mouse, and size of papillomas. Injection of methylnitrosourea i.p. into transgenic mice induced papillomas in the forestomach after 12 weeks (2 to 12 papillomas/mouse) at 100% incidence (K. Ando et al., Cancer Res., 52: 978-982, 1992). Administration of azatyrosine i.p. at a dose of 2 mg/mouse once every 2 days for 12 weeks after initiation with methylnitrosourea completely prevented the formation of forestomach papillomas. These results clearly indicated that azatyrosine inhibits chemical carcinogenesis in vivo.
9,10-Dimethyl-1,2-benzanthracene↗
Genetic alteration of catecholamine specificity in transgenic mice.
Epinephrine-producing cells are characterized by the presence of phenylethanolamine N-methyltransferase (PNMT), which catalyzes the formation of epinephrine from norepinephrine. We generated a line of transgenic mice carrying a chimeric gene containing human PNMT cDNA fused to the 4-kilobase fragment of the human dopamine beta-hydroxylase (DBH) gene promoter, to switch catecholamine phenotype in the nervous and endocrine systems. Human PNMT transcripts and immunoreactivity were mainly detected in norepinephrine neurons in brain and sympathetic ganglion as well as in norepinephrine-producing cells in adrenal medulla of transgenic mice, indicating that the human DBH gene promoter of 4 kilobases is sufficient to direct expression of the gene in norepinephrine-producing cells. Analysis of catecholamines in the various tissues showed that the expression of human PNMT in transgenic mice induced the appearance of epinephrine in sympathetic ganglion and dramatic changes in norepinephrine and epinephrine levels in brain, adrenal gland, and blood. These results indicate that the additional PNMT expression in norepinephrine-producing cells can convert these cells to the epinephrine phenotype, and suggest that norepinephrine-producing cells normally possess the basic machinery required for the synthesis of epinephrine except for PNMT. Thus it appears that the only major difference between norepinephrine- and epinephrine-producing cells is the expression of PNMT. Our transgenic animals provide an experimental model to investigate the functional differences between norepinephrine and epinephrine.
Animals↗
Overexpression of apolipoprotein E in transgenic mice: marked reduction in plasma lipoproteins except high density lipoprotein and resistance against diet-induced hypercholesterolemia.
Apolipoprotein E (apoE) has a high affinity to cell-surface low density lipoprotein (LDL) receptor. To determine the role of apoE in plasma lipoprotein metabolism, transgenic mouse lines with integrated rat apoE gene under the control of the metallothionein promoter were established. We found that a high expressor line produced rat apoE mainly in the liver, and the gene product was almost entirely associated with plasma lipoproteins. The plasma level of rat apoE in homozygotes for the transgene was 17.4 mg/dl after zinc induction (vs. 4.56 mg/dl of mouse apoE in controls). In this group, plasma cholesterol and triglyceride levels were 43% and 68% reduced as compared with controls, respectively. Heterozygotes showed decreases in both lipids to a lesser extent. Gel filtration chromatography showed that lipid reduction was mainly due to decreased very low density lipoproteins (VLDL) and LDL. Especially in zinc-treated homozygotes, VLDL had almost disappeared, and a remarkable decrease in LDL and a slight decrease in high density lipoprotein were also observed. Consistently, the plasma level of apoB, a structural protein of VLDL and LDL, was 78% lower than that of controls, indicating a marked reduction in lipoproteins containing apoB. Furthermore, the transgenic mice, in contrast to controls, did not develop hypercholesterolemia when fed a high cholesterol diet. These results demonstrated that overexpression of apoE reduces plasma cholesterol and triglyceride levels and prevents diet-induced hypercholesterolemia. From dramatic and dose-related decreases in plasma lipoproteins in transgenic mice, we conclude that apoE plays a key role in plasma lipoprotein metabolism.
Animals↗
Chemically induced forestomach papillomas in transgenic mice carry mutant human c-Ha-ras transgenes.
Forestomach papillomas and skin papillomas were induced very efficiently by a single dose administration of the chemical carcinogen methylnitrosourea (MNU) in transgenic mice (rasH2 line) carrying human hybrid c-Ha-ras genes, which encode the prototype p21 gene product. The incidence of forestomach papillomas was dose dependent; when 50 mg/kg of MNU were administered i.p., all of the transgenic mice (56 of 56) developed forestomach papillomas within 12 weeks after administration, whereas 5 and 0.5 mg/kg of MNU induced papillomas in 2 of 19 and 1 of 19 mice, respectively. Nine of 56 transgenic mice (16%) also developed skin papillomas at sites wounded by bites or scratches. Only 1 of 77 nontransgenic littermates developed forestomach papillomas after administration of 50 mg/kg of MNU, and no skin papillomas appeared within 12 weeks after MNU administration. The transgenes (integrated copy number, 5-6) in the tumors developed in 55 of 56 affected transgenic mice (98%) contained at least 1 copy of the transgene that was activated by somatic point mutation at the 12th codon, from GGC (Gly) to GAC (Asp). Because somatic point mutations at the 12th or 61st codon of transgenes have never been detected in normal tissues of transgenic mice thus far examined, these mutational activations of transgenes are tumor-specific events. RNA expression of these activated transgenes was also detected. From these results, it is suggested that somatic mutational activation of the human c-Ha-ras transgene plays a causative role in the occurrence of forestomach and skin papillomas induced by MNU administration in these transgenic mice. This transgenic mouse provides a unique screening system for chemicals that induce or suppress papillomagenesis.
Animals↗
De novo production of alpha 2-macroglobulin in cultured astroglia from rat brain.
Previous studies from our laboratory demonstrated that alpha 2-macroglobulin (alpha 2M) is one of the neurite-promoting factors in the conditioned medium of astroglia. In the present study, we further examined the de novo production of alpha 2M in cultured astroglia by determining the expression of alpha 2M mRNA, and the biosynthesis of [35S]methionine-labeled alpha 2M protein. We analyzed the mRNA of cultured astroglia by differential hybridization using specific probes to alpha 2M and its homologous protein, alpha 1-inhibitor 3 (alpha 1I3), after amplification of reverse-transcribed cDNA with the polymerase chain reaction. The result clearly showed that only alpha 2M mRNA is expressed in cultured astroglia. Northern blotting analysis revealed that alpha 2M mRNA is expressed mainly in the astroglia and is not detected in neurons, microglia and meningeal fibroblasts. Furthermore, the biosynthesis of alpha 2M protein in the astroglia was confirmed by an immunoprecipitation experiment after labeling of each type of cell with [35S]methionine. It was concluded that alpha 2M is produced in the cultured astroglia which is the major source of alpha 2M production among various types of cells in rat brain.
Animals↗
Analysis of the human tyrosine hydroxylase promoter-chloramphenicol acetyltransferase chimeric gene expression in transgenic mice.
To investigate cis-elements responsible for catecholaminergic (CAnergic) neuron-specific expression of the tyrosine hydroxylase (TH) gene, we produced lines of transgenic mice carrying 5.0-kb, 2.5-kb and 0.2-kb fragments from the 5'-flanking region of the human TH gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and designated them as TC 50, TC 25, and TC 02, respectively, and reporter gene expression in transgenic mice was analyzed by CAT assay by immunocytochemistry with anti-CAT antibody. High-level CAT expression was observed in the brain and adrenal gland using the 5.0-kb promoter of the TC 50 mice, but ectopic expression was consistently observed in several somatic tissues, e.g. thymus, colon, and testis. In brain, expression was achieved in CAnergic neurons with the largest construct (5.0 kb), but not with 2.5 kb or 0.2 kb of 5' flanking sequence. However, TC 50 mice also expressed CAT immunoreactivity in non-CAnergic neurons. In the TC 25 line CAT immunoreactivity was detected only in some non-CAnergic neurons. In the TC 02 line no CAT immunoreactivity was detected in any of the tissues examined. These results indicate that the 5.0-kb DNA fragment of the TH gene upstream region contains activity to express CAT in CAnergic neurons and surprisingly, lacks some regulatory elements attenuating ectopic expression, and that the 2.5-kb and 0.2-kb fragment are not sufficient for the proper expression. We discuss the presence of the tissue-specific regulatory elements in the structure portion of the TH gene and/or 3'-flanking region.
Animals↗
[Evaluation of aerosol therapy of streptomycin for tracheobronchial and pulmonary tuberculosis].
The clinical features of tracheobronchial tuberculosis are distinct from those of pulmonary tuberculosis in some aspects. Streptomycin (SM) is claimed by some investigators that it has a tendency to promote the development of bronchial stenosis due to scarred healing of the involved endobronchial mucosa, and, for that reason, they recommend not to use it in treatment of bronchial tuberculosis. In some patients with pulmonary tuberculosis, who have renal or hearing disturbance, SM avoided. Yet unless SM is used improvement of tuberculosis could be delayed. It is the purpose of the present investigation to point out that aerosol therapy of SM is useful for patients with respiratory tuberculosis. Furthermore, we wish to demonstrate that we can safely treat the patients with respiratory tuberculosis who suffer from renal function or hearing disorder by use of aerosol therapy of SM. Prior to clinical application of the inhalation therapy, we confirmed that the therapy was not harmful. Serum concentration of SM, when inhaled, was measured in 9 volunteers. Before and after administration of SM aerosol, spirograms were examined in 4 volunteers, nevertheless, no special abnormality was recognized. It seemed that serum concentration of SM after the administration was two low to evoke adverse reactions (less than 3.0 gamma). In 6 patients with pulmonary tuberculosis, blood gases were measured and no obvious change was observed. As a result, it was demonstrated that the endobronchial tuberculous lesions improved faster when treated by inhalation of aerosolized SM as compared with the conventional injection therapy, without evoking any apparent adverse reactions. In some of the patients with pulmonary tuberculosis, it seemed that the therapy was useful.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Plasma lipoprotein metabolism in transgenic mice overexpressing apolipoprotein E. Accelerated clearance of lipoproteins containing apolipoprotein B.
We have reported that transgenic mice overexpressing rat apo E shows marked reduction of plasma cholesterol and triglyceride levels due to the disappearance of VLDL and LDL. In this study, we investigated the metabolism of plasma lipoproteins in transgenic mice. After intravenous injection, the rates of clearance of 125I-VLDL and 125I-LDL were 3.0- and 2.4-fold greater in transgenic mice than in controls, respectively. Furthermore, clearance of chylomicron remnants estimated by oral retinyl palmitate-loading test was markedly enhanced in transgenic mice. The hepatic expression of LDL receptors by immunoblot analysis was similar in both groups. These data suggest that elimination of lipoproteins containing apo B was due to enhanced clearance of these lipoproteins enriched with apo E through hepatic LDL receptors. When fed a high cholesterol diet, controls showed twofold elevation of plasma cholesterol levels with marked increases in VLDL and LDL cholesterol on gel filtration chromatography. In contrast, cholesterol-fed transgenic mice showed resistance against these increases. High cholesterol feeding decreased the activity of hepatic LDL receptors and had no effect on enhancement of chylomicron remnant clearance in transgenic mice. Thus, overexpression of apo E facilitates metabolism of lipoproteins containing apo B presumably primarily via the LDL receptor pathway and possibly through an interaction with the chylomicron remnant receptor.
Animals↗
[A direct sequencing method for PCR products using lambda exonuclease].
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Base Sequence↗
A rapid and efficient protocol of the inverted PCR using two primer pairs.
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Animals↗
[A case of bronchocentric granulomatosis associated with uveitis].
A 32-year-old female was admitted to our hospital because of abnormal pulmonary shadows and a decrease in visual acuity. Analysis of peripheral blood revealed eosinophilia, and chest roentgenogram demonstrated multiple infiltrates in the right upper lung field. Pathological examination of transbronchial lung biopsy specimens revealed necrotizing granulomatous lesions in the walls of bronchioles, and a definitive diagnosis of bronchocentric granulomatosis was made. The cause of bronchocentric granulomatosis in this patient was suggested to be an allergic reaction to Aspergillus because of positive response to skin test for Aspergillus. Although it is reported that extrapulmonary involvement is rare in bronchocentric granulomatosis, the present case was associated with uveitis, and to our knowledge is the first reported case.
Adult↗
Enhanced expression of human tyrosine hydroxylase in the lower brainstem of transgenic mice.
We have previously reported the distribution of human tyrosine hydroxylase (TH) transgene expression in dopaminergic neurons (ventral tegmental area and substantia nigra), adrenal gland, and non-catecholaminergic neurons in the forebrain of transgenic (Tg) mice. In this paper, we analysed the transgene expression in catecholaminergic (CAergic) neurons in the lower brainstem of Tg mice, by in situ hybridization and immunocytochemistry at the light and electron microscopic levels. High-level hybridization signals of the human TH mRNA were observed in the locus ceruleus and nucleus tractus solitarii of the Tg brain. Intense TH immunoreactivity was expressed specifically in the Tg brainstem, as was observed in non-Tg mice. These results reveal that the human TH transgene contains the regulatory elements responsible for the expression in three kinds of CAergic (dopaminergic, noradrenergic and adrenergic) neurons of the mouse brain.
Animals↗
Expression of the human angiotensinogen gene in transgenic mice and transfected cells.
We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.
Angiotensinogen↗
Human renin in transgenic mouse kidney is localized to juxtaglomerular cells.
To examine whether expression of human renin in the transgenic mouse kidney is regulated in a cell-specific manner, we have characterized monoclonal antibodies against human renin and determined the renin-production site by immunohistochemistry. By using a monoclonal antibody specific for human renin, A6-11-6, we demonstrated that human renin in the transgenic mouse kidney is localized to the juxtaglomerular cells of afferent arterioles.
Animals↗
Hepatic and renal expression of rat apolipoprotein E under control of the metallothionein promoter in transgenic mice.
We created three lines of transgenic mice with an integrated rat genomic apolipoprotein E gene fused with the mouse metallothionein I promoter. These lines transcribed rat apoE mRNA in the liver and/or in the kidney and expressed significant amounts of rat apoE in plasma. Enhancement of the plasma level by treatment with Zn ion or Bi ion was observed.
Animals↗
Fate of haploid parthenogenetic cells in mouse chimeras during development.
The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic in equilibrium fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid-derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and less than 5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.
Animals↗
Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.
Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.
Adrenal Glands↗