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Biomedical subjects

M Nachtigal

Publications and source records attributed to M Nachtigal.

At least 19 recordsLinked to original sources
PubMed1998

A chromosome 4 satellite I DNA isolated from SV40-transformed human cells.

Analysis of cellular DNA insert isolated from a free replicative plasmid rescued from human cells transformed with an SV40 vector plasmid revealed the presence of two arrays of repetitive DNA arranged in tandem. One sequence was homologous to the consensus sequence of the human alpha satellite DNA and the adjoining sequence was a satellite DNA sequence which consisted of repetitive units of 42 base pairs (bp) and was designated HR42. The degree of homology between repetitive units was about 92%. By Southern analysis the HR42 sequence was detected in HHW416, a somatic cell hybrid containing human chromosome 4, but not in HDm-5, the somatic cell hybrid which has human chromosome 14. By fluorescence in situ hybridization this repetitive DNA was assigned uniquely to the centromeric region of human chromosome 4. These results show that HR42 belongs to a subfamily of satellite I DNA specific for human chromosome 4.

Base Sequence↗
PubMed1998

Galectin-3 expression in human atherosclerotic lesions.

The expression of galectin-3, a beta-galactoside-binding lectin, was studied in atherosclerotic lesions from specimens obtained from carotid endarterectomies, lower limb amputations, and thoracic aortas from autopsies of young adult trauma victims. Immunohistochemical staining with the monoclonal antibody M3/38 demonstrated the presence of galectin-3 in advanced atherosclerotic lesions from each of 13 cases of carotid endarterectomy and 16 lower limb amputations and in the thoracic aorta of 4 of 20 cases of trauma victim adults. Immunostaining did not detect galectin-3 in umbilical cord and normal thoracic aorta arteries and limb veins. Dual immunostaining with monoclonal antibodies M3/38 for galectin-3 and clone 1A4 for smooth muscle alpha-actin or HAM56 for human macrophage antigen showed that galectin-3 was localized predominantly in foam cells and macrophages and rarely (<5%) in the smooth muscle cells of atherosclerotic lesions. The incidence of galectin-3-positive cells was higher in the carotid artery atherosclerotic lesions, which are richer in foam cells, than in the lower limb atherosclerotic lesions, which are more fibrotic. Reverse transcription polymerase chain reaction showed a significantly higher ratio of galectin-3/beta-actin transcripts in 20 atherosclerotic arteries compared with that of 5 umbilical cord arteries. Western blot analysis confirmed a higher level of galectin-3 in atherosclerotic carotid and lower limb arteries compared with that of umbilical cord arteries. The increased expression of galectin-3 in atherosclerotic lesions suggests the involvement of this multifunctional protein in atherogenesis.

Actins↗
PubMed1997

Transformation of rabbit vascular smooth muscle cells by human cytomegalovirus morphological transforming region I.

The association of human cytomegalovirus with atherosclerosis and the monoclonal hypothesis of atherogenesis suggested that transformation of vascular smooth muscle cells may be an outcome of the virus-host cell interaction. To test this hypothesis, rabbit aorta smooth muscle cells were transfected with the morphological transforming region I (mtrI) of human cytomegalovirus (HCMV) linked to the neomycin resistance gene. Foci of neomycin-resistant and morphologically transformed cells were isolated and expanded into fourteen RCMV strains. Eight of these strains acquired immortalization, but only one strain (RCMV-21) retained recombined viral sequences integrated in the cellular DNA. RCMV strains were heterogeneous in their morphology, expression of smooth muscle alpha-actin, growth, and mitogenic response to serum and fibroblast growth factor (FGF)-2 and -4. All RCMV strains assayed except RCMV-3 showed DNA synthesis in low serum medium and, with the exception of RCMV-1 cells, all showed a significant mitogenic response to FGF-2 and FGF-4, Maintenance of the transformed phenotype appeared independent of the retention of the transforming viral sequences, which was suggestive of a "hit-and-run" mechanism. These results suggested that morphological transformation by HCMV DNA sequences could enhance the mitogenic response of vascular smooth muscle cells to fibroblast growth factors.

Animals↗
PubMed1996

Amplification of stromelysin-3 transcripts from carcinomas of the colon.

Stromelysin-3 has been recently described in association with the stroma of different types of cancer including colorectal carcinomas. This article reports the detection of transcripts for stromelysin-3 (matrix metalloproteinase-11 [MMP-11]) in extracts of tissue from colorectal carcinomas using the technique of reverse transcription-polymerase chain reaction (RT-PCR). In 12 cases of primary colon carcinoma, stromelysin-3 messenger RNA (mRNA) was detected after 25 cycles, whereas this procedure did not reveal stromelysin-3 mRNA expression in one rectal carcinoma micrometastasis to the liver or in normal colon tissue (controls) after 30 cycles of PCR. However, stromelysin-3 mRNA was detected in normal colon specimens after 45 cycles. The high sensitivity of this technique allows application for the investigation of the expression of stromelysin-3 in small amounts of tissue.

Adenocarcinoma↗
PubMed1996

Transformation but not ras-transfection increases the expression of galectin-3 in human HOS cells.

In ras-transfected NIH3T3 cells, the transcription of the Mr 34,000 beta-galactoside specific lectin galectin-3 depends on transformation phenotypes. This observation suggests that this lectin is associated with the transformation process and/or that the ras oncogene may modulate its expression; nevertheless the involvement of ras-gene product in galectin-3 gene expression still remains unclear. In the present study, we investigated the galectin-3 expression in human HOS cells transiently or stably transfected with a ras-containing vector. We observed an increase in galectin-3 mRNA and protein content in stably ras-transfected cells which had lost their anchorage dependence for growth but no increase in cells which needed anchorage for growth or in transiently ras-transfected cells. These results suggest that the galectin-3 up-regulation in ras-transfected HOS cells is the consequence of the cell transformation rather than a direct effect of the ras gene product on galectin-3 gene expression.

Adenocarcinoma↗
PubMed1995

A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture.

PURPOSE: A spontaneously arising, apparently transformed, cell line has been cloned from a primary culture of human retinal pigment epithelial (RPE) cells and has been subcultured more than 200 times. The similarities of these cells to human RPE cells in vivo have been determined. METHODS: The structure of the transformed cells has been determined by light and electron microscopy and by immunocytochemistry using antibodies that detect cytoskeletal and other proteins. The ability of the cell line to bind and phagocytose photoreceptor material has also been assessed by fluorescence and electron microscopy. The metabolism of all-trans-retinol has been investigated by incubation of the cells with 3H-all-trans-retinol and analysis of the metabolic products by high-performance liquid chromatography. RESULTS: The transformed cells possess an epithelial cobblestone morphology with intercellular junctional complexes containing N-cadherin. The cytoskeleton of these cells comprises cytokeratins that are characteristic of epithelial cells, together with actin, spectrin, and vimentin. The keratins expressed are those typical of RPE cells. The cells also express cellular retinaldehyde binding protein and retinol dehydrogenase activity but do not express retinoid isomerase or lecithin retinol acyl transferase activities. These cells also exhibit phagocytic activity. CONCLUSIONS: This cell line retains many of the metabolic and morphologic characteristics of RPE cells in vivo although there are some differences, particularly the loss of some enzymatic activities and cytoskeletal polarization. These cells should be useful in further studies of RPE cell metabolism and other functions.

Cell Line, Transformed↗
PubMed1991

Pregnancy termination because of chromosomal abnormalities: a study of 26,950 amniocenteses in the southeast.

A regional study was done to investigate the outcome of chromosomally abnormal pregnancies with respect to the parental decision to continue or to terminate the pregnancy. Fourteen medical centers in the southeastern United States contributed data on 26,950 amniocenteses. In 416 cases (1.54%), cytogenetic abnormalities were reported. Of 378 singleton pregnancies in which a cytogenetic abnormality was reported and for which information regarding pregnancy outcome was available, the decision to terminate the pregnancy was made in 276 (73.02%). When the chromosomal abnormality was autosomal in nature, 240 of the 293 pregnancies (81.91%) were terminated, as compared to 36 of the 85 pregnancies (42.35%) affected with a sex chromosomal abnormality. Pregnancies involving the most common autosomal trisomies (21, 18, and 13) were terminated at a rate of 92% to 95%. No significant difference in the rate of abnormality or in the rate of pregnancy termination existed between the 14 centers. Our findings reflect the largest series of amniocentesis results collected to date.

Abortion, Induced↗
PubMed1991

Homologous recombination of a plasmid containing the SV40 early region in rabbit cells.

The plasmid pSV3-neo that carries the SV40 early genetic region and the neor resistance gene underwent homologous recombination between SV40-duplicated sequences of the small t antigen intron and the polyadenylation site after transfection into rabbit vascular smooth muscle cells. This recombination yielded two free plasmids of 1.7 and 3.0 kb that replicated in transfected rabbit cells, while the vector plasmid did not. Replication of the two recombinant plasmids continued for several months in rabbit cells transformed by pSV3-neo, and their structure remained unchanged. Between 15 and 2,510 copies per cell of the 3.0-kb plasmid and between 370 and 1,565 copies per cell of the 1.7-kb plasmid were found in different pSV3-neo-transformed rabbit clones. Similar recombined plasmids were found 3 days after transfection of pSV3-neo onto SV40-transformed rabbit cells. By comparison, pSV3-neo was replicated in simian cells, but no recombined plasmids were detected by Southern analysis. No replication was detected after transfection of pSV3-neo into mouse cells. These results appear to reflect important differences between rabbit and simian cells in the efficiency of homologous recombination involving specific SV40 sequences and/or in the replication of the resulting recombinants. The role of the host cell in the evolution of SV40 sequences may influence the outcome of the infection with this virus.

Animals↗
PubMed1991

Collagen expression in mechanically stimulated cardiac fibroblasts.

The cardiac extracellular matrix, composed predominantly of collagenous fibers, forms a stress-tolerant network that facilitates the distribution of forces generated in the heart and provides for proper alignment of cardiac myocytes. Although considerable information exists regarding the morphological organization of the heart extracellular matrix, little is known about the regulation of the synthesis and accumulation of extracellular matrix components. A potentially significant factor in the cardiovascular system is mechanical stimulation including changes in physical tension and pressure. We recently have developed an in vitro model system to elucidate the effects of mechanical stretch on isolated populations of heart cells. In the present study, we have used biochemical and molecular biological techniques to analyze changes in collagen synthesis by cardiac fibroblasts in response to mechanical stretch. These studies show that the ratio of collagen type III to collagen type I increases in mechanically stretched cells. They also show that type III collagen mRNA levels are increased in response to cyclic mechanical stretch for durations as short as 12 hours. Type I collagen mRNA levels were not found to change under the stretch conditions used in this study. Our results emphasize the potential regulatory role of mechanical stimulation in the expression of specific genes in the heart and support previous studies indicating this to be an intriguing in vitro model of cardiac hypertrophy.

Animals↗
PubMed1991

Characterization of human vascular smooth muscle cells transformed by the early genetic region of SV40 virus.

Human arterial smooth muscle cells transfected with the plasmid pSV3-neo, which contains the SV40 virus early region and the neor gene, developed colonies of morphologically transformed cells. Five cell strains were initiated from these colonies and could be subcultivated for up to 9 months before entering a stage of crisis that ended their life span. Deoxyribonucleic acid (DNA) molecules containing viral sequences were found free and integrated in the transformed cells. The intranuclear SV40 large T antigen and the p53 cellular protein were expressed in the transformed cells. Most of the transformed cells were spindle shaped but some were large and multinucleated. The modal chromosome numbers were in the triploid range, and aberrations, particularly dicentrics, were common. The transcripts for smooth muscle actins were significantly reduced and there were less alpha-actin filaments detected by immunofluorescence. Cytochemical staining disclosed a large accumulation of lipid droplets in the transformed cells incubated with rabbit hypercholesterolemic beta-very-low-density lipoprotein. Chemical analysis showed that cholesteryl esters were significantly elevated in these cells. Phenotypic changes induced in human vascular smooth muscle cells by SV40 early genes are similar to those found in smooth muscle cells from atherosclerotic lesions and may indicate common pathogenetic mechanisms.

Actins↗
PubMed1990

Immortalization of rabbit vascular smooth muscle cells after transfection with a fragment of the BglII N region of herpes simplex virus type 2 DNA.

Rabbit arterial smooth muscle cells were transfected with the pBC24neo plasmid DNA which consists of a sequence of 790 base pairs from the BglII N fragment of herpes simplex virus type 2 DNA linked to the neo resistance gene. Selection in G418-containing medium resulted in eleven immortalized clones which showed increased growth rate and saturation densities. One of the eleven clones maintained the transforming DNA sequence integrated in the cellular genome and showed continuous resistance to G418, but Southern blot analysis of the other ten immortalized clones did not detect pBC24neo DNA sequences. Possible mechanisms of herpes simplex virus induced immortalization and their implications in the development of atheromatous plaques are discussed.

Animals↗
PubMed1990

Transformation of rabbit vascular smooth muscle cells by transfection with the early region of SV40 DNA.

Rabbit aortic smooth muscle cells (SMC) and Rb-1 cells, a continuous line of the same origin, were transformed by transfection with pSV3-neo DNA, a plasmid containing the SV40 early region linked to the neoR resistance gene. Transformed clones were selected in G418-containing medium at a rate of 10(-4) per cell. All transformed clones were immortalized and contained in the early passages two free recombined plasmids derived from pSV3-neo. At advanced passages pSV3-neo sequences were found integrated in the cellular genome. Transformed cells had an altered morphology and growth pattern that differed among clones. Some clones reached high density in low-serum medium. All the clones stained positively for the intranuclear T antigen. Some clones had distinct transcripts for the large T and small t antigens, while in others only larger or truncated transcripts were found. Alpha-actin filaments were visualized by immunofluorescent staining in all the clones, but Northern blot analysis revealed a significant reduction in transcripts for this actin. All the transformed clones accumulated, to a variable extent, cholesteryl esters after incubation with beta very low-density lipoprotein. Six of the eight transformed clones maintained a diploid chromosome number, but there was an increase in structural chromosome aberrations, predominantly dicentrics. Transfection of pSV3-neo into rabbit vascular SMCs is an efficient model for obtaining transformed clonal populations. These clones show some phenotypic changes that may be relevant to the study of atherogenesis.

Actins↗
PubMed1989

Characterization of a continuous smooth muscle cell line derived from rabbit aorta.

A spontaneously arising continuous cell line (Rb-1) derived from collagenase-elastase digested rabbit aorta has been propagated in vitro for over 100 passages. During this period, the Rb-1 cells remained spindle-shaped and formed regularly oriented parallel bundles. After Passage 50, Rb-1 cells were found to be serum-independent in their growth and reached higher saturation density than rabbit aorta smooth muscle cells. Alpha-actin and desmin filaments were detected by immunostaining in Rb-1 cells and early passage of rabbit aorta smooth muscle cells. The proportion of alpha-actin transcripts in Rb-1 cells was lower than that of transcripts for beta- and gamma-actins. The modal chromosome number was maintained at 44 between Passages 11 and 60, and two marker chromosomes were constantly present. Infection of Rb-1 cells with two strains of herpes simplex virus type 1 resulted in high titers of virus, whereas a herpes simplex virus type 2 temperature-sensitive mutant replicated only at the permissive temperature. The Rb-1 cell line could be used for the study of vascular smooth muscle cell proliferation and their interaction with viruses.

Actins↗
PubMed1988

Oncogenicity of rat prostate cells transformed in vitro with cadmium chloride.

Three rat ventral prostate (RVP) cell lines transformed after in vitro treatment with cadmium chloride (CdCl2) and one control untreated cell line were tested for tumorigenicity in newborn rats. All three cadmium-transformed RVP cell lines induced tumors at the site of inoculation in 95-100% of animals. The fibroblastoid RVP56Cd cell line induced sarcomas, whereas the epithelial cell lines RVP47-3G and RVP47-3F produced highly differentiated squamous cell carcinomas. About 20% of animals injected with RVP47-3G developed lung and splenic metastases. The tumors could be further passaged into young rats. The sarcomas had a hyperdiploid modal chromosome number similar to that of the RVP56Cd cell line. Carcinomas induced by the RVP47-3G cell line had a large proportion of stromal metaphases. The modal chromosome number of these carcinomas was in the hypertriploid-hypotetraploid range, similar to that of the parental cell line. These results demonstrate that treatment of RVP cells with CdCl2 in vitro results in neoplastic transformation. Since both fibroblastoid and epithelial prostate cells have undergone transformation, it seems possible that cadmium acted as a carcinogen without cell specificity. The susceptibility of these cells to the carcinogenic effect may be related to their resistance to cadmium. In the process of neoplastic transformation induced by CdCl2 in RVP epithelial cells changes of squamous metaplasia occur, and probably precede acquisition of tumorigenicity.

Animals↗
PubMed1988

Regulation of human chorionic somatomammotropin gene expression in rat pituitary tumour cells.

A gene (hCS-1) coding for human chorionic somatomammotropin (hCS) was introduced stably into rat anterior pituitary tumour (GC) cells. These cells were treated with hormones, growth and other factors implicated in hCS production in the placenta. Levels of hCS-1 gene mRNA and hCS release were increased by thyroid hormone, dexamethasone, a cyclic adenosine monophosphate (cAMP) analogue (8-bromo-cAMP), and phorbol ester, phorbol-12-myristate-13-acetate (PMA). No significant response to insulin, medium glucose levels, epidermal growth factor or insulin-like growth factor II was observed. A hybrid gene containing the 5'-flanking sequences from the intact hCS-1 gene was also stably introduced into GC cells. These cells were tested to determine the contribution made by 5'-flanking DNA on the responses observed with the intact gene. Although expression of this hybrid gene was stimulated by thyroid hormone, no response to 8-bromo-cAMP or PMA was observed. These data suggest that sequences downstream of the transcription initiation site might play an important role in hCS gene expression and regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Transformation of rabbit arterial smooth muscle cells with simian virus 40.

In vitro studies were carried out to induce viral transformation of vascular smooth muscle cells. Cultured rabbit arterial smooth muscle cells were infected with simian virus 40 (SV 40), and transformed cultures were produced that exhibit altered morphology, increased growth rate and plating efficiency, growth on semi-solid substrate, and chromosomal abnormalities. Nuclear SV 40 T-antigen was detected in all cells of these cultures. Muscle-specific actin was identified by a specific monoclonal antibody suggesting retention of smooth muscle cell characteristics by the transformed cells. Significant cytoplasmic lipid accumulation occurred in transformed cells incubated with beta-very low density lipoprotein, as revealed both by chemical analyses and Nile Red lipid staining of the culture. The transformed smooth muscle cells grow permanently in cell culture. Our investigations show that arterial smooth muscle cells transformed with SV 40 virus exhibit altered phenotypic properties distinct from that of normal arterial smooth muscle cells.

Animals↗
PubMed1987

Therapeutic efficacy of liposome-encapsulated ribavirin and muramyl tripeptide in experimental infection with influenza or herpes simplex virus.

Large, negatively charged, multilamellar liposomes were examined for their ability to improve the therapeutic activity of the broad-spectrum antiviral agent ribavirin (RIB) and the synthetic immunostimulant muramyl tripeptide (MTP-PE) in the treatment of viral pneumonitis. Liposome-encapsulated MTP-PE (L-MTP-PE) was superior to free MTP-PE in activating alveolar macrophages and in protecting mice against intranasal challenge with 10 LD50 (50% lethal dose) of herpes simplex virus type 1 (HSV-1). Mice treated with liposome-encapsulated or free MTP-PE had no detectable viremia and had lower pulmonary titers of virus than controls. Liposome-encapsulated RIB (L-RIB; 3 mg per mouse), administered several hours after infection, was more effective than was free RIB (10 mg per mouse) in protecting mice against intranasal challenge with 10 LD50 of influenza virus, but neither L-RIB nor free RIB protected mice against HSV-1 infection. In contrast, combination therapy with both L-RIB and L-MTP-PE was more effective than either agent used alone.

Acetylmuramyl-Alanyl-Isoglutamine↗