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T Mikkelsen

Publications and source records attributed to T Mikkelsen.

50 records · Page 3Linked to original sources
PubMed1988

An MuLV transmission vector system designed to permit recovery in E. coli of proviral and cellular flanking sequences.

We have introduced a bacterial suppressor gene (supF) into the long terminal repeat of a molecular clone of the murine leukemia virus (MuLV) SL3-3. A panel of replication competent virus was derived that replicates to high titers in NIH3T3 cells in culture. The tRNA gene is stably carried in the provirus. The supF and viral sequences are present in equimolar amounts in the RNA genome of the expressed recombinant virus. The proviral sequences containing supF can be recovered by cloning into a lambda vector carrying amber mutations. The DNA sequences in the recovered lambda recombinants show a high degree of stability. The presented system should facilitate the study of the interaction between proviral and cellular sequences flanking the integration site.

Bacteriophage lambda↗
PubMed1988

Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not myristylated.

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction.

Animals↗
PubMed1984

The nucleotide sequence of the Akv murine leukemia virus genome.

The nucleotide sequence of an infectious molecular clone of the Akv murine leukemia virus has been determined by the dideoxy chain termination method after subcloning in bacteriophage M13 vectors. The sequence predicts an RNA genome of 8371 nucleotides containing three large open reading frames corresponding to the gag, pol, and env genes. Signal sequences for transcription, splicing, and translation have been identified. The positions of 95 major RNase T1 resistant oligonucleotides of the Akv RNA genome have been located.

AKR murine leukemia virus↗
PubMed1982

Use of the Limulus test to determine the hygienic status of milk products as characterized by levels of gram-negative bacterial lipopolysaccharide present.

The Limulus test was used to investigate the occurrence of Gram-negative bacterial lipopolysaccharide (LPS) in milk and dairy products. This test specifically detects the extremely heat-resistant LPS, and so determines the total accumulated quantity of LPS in a product whether it has been heat-treated or not. A positive correlation between number of psychrotrophic bacteria and amount of LPS in fresh farm milk was found (r = 0.63). Fresh farm milk and freshly produced market milk contained on average less than 1 ng LPS/ml. The Limulus test is considered highly suitable for use in the dairy industry, being a method which will characterize quickly and specifically the hygienic level of a product by the extent of the cumulative contamination by Gram-negative bacteria.

Animals↗
PubMedDate not supplied

Brain tumor segmentation and characterization by pattern analysis of multispectral NMR images.

A major problem in tumor treatment planning and evaluation is determination of the tumor extent. This paper presents a pattern analysis methodology for segmentation and characterization of brain tumors from multispectral NMR images. The proposed approach has been used in 15 clinical studies of cerebral tumor patients who have been scheduled for surgical biopsy and resection. The tissue biopsy results, obtained at specific spatial coordinates determined in the analysis, have been utilized to validate the methodology. It was found that in all cases the lesion had extended into normal tissue, at least to the location where the sample was taken. In most cases, the proposed method suggested that the lesion had extended several millimetres beyond the point from where the biopsy sample was taken. In some cases, the extent of the lesion into normal tissue was well beyond the boundary seen on T1- or T2-weighted images. It is concluded that the proposed approach indicates brain tumor infiltration more precisely than what is visualized in the original NMR images and therefore its utilization facilitates proper treatment planning for the cerebral tumor patients.

Biopsy↗
PubMedDate not supplied

Increased SPARC expression promotes U87 glioblastoma invasion in vitro.

Our recent studies have focused on identifying invasion-promoting genes that are expressed early in brain tumor progression. We identified and characterized SPARC (secreted protein acidic and rich in cysteine) as a potential candidate. To determine whether increased SPARC expression functionally promotes brain tumor invasion, SPARC was transfected into U87MG glioblastoma cells using the tetracycline-off gene expression system. The parental cell line (U87MG), the parental transactivator-transfected clone (U87T2) and three selected U87T2-SPARC-transfected clones (A2bi, A2b2 and C2a4) were characterized for endogenous and transfected SPARC expression. In comparison to the parental or U87T2 cell lines, the SPARC-transfected clones demonstrated: (1) morphological changes, (2) increased SPARC transcript and protein abundances that were down-regulated by the tetracycline analog doxycycline, (3) perinuclear localization of the transfected SPARC (consistent with reported localization of SPARC in normal cells in culture) and (4) altered adhesion and increased invasion as assessed by the spheroid confrontation assay. These data indicate that increased SPARC expression contributes to U87 glioblastoma tumor invasion in vitro and that these cell lines will serve as useful reagents to investigate the mechanism(s) by which SPARC promotes this phenotype in vitro and in vivo.

Animals↗
PubMedDate not supplied

Cathepsin B and glioma invasion.

Increased expression of cathepsin B has been reported in a number of human and animal tumors. This has also been observed in human gliomas where increases in cathepsin B mRNA, protein, activity and secretion parallel malignant progression. In the present study, we showed that cathepsin B was directly involved in glioma cell invasion. Activity of cathepsin B was an order of magnitude higher in glioma tissue than in matched normal brain. Inhibitors of cysteine proteases reduced invasion of glioma cells in two in vitro models: invasion through Matrigel and infiltration of a glioma spheroid into a normal brain aggregate. Glioma spheroids expressed higher levels of cathepsin B than did monolayers and the ability of subclones differing in cathepsin B activity to infiltrate normal brain aggregates paralleled their cathepsin B activity. We confirmed that intracellular staining for cathepsin B occurs at the cell periphery and in cell processes and observed extracellular staining on the cell surface. In addition, we demonstrated that intracellular cathepsin B located at the cell periphery and in processes was active. The cell surface cathepsin B colocalized with areas of degradation of an extracellular matrix component. We hypothesize that the increased expression of active cathepsin B in gliomas leads to increases in invasion in vitro and in vivo and have developed a xenotransplant model in which this hypothesis can be tested.

Animals↗
PubMedDate not supplied

The role of protein kinase Calpha in U-87 glioma invasion.

To investigate the hypothesis that protein kinase Calpha (PKCalpha) is functional glial tumor cell invasion, stable PKCalpha sense and antisense transfected U-87 cell lines were established and PKCalpha expression characterized by Western blot and PKC activity assays. Invasion assays including barrier migration (Koochekpour et al., Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma lines. Eur. J. Cancer, 1995, 31, 375-380; Merzak et al., CD44 mediates human glioma cell adhesion and invasion in vitro. Cancer Res., 1994, 54, 3988-3992; Merzak et al., Cell surface gangliosides are involved in the control of human glioma cell invasion in vitro. Neurosci. Lett., 1994, 177, 11-16), and spheroid confrontation were used to study the relationship between PKCalpha expression and invasiveness. PKCalpha overexpressing clones show increased barrier migration (1.5x) relative to the control transfected clones. PKCalpha inhibited clones exhibited reduced invasiveness, to < 50%. In coculture with PKCalpha overexpressing clones, the remaining normal fetal rat brain aggregate volume was significantly decreased (up to 200%) but 90% of the initial brain volume was left in PKCalpha inhibited clone in the rat brain aggregate tumor spheroid confrontation. This effect was not associated with significant growth inhibition. We conclude that expression of PKCalpha in glioma-derived cell lines appears to be central to glioma invasion in vitro.

Animals↗