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M Christensen

Publications and source records attributed to M Christensen.

At least 19 recordsLinked to original sources
PubMed1992

Generation of Gla-domainless FVIIa by cathepsin G-mediated cleavage.

Coagulation factor VII contains ten gamma-carboxyglutamic acid residues in the N-terminal region (Gla-domain) which are essential for the hemostatic function of FVII. The present study shows that granulocyte cathepsin G degrades the Gla-domain of FVIIa in vitro. Characterization of the truncated FVIIa by SDS-PAGE and N-terminal amino acid sequence analysis revealed that cleavage had occurred between Tyr-44 and Ser-45 and that further cleavage was only obtained on extensive cathepsin G exposure. Cleavage of vitamin K-dependent coagulation factors by cathepsin G may play a role in vivo, and it offers a convenient way of obtaining proteins deprived of their Gla-domain for functional and structural studies.

1-Carboxyglutamic Acid↗
PubMed1992

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2-13.3.

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene (LIG1) is localized to 19q13.2-13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.

Blotting, Southern↗
PubMed1992

Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting.

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).

Chromosomes, Human, Pair 19↗
PubMed1992

Altered expression of ABO (H) carbohydrate antigens is seen in pleomorphic adenomas.

Cell surface carbohydrate antigens show changes in relation to differentiation, maturation and malignant transformation. The expression of type 2 chain ABH carbohydrate structures of the ABO histo-blood group system was investigated in 28 pleomorphic adenomas (PA) and normal parotid glands in order to study possible changes in the glycosylation pattern. The distribution of carbohydrate structures was investigated by immunohistological stainings of formalin-fixed paraffin-embedded material using monoclonal antibodies (MAbs) with well-defined specificity. A strong interindividual variation was found in the normal tissue as well as in the tumors. In normal tissue, acinus and duct cells all expressed elongated carbohydrate structures. The yoepithelial cells did not stain with any of the MAbs investigated. In the PAs, staining was seen in the ductular structures and myoepithelial cells. In contrast to normal tissue, the tumors expressed the short precursor molecule sialylated N-acetyllactosamine. Furthermore, the PAs showed loss of H and A antigens, and a reduced expression of Le(y) compared to normal tissue. The ductular structures as well as the modified myoepithelial cells expressed binary N-acetyllactosamine, which in the normal tissue could only be found in the striated and excretory ducts. Thus our study has shown that aberrant glycosylation is not only a feature of malignant neoplasms but also occurs in pleomorphic adenomas.

ABO Blood-Group System↗
PubMed1992

Variability in teniposide plasma protein binding is correlated with serum albumin concentrations.

Teniposide is a widely used anticancer drug that is extensively bound to plasma proteins (greater than 95%). We evaluated the drug's plasma protein binding in nine patients with acute lymphocytic leukemia who were in their first complete remission, and in a second group of nine patients at the time of relapse and subsequently after achieving another complete remission. Plasma protein binding was assessed by equilibrium dialysis, with direct high-performance liquid chromatographic measurement of total and free teniposide. The mean unbound fraction was 0.44% (0.21-0.88%) in the plasma of patients in first remission. It was significantly higher in patients at the time of relapse (mean = 0.86%; range 0.68-1.08%) and after achieving another complete remission (mean = 1.25%; range 0.51-2.11%). Serum albumin values were significantly lower at the time of relapse (mean = 4.6 vs 4.0 mg/dl; p less than 0.014), and decreased further during intensive postremission therapy containing L-asparaginase (mean = 3.2; p less than 0.05). For all 18 patients, a significant negative correlation (r2 = 0.667; p less than 0.001) was found between serum albumin and unbound teniposide, with low albumin being associated with higher unbound fraction. Such patients have higher systemic exposure to unbound (presumably active) teniposide at any given total plasma concentration of the agent.

Adolescent↗
PubMed1992

Halothane 2% for caesarean section.

During the induction-delivery interval for Caesarean section delivery, 2% halothane in pure oxygen was administered. The technique was compared to a 0.5% halothane in 50% oxygen/50% nitrous oxide anaesthesia. When a continuous infusion of oxytocin was administered, no excessive haemorrhage was seen. No maternal reminiscence was seen using 2% halothane, but awareness was recorded using 0.5% halothane in 50% nitrous oxide in 15% of the mothers. When there were no signs of preoperative fetal distress, the neonates were unaffected by the halothane concentration provided the induction-delivery interval was short. In cases of fetal distress, the administration of 2% halothane further aggravated the condition of the neonates, as indicated by lowered 1-min Apgar scores, umbilical oxygen tensions, pH and base excess values.

Adult↗
PubMed1991

Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine.

Factor VII is a multidomain, vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol. Chem. 264, 20320-20325). In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation. Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis. Using a combined strategy of amino acid sequencing, carbohydrate and amino acid composition analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII. Approximately equal amounts of the three glycan structures were observed in plasma factor VII, whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated. In addition to the O-linked glycan structures observed at serine 52, a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII. Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative. Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60. In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII, we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue. Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay. The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa. In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa. These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor, or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗
PubMed1991

Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescence in situ hybridization.

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequence as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.

Animals↗
PubMed1991

Sequences required for coordinate induction of adeno-associated virus p19 and p40 promoters by Rep protein.

A series of contiguous 30-bp deletions were introduced into the regions upstream of the p19 and p40 promoters of adeno-associated virus (AAV), and the effects of these deletions on induction of AAV transcription by the rep gene products was evaluated. A novel complementation system was devised for supplying wild-type Rep protein when mutations disrupted the trans activation activity of the Rep protein. Transcription from the p40 promoter was eliminated upon deletion of the TATA sequence located between -4 and -33 from the cap site. Deletions which removed sequences from -34 to -123 bp from the p40 mRNA start site substantially reduced Rep induction of p40 transcription. p19 transcription was also undetectable when the p19 TATA sequence between -4 and -33 was deleted. In contrast to the p40 region, two types of cis-active sequences were found associated with the p19 promoter. Sequences between -4 and -63 bp relative to the p19 cap site were essential for Rep induction only from the p19 promoter. Deletions between -94 and -153 bp relative to the p19 cap site reduced Rep induction of both the p19 and p40 promoters coordinately. These two noncontiguous regions were separated by a 30-bp sequence that was not essential for transcription control. Further deletion analysis delineated a second cis-active element, associated with the p5 promoter (AAV nucleotides 191 to 320), which was also necessary for coordinate Rep activation of both the p19 and p40 promoters. Finally, the dependence of p40 transcription on the Rep-responsive elements within the p5 and p19 regions could be overcome by the presence of the AAV terminal repeats, suggesting that the terminal repeats contained redundant Rep-responsive elements. These results implied an interdependence in cis between the three AAV promoters and suggested a novel mechanism for coordinate regulation of gene expression in response to the trans-activating Rep protein. Coordinate induction appeared to be the result of a simultaneous interaction between the Rep protein and sequence elements associated with two or all three of the AAV promoters.

Adenoviruses, Human↗
PubMed1990

Marketers hone their skills to reach target markets.

New challenges lie ahead for health care marketers. Marketers must communicate with several important groups: their own administration, the general public, employers, hospital employees, and, of course, physicians. And as budgets tighten, the methods used to communicate must become more creative and more efficient.

Attitude of Health Personnel↗
PubMed1989

Revised amino acid sequence of pancreatic spasmolytic polypeptide exhibits greater similarity with an inducible pS2 peptide found in a human breast cancer cell line.

The published amino acid sequence of pancreatic spasmolytic polypeptide (Thim, L., Thomsen, J., Christensen, M. and Jørgensen, K.H. et al. (1985) Biochim. Biophys. Acta 827, 410-418) has been checked by a combination of mass spectroscopy and Edman degradation. The pyroglutamyl blocking group was positively identified, and residue assignments at four positions were corrected: Lys48 (not Ser), Ser63 (not Lys), Cys68 (not Ser) and Ser74 (not Cys). The revised sequence exhibits greater similarity with pS2 peptide, a 60 residue polypeptide which is induced by oestrogen in the human breast cancer cell line MCF-7 and found in malignant but not in non-malignant breast tissue.

Amino Acid Sequence↗
PubMed1989

Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Amino Acids↗
PubMed1989

Effects of selenium on toxicity and ultrastructural localization of mercury in cultured murine macrophages.

The effects of selenium on cellular toxicity and histochemical distribution of mercury were examined in a cell culture system of mouse peritoneal macrophages. Selenium protected against the toxicity of mercury in cultures exposed to 4 microM of mercuric chloride. Selenomethionine caused a significant increase in cell survival throughout the experiments, while sodium selenite delayed the toxicity of mercury for a while, after which selenite itself had a toxic effect. The amount of mercury visualized by autometallography was increased in macrophage cultures pre-exposed to sodium selenite or selenomethionine. The additional mercury made visible by this histochemical demonstration was located in the cytoplasm as well as in the lysosomes.

Animals↗