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T Kristensen

Publications and source records attributed to T Kristensen.

At least 91 records · Page 5Linked to original sources
PubMed1991

Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA.

Human beta 2-glycoprotein (beta 2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature beta 2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the beta 2gpI cDNA. Upon isoelectric focusing, recombinant beta 2gpI obtained from expression of beta 2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as beta 2gpI isolated from plasma, and at least 5 polypeptides were visible.

Amino Acid Sequence↗
PubMed1991

Porcine SINEs: characterization and use in species-specific amplification.

A porcine repetitive DNA sequence has been isolated from an intron of the glucose phosphate isomerase gene. The copy number of this and related sequences was estimated to be approximately 10(5) copies per genome. The sequence possesses all the characteristics of short interspersed elements (SINEs) described in other mammals: The repeat is 300 bp in length, has an poly(A)stretch, and contains insertion duplication sites. Homology to seven other porcine sequences, which also have the characteristics of SINEs, has been demonstrated. Primer oligonucleotides, based on conserved regions in the SINE sequences, have been synthesized. Using these primers, PCR-mediated specific amplification of porcine sequences was demonstrated from pig x mouse and pig x hamster hybrid cell lines. Cloning and sequencing of some amplified porcine sequences verify that the sites of priming are SINE sequences.

Animals↗
PubMed1991

Monoclonal antibody treatment (anti-CD4 and anti-interleukin-2 receptor) combined with cyclosporin A has a positive but not simple dose-dependent effect on rat renal allograft survival.

The use of monoclonal antibodies (MoAbs) in experimental and clinical organ transplantation is of increasing interest since this treatment seems to offer an opportunity for specific immunomodulation. In a rat kidney allograft model, Cyclosporin A (CyA) treatment (12.5 mg/kg/d, day 0-14) was combined with murine anti-rat CD4 (MRC OX-38) and murine anti-rat IL-2R (MRC OX-39) MoAbs at doses of 100 or 300 micrograms/kg/d (day 0-7) and plasma concentrations of the murine MoAb were determined. In both groups receiving combined treatment with CyA and MoAb, graft survival was prolonged to an average of 65 days, compared to a graft survival of 9-10 days in non-treated recipients. Further, the data showed a beneficial effect of CyA + MoAb treatment versus CyA alone (graft survival 32 days). The threefold increased MoAb dose did not seem to improve graft survival or function. Treatment with OX-38 + OX-39 at a dose of 100 micrograms/kg/d each resulted in plasma levels of 280 ng/ml 14 days after transplantation. Corresponding values after the administration of 300 micrograms/kg/d were 1800 ng/ml in graft recipients as well as controls. These findings indicate that the effect of MoAbs in complex organ transplantation models is not simply dose dependent and that in vitro assays are of limited value in predicting the effect of a given MoAb when used in vivo. The determination of MoAb plasma levels, however, may be a useful tool in defining optimal MoAb administration and to monitor therapeutically effective plasma levels.

Animals↗
PubMed1991

Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes.

Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.

Base Sequence↗
PubMed1990

Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the alpha 2-macroglobulin receptor.

The human placental receptor (alpha 2MR) for alpha 2-macroglobulin-proteinase complexes contains 3 polypeptides of approx. 500 kDa, 85 kDa, and 40 kDa. N-terminal sequence analysis of the 500 kDa and 85 kDa polypeptides, analysis of a random selection of peptides convering 536 residues from these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the alpha- and beta-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119-4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769-1776). N-terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested that LRP is the functional receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) and in addition may have as yet unsettled functions in lipoprotein metabolism.

Amino Acid Sequence↗
PubMed1990

Expression of human alpha 2-macroglobulin cDNA in baby hamster kidney fibroblasts: secretion of high levels of active alpha 2-macroglobulin.

Human alpha 2-macroglobulin (alpha 2M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase, alpha 2M entraps the proteinase molecule in a reaction that involves large conformational changes in alpha 2M. We describe the molecular cloning of alpha 2M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human alpha 2M with a specific enzyme-linked immunosorbent assay. Human recombinant alpha 2M (r alpha 2M), secreted and purified from isolated transfected BHK cell lines, was structurally and functionally compared to alpha 2M purified from human serum. The results show that r alpha 2M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r alpha 2M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference. Moreover, r alpha 2M-trypsin complex bound to purified human placental alpha 2M receptor with an affinity indistinguishable from that of a complex formed from serum-derived alpha 2M and trypsin.

Animals↗
PubMed1990

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene.

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Molecular analysis of the murine C4b-binding protein gene. Chromosome assignment and partial gene organization.

Murine C4b-binding protein (C4BP) is a regulatory molecule in the classical pathway of complement. C4BP is composed predominantly of short consensus repeats (SCRs) approximately 60 amino acids in length, which contain a framework of conserved residues. The SCRs are found in many complement molecules and a growing number of noncomplement molecules as well and are a major structural feature of some of these molecules. To characterize the structure of the murine C4BP gene, a cosmid library constructed from Balb/c liver DNA was screened. Several nearly identical, overlapping clones were identified; however, none of the clones, alone or in combination, covered the entire C4BP gene. One clone (D26) was chosen for detailed analysis and found to contain all but the leader region, the first SCR, and the first half of the second SCR. The SCRs three through six were each encoded by single exons. Only the latter half of the second SCR was present on the clone, and it was encoded by a single exon, demonstrating that murine C4BP has a split SCR at the genomic level. Structural mapping of this portion of the gene demonstrates that the region extending from the second half of the second SCR through the nonrepeat and untranslated region spans approximately 12 kb; however, genomic Southern blot analysis suggests that the gene is between 20 and 30 kb in length. Analysis of the 3' genomic sequence demonstrates that this region of the gene has homology with SV-40 late (class II) RNA sequences. These sequences may play a role in 3' cleavage of the precursor RNA prior to polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

[Gene technology in clinical medicine--DNA sequencing].

There are two established procedures for sequencing cloned DNA fragments. In Maxam-Gilbert sequencing, or sequencing by chemical degradation, end-labeled DNA is treated with various chemicals which induce base specific chain breaks with a low frequency. In dideoxy sequencing, or sequencing by the Sanger procedure, a single-stranded DNA is used as a template for synthesis of a labeled complementary strand by a DNA polymerase. Addition of dideoxynucleoside triphosphates will induce base-specific chain termination. In both procedures the nucleotide sequence of the cloned DNA can be deduced after fractionation of the labeled products by polyacrylamide gel electrophoresis. As yet, radioactive labeling of the reaction products is most common, but fluorescence labeling and computer-assisted automated sequence interpretation has become as a powerful alternative during the last years. Further automation of the various processes involved in DNA sequencing will be necessary for the planned sequencing of large genomes, such as the Escherichia coli genome, the yeast genome, and the human genome.

Animals↗
PubMed1989

Immunotherapy with dog and cat extracts in children.

Hypersensitivity to dogs and cats causes asthma in many children. In this open controlled clinical study we wanted to establish whether immunotherapy can be recommended as a supplement to conventional medical therapy in dog- and cat-induced asthma in children. Twenty-seven children with mean age 10 7/12 years and with allergy to dog or cat according to anamnesis, skin prick test (SPT), specific IgE (RAST) and bronchial provocation test (BPT) entered the study. On the basis of age and bronchial sensitivity they were randomly allocated to either immunotherapy with dog or cat extract (active group, n = 14) or conventional medical therapy (control group, n = 13). Immunotherapy comprised subcutaneous injections with an alum-adsorbed depot extract (Alutard-SQ), and a maintenance dose of 100,000 SQ-U or the maximum tolerated dose. Before entering the study and after 9 months' therapy the patients were evaluated by BPT, SPT, RAST - and IgG4 analyses. The active group showed statistically significant change in bronchial tolerance (P less than 0.02), whereas no such change was found in the control group (P greater than 0.05). The change in skin sensitivity was significantly greater in the active group than in the control group (P less than 0.05), whereas no significant differences were found in RAST and IgG4. However, a significant (P = 0.05) increase in IgG4 was seen in the active but not in the control group. Frequency and degree of side effects in this study were acceptable.

Adolescent↗
PubMed1989

Postimmunization activity of oligoadenylate synthetase in peripheral blood lymphocytes from healthy individuals.

The effect of immunizing healthy individuals with either tetanus toxoid or yellow fever live attenuated vaccine was examined by measuring interferon (IFN)-dependent oligoadenylate synthetase (2-5A) activity. This enzyme converts ATP into oligonucleotides coupled together in 2'-5'diester bonds. The synthetized products possess among other effects growth inhibiting properties and stimulate a latent RNase, thus playing an important role in the defense against viral infections. Although 2-5A activity is known to increase following virus infections and perhaps therefore to reflect a previous IFN exposure, little is known about the ability of vaccines to activate 2-5A in healthy individuals. Controlled dosages of commercially available vaccine preparations were therefore administered to 17 healthy Danish volunteers. In one study, the effect of a primary stimulus, yellow fever, was tested. It was found that the 2-5A activity increased to reach a peak by 1,000% by day nine. In another study, the effect of a secondary stimulus or booster, Tetanus toxoid, was tested. The response to this antigen was a 40% decrease in 2-5A activity from day 1 to day 18. Thus, the 2-5A activity highly reflects the type of antigen used for immunization and possibly even whether the individuals previously had been exposed to the given antigen. As IFNs are very shortlived in vivo measuring 2-5A activity is a sensitive way of estimating changes in blood immune cells to exogenous antigens.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

T7 DNA polymerase in automated dideoxy sequencing.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.

Base Sequence↗