PubMed Health⌕ Search

Biomedical subjects

F Larsen

Publications and source records attributed to F Larsen.

At least 55 records · Page 3Linked to original sources
PubMed1997

Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

Chromatography, Affinity↗
PubMed1995

Polyadenylic-polyuridylic acid enhances the natural cell-mediated cytotoxicity in patients with breast cancer undergoing mastectomy.

BACKGROUND: Surgical procedures suppress host antitumor defense mechanisms, which may increase the risk of metastatic tumor dissemination. We have evaluated the effects of the biologic response modifier polyadenylic-polyuridylic acid (PAPU) on natural cytotoxicity in patients with breast cancer undergoing operation. METHODS: PAPU (150 mg) or placebo was given intravenously during the perioperative period (preoperative, days -1 and 0; postoperative, days 1, 3, 5, 7, and 14). The function (chromium release assay) and number (flow cytometry) of natural killer (NK) cells were measured before operation (days -2 and -1), on the day of operation (day 0), and after operation (days 1, 2, 4, 6, and 18). RESULTS: Surgical procedures suppressed NK cell cytotoxicity in the placebo group on postoperative days 1 (p < 0.001), 4, 6, and 18 (p < 0.05), whereas inhibition on postoperative day 2 failed to reach significance. PAPU abolished this immunosuppression after operation. The NK cell activity was elevated when compared with the control group; it was significant (p < 0.05) on postoperative days 1, 2, 4, 6, and 18. Surgical procedures also reduced circulating NK cell numbers during the first postoperative week in the placebo group; the decrease was statistically significant on day 4. The decrease in NK cell numbers in the PAPU group was insignificant. CONCLUSIONS: PAPU prevented the decrease in the circulating number and cytotoxic activity of NK cells that occurred after operation and enhanced NK cell cytotoxicity. This may have important implications for patients with cancer undergoing major operation.

Adult↗
PubMed1995

Physical linkage of the gene cluster containing the LCAT gene to the DNA marker D16S124 at human chromosome region 16q22.1.

Pulsed-field gel electrophoresis has been used to construct a long-range restriction map spanning more than 900 kb in the q22.1 region of human chromosome 16. The gene cluster containing the lecithin:cholesterol acyl transferase (LCAT) gene is located less than 480 kb from the anonymous DNA marker D16S124 in this map. The results suggest three putative CpG islands within 125 kb, in addition to the island previously shown to be located within the gene cluster. This implies a clustering of both genes and CpG islands in this chromosomal region.

Chromosome Mapping↗
PubMed1994

Evaluation of the exon predictions of the GRAIL software.

Identification of transcribed sequences within genomic regions has been a major rate-limiting step in the pursuit of genes. We have tested the ability of GRAIL 1 and GRAIL 2 to locate coding exons in the 41 longest human genomic sequences available in release 38 of the EMBL database. Of the total annotated exons GRAIL 1 correctly identified 68% of coding exons. GRAIL 2 correctly identified 71%. Of the 686 predictions made by GRAIL 1, 46.4% did not correspond to an annotated exon. The corresponding figures for GRAIL 2 were 556 and 30.5%. The GRAIL 2 software predicted as excellent a significantly higher proportion of the coding exons than did GRAIL 1, and also gave a significantly lower figure for false predictions. GRAIL 2 thus represents a significant improvement over GRAIL 1.

Exons↗
PubMed1993

A tight cluster of five unrelated human genes on chromosome 16q22.1.

A locus on human chromosome 16q22.1 contains at least five tightly clustered genes which are unrelated by sequence homology and apparently unrelated by function. The genes for a putative proteasome subunit (MECL 1), a chymotrypsin-like protease (CTRL), a protein serine kinase (PSKH1), the previously cloned lecithin:cholesterol acyl transferase (LCAT) and a protein of unknown function are found within 40 kb of genomic DNA. Exons from the former four genes are located within a 12 kb region including a CpG island associated with the putative proteasome gene. Three of the genes are widely expressed, whereas the genes for the protease and LCAT are highly tissue specific. The distance between the transcriptional units of the gene upstream of LCAT and LCAT is only 199 bp. Alternative polyadenylation of the protease transcripts creates a transcription unit which overlaps with the oppositely oriented kinase gene. The selective advantage of this unusual gene clustering may involve transcriptional interference(s) and coregulatory events not yet understood. Given the current estimate of about 100,000 genes in the human genome, our findings support the notion that genes are not evenly distributed.

Amino Acid Sequence↗
PubMed1993

A methylated CpG island 3' in the apolipoprotein-E gene does not repress its transcription.

CpG islands are always associated with the 5' end of housekeeping genes, covering their promoters and transcription start sites. CpG islands associated with genes of limited expression are less uniformly localized; the genes for apolipoprotein-E and -AI contain CpG islands corresponding to their last exons. As expected, the CpG island in the apo-AI gene is unmethylated in DNA from all tissues analyzed, expressing as well as non-expressing apolipoprotein-AI. In contrast, the apo-E CpG island is methylated in DNA from all tissues analyzed except sperm. The apo-E gene is transcribed in many tissues and is not repressed by this methylation. This establishes a functional difference between 5' and 3' CpG islands, because methylation of the former invariably leads to transcriptional repression. A similar methylation pattern was seen in the rat apo-E gene, which implies that this pattern probably was established before the divergence of rodents and primates. The numerous human apo-E alleles resulting from CpG to TpG/CpA mutations in the CpG island (i.e. deamination of methylated cytosine to thymine) suggest that this island is less protected from methylation in germ line than typical CpG islands.

Alleles↗
PubMed1993

Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase.

Glutamic acid decarboxylase autoantibodies may aid in rapid screening strategies predicting IDDM before clinical onset. Rat islets contain GAD65 and GAD67 autoantibody targets, but human islets express only GAD65, now confirmed by direct immunoprecipitation from radiolabeled rat and human islets. Because human IDDM involves beta-cell-specific autoimmunity, we tested 190 new IDDM patients and 51 healthy control subjects for antibodies to recombinant human islet GAD65, rat islet GAD67, or human insulinoma/cerebellum GAD67, each expressed separately in hamster fibroblasts. By using immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometric fluorogram scanning, 132 of 190 (70%) of new IDDM patients had GAD65 autoantibodies, whereas only 17 of 190 (9%) had antibodies to rat GAD67 (P < 0.001). Of healthy control subjects, 2 of 51 (3.9%) and 1 of 51 (1.9%) had antibodies to GAD65 and GAD67, respectively. All 17 GAD67 antibody-positive patients also had GAD65 antibodies; 14 of 17 with greater GAD65 than GAD67 index. Control studies showed comparable reactivity between recombinant rat and human GAD67 and between different subcellular preparations of recombinant GAD67 of either species. In conclusion, only GAD65 is expressed in human islets, the autoantibody response is primarily to this isoform, and GAD67 antibodies add little to IDDM detection.

Adolescent↗
PubMed1992

Tissue-specific methylation of a CpG island in transgenic mice.

Clustering of CpG dinucleotides in CpG-rich islands is a characteristic feature of mammalian genomes. Such CpG islands are frequently associated with genes and usually hypomethylated, regardless of the gene activity. This is the case for the CpG island of the murine Thy-1 gene. A transgenic line containing multiple copies of a truncated, concatemeric CpG island from the Thy-1.1 allele (Thy-1.2 background) showed that a stable fraction (approx. 0.20) became fully methylated in somatic tissues of homozygous mice with respect to testable restriction sites, while the remaining copies were methylation-free, i.e., this methylation appears to be an 'all-or-none' phenomenon. DNA from extraembryonic tissues (placenta and yolk sac) and epididymal sperm showed, however, an even higher degree of methylation in two distinct patterns. In the extraembryonic tissue, partial methylation of each copy was seen, whereas in sperm a high degree of 'all-or-none' methylation (greater than 0.35) was observed.

Animals↗
PubMed1992

CpG islands as gene markers in the human genome.

CpG islands are short, dispersed regions of unmethylated DNA with a high frequency of CpG dinucleotides relative to the bulk genome. We have analyzed 375 genes and 58 pseudogenes from the human entries in the EMBL Database for the presence of CpG islands. All 240 islands identified are associated with genes, and almost all cover at least a part of one exon; i.e., they are useful landmarks in the genome for identifying genes. More than half of the genes analyzed were associated with islands. All housekeeping and widely expressed genes have a CpG island covering the transcription start, whereas 40% of the genes with a tissue-specific or limited expression are associated with islands. In this latter group of genes, the position of the islands was not biased toward the 5' end of the transcription unit.

Dinucleoside Phosphates↗
PubMed1992

Choice of enzymes for mapping based on CpG islands in the human genome.

The frequencies of sites for rare-cutting restriction enzymes in 2.9 million bp of human genomic DNA sequence in the EMBL database have been determined and compared with the expected frequencies. Rare cutters can be divided into four groups based on certain features of their recognition sites. Mlu, I, Nru I, Spl I, and Pvu I are predicted to cleave genomic DNA most infrequently, which is borne out by the fragment lengths observed for Mlu I and Nru I. Thus, these four enzymes are ideal for making long-range maps based on pulsed-field electrophoresis. Other enzymes like Not I are useful for making more detailed maps. Finer maps for identification of CpG islands and associated genes should involve several rare cutters including Eag I, Sac II and Bss HII. A cluster of sites for at least two such enzymes is a good indicator of a CpG island, and 78% of the island-associated genes can be located in this way.

Base Composition↗
PubMed1992

Multiple oral administration of a ketoprofen-dextran ester prodrug in pigs: assessment of gastrointestinal bioavailability by deconvolution.

Deconvolution has been applied to estimate the in vivo dissolution/release process of ketoprofen from a ketoprofen-dextran ester prodrug in pigs. The prodrug was given to three pigs at intervals of 12 hr and in seven doses corresponding to 4 mg ketoprofen/kg body weight. Frequent blood sampling was carried out at the first, third, and seventh intervals. Plasma steady-state concentrations of ketoprofen following the prodrug administration were between 2 and 4 micrograms/ml. The reference consisted of a single p.o. dose of parent ketoprofen (4 mg/kg body weight). For each pig the response following the multiple dosing was deconvolved with the reference response using an algebraic deconvolution procedure adopted from the literature. The obtained cumulated in vivo dissolution/release profiles revealed similar release rates for the three pigs and similar extents of release (59, 70, and 65%). The mean in vivo dissolution/release times (MDT) were calculated to be 5.4, 6.1, and 5.7 hr, respectively. In conclusion, following administration of the dextran prodrug the plasma concentration curves and the dissolution/release profiles are uniform, with small interindividual variations.

Administration, Oral↗
PubMed1992

Late embryogenesis-abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress.

The late embryogenesis-abundant (Lea) genes, which are suggested to act as desiccation protectants during seed desiccation and in water-stressed seedlings, can be induced by abscisic acid (ABA) and various kinds of water-related stress. Using cotton Lea cDNAs as probes it was found that several of the Lea genes are conserved at the mRNA level in dicots and monocots. By screening a barley cDNA library with a cotton Lea D19 cDNA a family of three members was isolated. The putative B19 proteins have strong similarities to the Em protein in wheat and to LEA proteins from several dicots. However, the middle part of the B19 proteins consists of a 20-amino acid motif repeated three and four times in B19.3 and B19.4, respectively, but only once in B19.1. The gene products are strongly hydrophilic, the internal 20-amino acid motif being the most hydrophilic part. This motif is found once in cotton Lea D19 but is repeated twice in cotton Lea D132, indicating that the repeats are universal among monocot and dicot B19-like genes. The B19 genes are regulated similarly during embryo development, but to very different levels. In contrast, they are differentially regulated by ABA and various types of osmotic stress. In immature embryos all three genes are responsive to ABA and mannitol. However, B19.1 is also responsive to salt. Cold stress does not induce B19 mRNAs; only a stabilization of the transcript levels is seen. These results suggest that the responses to salt stress and exogenous ABA operate through different pathways.

Abscisic Acid↗