Inge Nilsen | Nofima - Academia.edu (original) (raw)

Papers by Inge Nilsen

Methods in …, Jan 1, 1995

This chapter describes a technique that aims at using blood directly in polymerase chain reaction... more This chapter describes a technique that aims at using blood directly in polymerase chain reaction (PCR) and minimizing sample size and manipulation, as well as the risk of contamination and exposition to health hazards. It is particularly suitable for the screening of large numbers of blood samples for the detection of specific DNA sequence alterations. Direct use of washed and boiled blood cells in the PCR reaction is a simple and reliable method for DNA amplification. The technique offers a tool for qualitative DNA studies and is feasible for the analysis of specific DNA sequences. Washed blood cells from 2–100μl of blood can be used in 50 or 100μl PCR reactions. The technique described in the chapter eliminates time-consuming DNA purification with the use of toxic chemicals and enzymes for protein digestion. The samples are handled in a single tube, reducing the risk for the contamination or intermixing of tubes. Exposure to health hazards is minimized and the expenses can be kept low by optimizing the concentrations of individual reactants in the PCR. Amplified DNA may be utilized in different analyses, such as restriction enzyme digestion, sequencing cloning, and probe blotting.

Journal of molecular …, Jan 1, 2008

Biochemical …, Jan 1, 1992

Journal of molecular …, Jan 1, 1996

By expressing a mutanttrpRgene in anEscherichia colistrain that istrpR−and has β-galactosidase ac... more By expressing a mutanttrpRgene in anEscherichia colistrain that istrpR−and has β-galactosidase activity fused to thetrppromotor/operator, thus putting the β-galactosidase activity under the control of the Trp repressor, we can determine quantitatively the relative repression activity of such mutant(s). We used this technique to analyse the biological consequences of substituting certain amino acid residues in only one of the two corepressor binding pockets. By combining two compatible plasmids in this strain, one expressing the mutant T44M and the other expressing only one substitution at a time at position 85, we analysed the repression activity of the resulting interactionsin vivo. This approach allowed us to engineer active dimer repressors made of two inactive or partially active monomers. Amino acid substitutions at position 85 with a positive or with an indole ring (W) appeared to complement T44M, while amino acids with a negative charge did not. Only L substitution at position 85 appeared to restore activity among the hydrophobic amino acids tested. Similar to the wild-type repressor activity, the successful mutant – mutant interactions wereL-tryptophan dependent.In vivoregulation by three knownL-tryptophan analogues demonstrated the same trend of regulation among the wild-type repressors and the active mutant – mutant combinations.

… and Physiology Part B: …, Jan 1, 2010

Atlantic salmon goose-type lysozyme (SalG) was previously shown to display features of cold-adapt... more Atlantic salmon goose-type lysozyme (SalG) was previously shown to display features of cold-adaptation as well as renaturation following heat treatment. In this study differential scanning calorimetry (DSC) was carried out to investigate unfolding and potential refolding, while X-ray crystallography was used to study structural factors contributing to the temperature-related characteristics. The recombinant SalG has a melting temperature (Tm) of 36.8 °C under thermal denaturation conditions and regains activity after returning to permissive (low) temperature. Furthermore, refolding is dramatically reduced in solutions with high SalG concentrations, coupled with significant protein precipitation. The structural features of SalG closely resemble those of other g-type lysozymes. However, the N-terminal region of SalG is less anchored to the rest of the molecule due to the absence of disulphide bonds, thus, contributing significantly to the low Tm of SalG. The absence of disulphide bonds and the distribution of salt bridges may at the same time ease refolding leading to renaturation.

Comparative …, Jan 1, 2001

PCR in Neuroscience, Jan 1, 1995

Comparative Biochemistry and Physiology Part C: …, Jan 1, 2007

Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic s... more Glutathione S-transferase from the digestive gland of the cold-adapted marine bivalve Icelandic scallop was purified to apparent homogeneity by single GSTrap chromatography. The enzyme appeared to be a homodimer with subunit Mr 22,000 having an optimum catalytic activity at pH 6.5–7. Enzymatic analysis of scallop GST using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione resulted in apparent values for KmGST and KmCDNB of 0.3 mM and 0.4 mM, respectively. The scallop GST lost activity faster than porcine GST when exposed to increased temperatures, but both enzymes needed 10 min incubation at 60 °C for complete inactivation. A partial coding sequence was identified in cDNA synthesised from digestive gland mRNA. Comparison to known sequences indicates that the gene product is a glutathione S-transferase, and the predicted Icelandic scallop GST protein scores 40% sequence identity and 60% sequence similarity to mu-class proteins.

…, Jan 1, 1994

The ability of acetaldehyde, a respiratory carcinogen present in tobacco smoke and automotive emi... more The ability of acetaldehyde, a respiratory carcinogen present in tobacco smoke and automotive emissions, to affect cell viability, thiol status and intracellular Ca2+ levels and to cause DNA damage and mutations has been studied using cultured human cells. Within a concentration range of 3-100 mM, a 1 h exposure to acetaldehyde decreases colony survival and inhibits uptake of the vital dye neutral red in bronchial epithelial cells. Acetaldehyde also causes both DNA interstrand cross-links and DNA protein cross-links whereas no DNA single strand breaks are detected. The cellular content of glutathione is also decreased by acetaldehyde, albeit, without concomitant changes in the glutathione redox status or in the content of protein thiols. Transient or sustained increases in cytosolic Ca2+ occur within minutes following exposure of cells to acetaldehyde. Moreover, acetaldehyde significantly decreases the activity of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Finally, a 5 h exposure to acetaldehyde causes significant levels of 6-thioguanine resistance mutations in an established mutagenesis model involving skin fibroblasts. The results indicate that mM concentrations of acetaldehyde cause a wide range of cytopathic effects associated with multistep carcinogenesis. The fact that acetaldehyde, in relation to its cytotoxicity, causes comparatively higher genotoxicity and inhibits DNA repair more readily than other major aldehydes in tobacco smoke and automotive emissions is discussed.

Biochemical Pharmacology, Jan 1, 1992

Alteration in gene expression of the proto-oncogene c-myc in HL-60 cells is associated with diffe... more Alteration in gene expression of the proto-oncogene c-myc in HL-60 cells is associated with differentiation of these cells. We have studied the steady state levels of c-myc transcripts, the levels of transmethylation metabolites S-adenosylmethionine and S-adenosyl-homocysteine and the methylation pattern of the c-myc gene after treatment of HL-60 cells with the transmethylation inhibitor and granulocytic inducer, 3-deaza-(±)-aristeromycin. A transient increase in c-myc RNA levels after 45 min of drug exposure was observed which was accompanied by changes in the ratio of transmethylation metabolites in both whole cells and nuclei. The changes in transmethylation metabolites in whole cells, although compatible with levels frequently associated with hypomethylation of cellular components, caused no changes in methylation of c-myc DNA sequences of the HL-60 cells as detected by HpaII or MspI digestion and Southern blotting.

… and Physiology Part B: Biochemistry and …, Jan 1, 2001

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp ... more Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.

Experientia, Jan 1, 2003

Genome clones and expressed sequence tags (ESTs) from the ascidian Ciona intestinalis and from th... more Genome clones and expressed sequence tags (ESTs) from the ascidian Ciona intestinalis and from the larvacean Oikopleura dioica were analysed for the presence of lysozyme-encoding genes. Two genes were found to potentially code for goose-type lysozymes in Oikopleura, while three or possibly more g-type proteins form the lysozyme complement of C. intestinalis, and at least one of these genes from each species is expressed based on EST data. No genes for chicken- or invertebrate-type lysozymes were found in either urochordate species. Consistent with this finding, extracts of Oikopleura animals possessed hydrolysing activity on bacterial cell walls, and this activity was not inhibited in the presence of a known inhibitor of chicken-type lysozyme. A wide range of isoelectric points for the predicted lysozymes from Ciona (pI 4.4, 6.4 and 9.9) and from Oikopleura (pI 5.0 and 8.0) suggests tissue-specific adaptations as well as specific functional roles of the lysozymes. Comparisons of gene structures, encoded sequences, cysteine residue content and their positions in the proteins indicate that the g-type lysozymes of Ciona intestinalis are more closely related to those of vertebrates than are the g-type lysozymes of Oikopleura. Multiple genes from each species may result from separate and lineage-specific duplications followed by functional specialisation.

Comparative Biochemistry and …, Jan 1, 2003

Initial analyses of lysozyme activities in individual blue mussels Mytilus edulis indicated varia... more Initial analyses of lysozyme activities in individual blue mussels Mytilus edulis indicated variations in features of activity from the crystalline style to the remaining body parts (the soft body). Two separate larger scale lysozyme isolations were performed employing extracts from 1000 styles and 50 soft bodies, respectively. The soft body origin contained one, or one major, lysozyme that was purified to homogeneity. This 13 kDa protein, designated bm-lysozyme, was sequence-analysed and found to represent the product of a recently published invertebrate-type lysozyme gene from M. edulis. Three additional lysozymes were isolated from the style extract and one of them was fully purified. All four lysozymes showed different profiles of enzymatic features such as responses to pH, ionic strengths and divalent cations. From the results and the profound differences demonstrated we believe that the observed multiple forms of lysozyme activities in blue mussel reflect multiple genes instead of individual lysozyme variants and that the lysozymes serve different functions in the blue mussel.

Journal of …, Jan 1, 1996

Gene, Jan 1, 2001

In a recent publication we reported the protein purification, characterization, and the gene isol... more In a recent publication we reported the protein purification, characterization, and the gene isolation of a cDNA encoding the antibacterial cold-active lysozyme-like protein chlamysin from the marine bivalve Chlamys islandica. A 4.2 kb genomic chlamysin gene has now been amplified and sequence-analyzed. By comparison to the cDNA sequence and its translation product, the coding region was found separated in four exons of 38-252 bp. The introns range in size from 0.8 to 1.5 kb, and have traditional spliceosomal intron 5′-GT donor and 3′-AG acceptor sites for splicing. Two of the introns contain multiple copies of three sequence motifs not found repeated in other published genes. The over-all gene organization of chlamysin resembles chicken-type (c-type) lysozyme genes in vertebrates, but is different from the three-exon structure in invertebrate c-type lysozyme genes. A phylogenetic analysis of invertebrate-type (i-type) and c-type lysozyme proteins demonstrated a large evolutionary distance between the i-type and the c-type enzyme classes. Exons of the i-type genes are not equally organized according to their homolog protein domains.

FEBS letters, Jan 1, 1999

An antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine biv... more An antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5–6.2) and at low temperature (4–35°C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70°C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.