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Biomedical subjects

J Nilsson

Publications and source records attributed to J Nilsson.

At least 73 records · Page 4Linked to original sources

Matrilinear phylogeography of Atlantic salmon (Salmo salar L.) in Europe and postglacial colonization of the Baltic Sea area.

Sixty-four samples from 46 salmon populations totalling 2369 specimens were used for polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial ND1 region. The final analyses included 3095 specimens from 60 populations in Northern Europe. A subsample was analysed by RFLP of ND3/4/5/6. Representative RFLP haplotypes from different parts of the distribution area were sequenced and the phylogeny of European haplotypes and their relations to the North American lineage was described. The four common European haplotypes derive from the ancestral ND1-BBBA (rooting the European clade to the North American) by one-step substitutions: AAAA < AABA < BBBA > BBBB. The Swedish west-coast populations differ from the geographically close southern Baltic, indicating absence of inward and limited outward gene flow through the Danish straits during the last 8000 years. Within the Baltic Sea, only three ND1 haplotypes were detected and there was no variation for ND3/4/5/6. In the whole southern Baltic and in lakes Vänern, Ladoga and Onega the haplotype AABA dominated. Proposed postglacial colonization routes to the Baltic Sea are discussed in relation to the haplotype distribution pattern.

Animals↗

Identification of the origin of an Atlantic salmon (Salmo salar L.) population in a recently recolonized river in the Baltic Sea.

The founder event in a recently recolonized salmon population in the Baltic Sea (Gulf of Finland) was investigated. To identify the origin of the founders, four wild populations and two hatchery stocks were analysed using six microsatellite loci. The results of assignment tests and factorial correspondence analysis suggest that the initial recolonizers of the river Selja originated from the geographically nearest (7 km) wild population (river Kunda) but as the result of stocking activities, interbreeding between recolonizers and hatchery individuals has occurred in subsequent years. Although the hatchery releases are outnumbering the wild salmon recruitment in the Baltic Sea at present, our results suggest that the native populations may still have an important role in colonization processes of the former salmon rivers.

Animals↗

Generalization of the normalized dose-response gradient to non-uniform dose delivery.

A generalization of the standard dose-response gradient to arbitrarily heterogeneous dose distributions has been developed. The generalized dose-response gradient is the scalar product of the vector representing the dose distribution and the gradient of the dose-response relation with respect to that dose vector. It is shown that, for a tumor, the individual gamma-values for each portion of the tumor divided by the corresponding local tumor control probability should be added to get the total value for the heterogeneously irradiated tumor. This corresponds to summing up the contributions of all tumor volumes so that the total value of the gradient is related to the logarithm of the total tumor clonogen number. General expressions are also derived for the change in the dose-response relation as a function of a change in the delivered dose distribution.

Dose Fractionation, Radiation↗

Biologically optimized radiation therapy.

Advanced treatment optimization is possible using quantitative radiobiological dose response models. Although all present models are necessarily linked to a certain degree of uncertainty, this will only have a small influence on the relative shape of the resultant optimal dose distribution. However the exact dose level should perferably be determined clinically by dose escalation with the optimized dose plan as a control arm. It is shown that a large part of the biological effect of high linear energy transfer radiation is due to the spectrum of low-energy delta-electrons that can generate dense clusters of complex DNA damage. Such electrons are efficiently generated by low-energy photons or densely ionizing ion beams and to a considerably smaller degree by high-energy electrons, photons and protons. A new analytical expression is developed for the effective radiation resistance of heterogeneous tumors, making it possible to approximate the response of such tumors by the effective clonogen number N0,eff and the effective D0 value D0,eff. It is shown that a relatively small number of resistant tumor cells may well be sufficient to dominate the response of hypoxic or otherwise heterogeneous tumors. Finally, several examples are given of intensity-modulated dose distributions generated by multiple radiation modalities, the total effect of which is biologically optimized.

Cell Survival↗

Endoscopic surveillance of columnar-lined esophagus: frequency of intestinal metaplasia detection and impact of antireflux surgery.

OBJECTIVE: To quantify the occurrence of intestinal metaplasia in columnar-lined esophagus (CLE) during endoscopic surveillance and to evaluate the impact of antireflux surgery on the development of intestinal metaplasia. SUMMARY BACKGROUND DATA: The malignant potential in segments of CLE is mainly restricted to those containing intestinal metaplasia. Patients with segments of CLE in which no intestinal metaplasia can be detected are rarely enrolled in a surveillance program but may still be at increased risk of developing esophageal adenocarcinoma because intestinal metaplasia may be missed or may develop with time. METHODS: The occurrence of intestinal metaplasia on biopsy samples was determined on repeated endoscopies in 177 patients enrolled in a surveillance program for CLE. The incidence of intestinal metaplasia in patients with no evidence of intestinal metaplasia on the two first endoscopies was evaluated on the subsequent endoscopies and compared in patients with medically and surgically treated gastroesophageal reflux disease. RESULTS: Intestinal metaplasia was found in 53% of the patients (94/177) on their first surveillance endoscopy and was more prevalent in long segments of CLE. The prevalence of intestinal metaplasia increased markedly with increasing number of surveillance endoscopies. Intestinal metaplasia tended to be detected early in patients with long segments of CLE; in patients with shorter segments, intestinal metaplasia was also detected late in the course of endoscopic surveillance. Patients with surgically treated reflux disease were 10.3 times less likely to develop intestinal metaplasia compared with a group receiving standard medical therapy. CONCLUSION: Biopsy samples from a single endoscopy, despite an adequate biopsy protocol, are insufficient to rule out the presence of intestinal metaplasia. Patients in whom biopsy specimens from a segment of CLE show no intestinal metaplasia have a significant risk of having undetected intestinal metaplasia or of developing intestinal metaplasia with time. Sampling error is probably the reason for the absence of intestinal metaplasia in segments of CLE longer than 4 cm, whereas development of intestinal metaplasia is common in patients with shorter segments of CLE. Antireflux surgery protects against the development of intestinal metaplasia, possibly by better control of reflux of gastric contents.

Aged↗

CRM1 mediates the export of ADAR1 through a nuclear export signal within the Z-DNA binding domain.

RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

Active Transport, Cell Nucleus↗

Increased rate of apoptosis in intimal arterial smooth muscle cells through endogenous activation of TNF receptors.

Intimal proliferation of smooth muscle cells (SMCs) is a key event in the vascular response to injury, including the early stages of atherosclerosis and restenosis after angioplasty. Tumor necrosis factor-alpha (TNF-alpha) has been reported to stimulate growth of cultured human SMCs, but activation of TNF receptors is also known to induce cell death by apoptosis. We report here that SMCs isolated from the neointima of injured rat aortas are characterized by increased expression of TNF-alpha in response to interleukin-1beta and gamma-interferon compared with medial SMCs. Basal and serum-stimulated DNA synthesis was higher in intimal than in medial SMCs. In contrast to previous findings on human SMCs, exposure to interleukin-1beta/gamma-interferon or TNF-alpha did not affect the growth of rat medial SMCs, inhibited DNA synthesis, and decreased cell numbers in cultures of intimal SMCs. Incubation of intimal SMCs with these cytokines also resulted in induction of terminal dUTP nick end-labeling positivity and caspase-3 expression, suggesting cell death by apoptosis, whereas medial cells were markedly less sensitive in this respect. Cytokine-induced apoptosis in intimal cells was effectively inhibited by treatment with antibodies against TNF receptors. These findings suggest that endogenous activation of TNF receptors may represent a way to limit accumulation of SMCs in injured arteries. This mechanism may also be important in SMC death in advanced atherosclerotic plaques.

Animals↗

Protein chips based on recombinant antibody fragments: a highly sensitive approach as detected by mass spectrometry.

With the human genome in a first sequence draft and several other genomes being finished this year, the existing information gap between genomics and proteomics is becoming increasingly evident. The analysis of the proteome is, however, much more complicated because the synthesis and structural requirements of functional proteins are different from the easily handled oligonucleotides, for which a first analytical breakthrough already has come in the use of DNA chips. In comparison with the DNA microarrays, the protein arrays, or protein chips, offer the distinct possibility of developing a rapid global analysis of the entire proteome. Thus, the concept of comparing proteomic maps of healthy and diseased cells may allow us to understand cell signaling and metabolic pathways and will form a novel base for pharmaceutical companies to develop future therapeutics much more rapidly. This report demonstrates the possibilities of designing protein chips based on specially constructed, small recombinant antibody fragments using nano-structure surfaces with biocompatible characteristics, resulting in sensitive detection in the 600-amol range. The assay readout allows the determination of single or multiple antigen-antibody interactions. Mass identity of the antigens, currently with a resolution of 8000, enables the detection of structural modifications of single proteins.

Antibodies↗

Involvement of p38-mitogen-activated protein kinase in Staphylococcus aureus-induced neutrophil apoptosis.

Apoptosis occurred in human neutrophils within an hour of exposure to viable serum-opsonized Staphylococcus aureus, as indicated by appearance of cells with condensed nuclei, fragmented DNA, and increased phosphatidylserine exposure. In contrast, serum-opsonized, heat-killed S. aureus did not induce apoptosis. This discrepancy could not be explained by differences in bacterial uptake or total NADPH-oxidase activity. Suppressing phagocytosis by pretreating the neutrophils with cytochalasin b or by using nonopsonized bacteria did not prevent apoptosis. A supernatant from bacteria grown for 2 h in nutrient broth had a strong proapoptotic influence that was abrogated by heat treatment. Exposure to viable S. aureus or supernatant also led to activation of p38-mitogen-activated protein kinase in the neutrophils. Inhibition of this kinase with SB203580 reduced the apoptosis-inducing capacity of both bacteria and supernatant. We conclude that S. aureus activates p38-mitogen-activated protein kinase in neutrophils and induces apoptosis, probably mediated by a bacteria-derived soluble factor(s).

Apoptosis↗

Consensus predictions of membrane protein topology.

We have explored the possibility that consensus predictions of membrane protein topology might provide a means to estimate the reliability of a predicted topology. Using five current topology prediction methods and a test set of 60 Escherichia coli inner membrane proteins with experimentally determined topologies, we find that prediction performance varies strongly with the number of methods that agree, and that the topology of nearly half of all E. coli inner membrane proteins can be predicted with high reliability (>90% correct predictions) by a simple majority-vote approach.

Bacterial Proteins↗

Skin fibroblasts from spermine synthase-deficient hemizygous gyro male (Gy/Y) mice overproduce spermidine and exhibit increased resistance to oxidative stress but decreased resistance to UV irradiation.

Hemizygous gyro male (Gy/Y) mice are a model for X-linked hypophosphataemic rickets. As in humans, the disease is caused by deletions in the Phex gene, a phosphate-regulating gene having homologies with endopeptidases on the X chromosome. Some phenotypic abnormalities in Gy/Y mice have recently been attributed to the fact that the Gy deletion also includes the neighbouring spermine synthase gene, resulting in spermine deficiency. Spermine and its precursors spermidine and putrescine are essential for cell growth and differentiation. As a novel method for studying the function of spermine, we established primary cultures of skin fibroblasts from hemizygous Gy/Y mice. The Gy/Y cells contained no detectable spermine. In view of the fact that spermine is a free-radical scavenger in vitro, we were surprised to find that Gy/Y cells were more resistant to oxidative stress than their normal (X/Y) counterparts. However, our finding that spermidine accumulates markedly in the spermine-deficient Gy/Y cells can probably explain this increased resistance. It is the first indication that spermidine can serve as a free-radical scavenger in vivo and not only in vitro. When subjecting the Gy/Y cells to UV-C irradiation we made another interesting finding: the mutant cells were more sensitive than the normal X/Y cells. This finding indicates that spermine, probably because of its high-affinity binding to DNA, is important in protection against chromatin damage.

Animals↗

Tailored fluorouracil, epirubicin, and cyclophosphamide compared with marrow-supported high-dose chemotherapy as adjuvant treatment for high-risk breast cancer: a randomised trial. Scandinavian Breast Group 9401 study.

BACKGROUND: Chemotherapy drug distribution varies greatly among individual patients. Therefore, we developed an individualised fluorouracil, epirubicin, cyclophosphamide (FEC) regimen to improve outcomes in patients with high-risk early breast cancer. We then did a randomised trial to compare this individually tailored FEC regimen with conventional adjuvant chemotherapy followed by consolidation with high-dose chemotherapy with stem-cell support. METHODS: 525 women younger than 60 years of age with high-risk primary breast cancer were randomised after surgery to receive nine cycles of tailored FEC to haematological equitoxicity with granulocyte colony-stimulating factor (G-CSF) support (n=251), or three cycles of FEC at standard doses followed by high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin (CTCb), and peripheral-blood stem-cell or bone-marrow support (n=274). Both groups received locoregional radiation therapy and tamoxifen for 5 years. The primary outcome measure was relapse-free survival, and analysis was by intention to treat. FINDINGS: At a median follow-up of 34.3 months, there were 81 breast-cancer relapses in the tailored FEC group versus 113 in the CTCb group (double triangular method p=0.04). 60 deaths occurred in the tailored FEC group and 82 in the CTCb group (log-rank p=0.12). Patients in the CTCb group experienced more grade 3 or 4 acute toxicity compared with the tailored FEC group (p<0.0001). Two treatment-related deaths (0.7%) occurred in the CTCb group. Six patients in the tailored FEC group developed acute myeloid leukaemia and three developed myelodysplastic syndrome. INTERPRETATION: Tailored FEC with G-CSF support resulted in a significantly improved relapse-free survival and fewer grade 3 and 4 toxicities compared with marrow-supported high-dose chemotherapy with CTCb as adjuvant therapy of women with high-risk primary breast cancer.

Acute Disease↗

The tissue-specific regulation of the carboxyl ester lipase gene in exocrine pancreas differs significantly between mouse and human.

The carboxyl ester lipase (CEL) gene is highly expressed in exocrine pancreas and expression of the human CEL gene is mediated by a strong tissue-specific enhancer, which is absolutely necessary for high-level expression. The mouse promoter, on the other hand, does not contain a corresponding enhancer element, but instead is totally dependent on another pancreas-specific element. This element is identified as a pancreatic transcription factor 1 (PTF 1)-binding site. The human CEL promoter also contains a putative PTF 1 element located at a position corresponding to the essential PTF 1 site in the mouse promoter. However, nucleotide changes in the human promoter 5' flanking this PTF 1 site have created an overlapping CCAAT/enhancer-binding protein (C/EBP)-like binding motif, interfering with the binding of PTF 1. Hence, our findings provide an example of genetic divergence between species not accompanied by difference in function.

Amino Acid Motifs↗

Airborne cell analysis.

A miniaturized analysis system for the study of living cells and biochemical reactions in microdroplets was developed. The technique utilizes an in-house-developed piezoelectric flow-through droplet dispenser for precise reagent supply and an ultrasonic levitator for contactless sample handling. A few-cell study was performed with living primary adipocytes. Droplets (500 nL) containing 3-15 individual cells were acoustically levitated. The addition of beta-adrenergic agonists into the levitated droplet using the droplet dispenser stimulated adipocyte lipolysis, leading to free fatty acid release and a consequent pH decrease of the surrounding buffer. The addition of insulin antagonized lipolysis and hence also the decrease in pH. The changes in pH, i.e., the cell response in the droplet, were followed using a pH-dependent fluorophore continuously monitored by fluorescence imaging detection. An image analysis computer program was employed to calculate the droplet intensities. To counteract droplet evaporation, found to affect the fluorescence intensities, a separate dispenser was used to continually add water, thus keeping the droplet volume constant.

Adipocytes↗

Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol.

Amino Acid Sequence↗

Reactive-site design in folded-polypeptide catalysts--the leaving group pKa of reactive esters sets the stage for cooperativity in nucleophilic and general-acid catalysis.

The second-order rate constants for the hydrolysis of nitrophenyl esters catalysed by a number of folded designed polypeptides have been determined, and 1900-fold rate enhancements over those of the 4-methylimidazole-catalysed reactions have been observed. The rate enhancements are much larger than those expected from the pKa depression of the nucleophilic His residues alone. Kinetic solvent isotope effects were observed at pH values lower than the pKa values of the leaving groups and suggests that general-acid catalysis contributes in the pH range where the leaving group is predominantly protonated. In contrast, no isotope effects were observed at pH values above the pKa of the leaving group. A Hammett rho value of 1.4 has been determined for the peptide-catalysed hydrolysis reaction by variation of the substituents of the leaving phenol. The corresponding values for the imidazole-catalysed reaction is 0.8 and for phenol dissociation is 2.2. There is therefore, very approximately, half a negative charge localised on the phenolate oxygen in the transition state in agreement with the conclusion that transition-state hydrogen-bond formation may contribute to the observed catalysis. The elucidation at a molecular level of the principles that control cooperativity in the biocatalysed ester-hydrolysis reaction represents the first step towards a level of understanding of the concept of cooperativity that may eventually allow us to design tailor-made enzymes for chemical reactions not catalysed by nature.

Amino Acid Sequence↗

Antizyme inhibitor is rapidly induced in growth-stimulated mouse fibroblasts and releases ornithine decarboxylase from antizyme suppression.

Ornithine decarboxylase (ODC) catalyses the first step in the synthesis of the polyamines putrescine, spermidine and spermine. The polyamines are essential for cell growth, but at elevated levels they may be tumorigenic, toxic, or may induce apoptosis. Therefore, ODC activity is highly regulated. It is induced when cells are stimulated to grow, and it is subjected to feedback inhibition by the polyamines. By causing ribosomal frameshifting, polyamines induce the synthesis of antizyme, a 23-kDa protein, which binds to ODC, inhibits its activity and promotes its degradation by the 26 S proteasome. Antizyme, in turn, is inhibited by antizyme inhibitor (AZI). We describe the cloning of a mouse AZI cDNA, encoding a protein with high homology to mouse ODC. Using purified recombinant proteins, we show that AZI (which has no ODC activity) can release enzymically active ODC from antizyme suppression in vitro. We also show that ODC reactivation takes place in mouse fibroblasts upon transient transfection with an AZI-expressing plasmid construct. Finally we demonstrate that the AZI mRNA content of mouse fibroblasts increases significantly within an hour of growth stimulation, i.e. much earlier than ODC transcripts. Our results indicate that induction of AZI synthesis may represent a means of rescuing ODC molecules that have been inactivated and tagged for degradation by antizyme, when culture conditions improve and polyamine production is needed for cell growth and proliferation.

3T3 Cells↗