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V Nielsen

Publications and source records attributed to V Nielsen.

At least 19 recordsLinked to original sources
PubMed1998

Hypoxia-reoxygenation is as damaging as ischemia-reperfusion in the rat liver.

OBJECTIVE: We hypothesized that the extent of injury and release of xanthine oxidase, an oxidant generator, into the circulation would be less in normal-flow hypoxia-reoxygenation than in equal duration no-flow ischemia-reperfusion. DESIGN: Randomized study. SETTING: University-based animal research facility. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: The livers were isolated, perfused, and then randomly subjected to 2 hrs of hypoxia (normal flow, low oxygen) or ischemia (no flow, no oxygen), and 2 hrs of reperfusion. Hepatocytes were also isolated, and were subjected to either: a) hypoxia (0, 2, 4, and 6 hrs); or b) hypoxia (2 and 4 hrs) with reoxygenation (2 hrs). MEASUREMENTS AND MAIN RESULTS: The extent of liver injury (as assessed by release of hepatocellular enzymes) and the release of xanthine oxidase were measured from isolated-perfused rat livers and cultured hepatocytes. The pattern of release of xanthine oxidase in isolated-perfused liver effluent was different in hypoxia-reoxygenation compared with ischemia-reperfusion. During hypoxia, xanthine oxidase gradually increased in the effluent; then, the xanthine oxidase decreased to low concentrations during reoxygenation. After ischemia, there was a sharp spike in xanthine oxidase at 1 min of reperfusion, with a rapid decrease to low concentrations. The total release of xanthine oxidase during hypoxia-reoxygenation was similar to that during ischemia-reperfusion. Lactate dehydrogenase and other markers of liver injury showed a pattern of release that was similar to that of xanthine oxidase, but the total release of markers was not different between the two groups. In hepatocytes, most of the release of enzymes occurred in hypoxia, and the rate of release was not different between hypoxia and hypoxia-reoxygenation. CONCLUSIONS: Hypoxia-reoxygenation results in as much damage to the liver as ischemia-reperfusion, and results in the release of a similar amount of oxidant-producing xanthine oxidase into the circulation.

Alanine Transaminase↗
PubMed1997

Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner.

When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi x HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype.

B-Lymphocytes↗
PubMed1996

Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells.

To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cell surface is quantitated. The receptor activity is measured by incubating the cells with fluorescence labeled LDL (Dil-labelled LDL) at 37 degrees C and 4 degrees C. The transfected cells stained with anti-LDL-R antibodies can also be analysed by immunofluorescence microscopy allowing the study of the intracellular location of variants of the receptor. To evaluate these methods, we are analyzing four previously well-characterized LDL-R mutations, belonging to each of the classes 2 to 5. Preliminary data show that mutant genes belonging to class 3 and 4A give rise to receptor protein on the cell surface, but impaired LDL uptake, while mutant receptors belonging to class 2A and 5 can only be detected intracellularly. Expression of the class 2A mutation results in an ER staining pattern, whereas the class 5 mutation gives rise to an intracellular staining compatible with localization in the endosomal/lysosomal compartments. We conclude that this system is useful for a rapid functional analysis of newly discovered mutations in the LDL-R gene.

Cell Line↗
PubMed1996

Flow cytometric DNA analyses of 105 fresh hydatidiform moles, with correlations to prognosis.

Cellular DNA contents of consecutive hydatidiform moles were analysed by flow cytometry of unfixed samples, freed of maternal tissue and treated with detergent and trypsin, using two external controls. DNA-diploidy was found in 62 moles, DNA-triploidy in 42 and DNA-tetraploidy in one. Two non-molar placentas and two invasive moles were found to be DNA-diploid. In 28 of 42 DNA-triploid moles, trophoblastic hyperplasia was not noted, making the discrimination between vesicular abortions with or without trophoblastic hyperplasia unwarranted on genetic grounds. A good fit was obtained to a distribution with one G0-1 peak in 49 cases, whereas 60 cases showed two peaks. Technical artifacts, created by flow cytometry, were excluded. Differences in the nuclear accessibility for the dye and degradation of DNA are unlikely causes of this heterogeneity. The existence, in vivo, of two or more cell populations with different DNA contents is a reasonable explanation. The criteria for persistent trophoblastic disease were standardised. Eight cases of persistent trophoblastic disease were observed among the 62 DNA-diploid moles (13%); no case was observed after DNA-triploid or DNA-tetraploid moles. Combining these data, with those of other studies where ploidy was determined using optimal techniques [10,13], allows the calculation of 95%-confidence-limits for the risk of persistent trophoblastic disease after a triploid mole to 0-2.7%.

Aneuploidy↗
PubMed1996

[Regeneration of tree cover in a small urban forest reserve from the moist Premontane region].

The tropical premontane moist forest is one of the most destructed life zones in Costa Rica. For such regions, a small natural preserve system has been proposed. An inventory of trees greater than 3.5 cm in diameter at breast height was done in one hectare urban forest patch that was left under natural regeneration for 30 years. The inventory included 940 individuals of at least 55 species and 32 families. A Holdridge Complexity Index of 58 was obtained. There was a strong dominance by small-diameter trees of successional and exotic species.

City Planning↗
PubMed1994

Characterization of a disease-causing Glu119-Lys mutation in the low-density lipoprotein receptor gene in two Danish families with heterozygous familial hypercholesterolemia.

Mutations in the gene for the low-density lipoprotein receptor (LDL receptor) cause the autosomal dominant inherited disease familial hypercholesterolemia (FH). In 15 Danish patients with heterozygous FH we have screened exon 4 of the LDL receptor gene for point mutations and small rearrangements employing genomic DNA amplification and bidirectional solid-phase sequencing. Two subjects were found to be heterozygous for a guanine to adenine base substitution at nucleotide position 418 of the LDL receptor cDNA. This point mutation results in an amino acid change from glutamic acid to lysine at amino acid residue 119 in the third repeat of the cysteine-rich ligand binding domain of the mature LDL receptor. Disruption of LDL receptor function by the Glu119-Lys mutation was confirmed by site-directed mutagenesis and expression in COS-7 cells. By Western blotting the mutation was found to affect the processing of the LDL receptor protein. Using flow cytometric analysis of the transfected cells a decreased binding and internalization of LDL by the mutant receptor was documented. By means of a mutation-specific PCR-based assay the Glu119-Lys mutation was not detected in another 85 apparently unrelated Danish heterozygous FH patients. We identified six persons in the index families with the Glu119-Lys mutation cosegregating with the clinical syndrome of FH in these families. Furthermore, haplotype analysis revealed that the haplotype [SfaNI+, StuI+, AvaII-, (dTA)7] of the mutation carrying allele was the same in the two apparently unrelated patients. This indicates that the mutation has been inherited from a common ancestor.

Adult↗
PubMed1992

Phenotypic determination of T-lymphocytes responding to chemotactic stimulation from fMLP, IL-8, human IL-10, and epidermal lymphocyte chemotactic factor.

Human T lymphocytes were collected after they had migrated towards N-formyl-methionyl-leukylphenylalanine (fMLP), rIL-8, human IL-10 (hIL-10), and epidermal lymphocyte chemotactic factor (ELCF). They were stained for determination of their phenotype by FACS analysis using anti-CD4, -CD8, -CD18, -CD45R0 and OPD4 antibodies. Human IL-10 increased the percentage of CD8+ T lymphocytes in the migrating cell population by 152% compared with cells migrating towards the medium and decreased the number of CD4+ T lymphocytes by 79%. ELCF increased the number of CD4+ T lymphocytes by 18%, and the number of CD45R0+ T lymphocytes by 52%, while the number of CD8+ T lymphocytes was decreased by 20%. rIL-8 increased the number of CD4+ T lymphocytes and decreased the CD8+ T lymphocytes. The distribution of the different subpopulations of T lymphocytes was not changed significantly by fMLP. The observed changes in the phenotypes did not occur when incubating T lymphocytes with the chemotaxins. Our observations demonstrate that individual chemotactic factors will attract specific subsets of T lymphocytes. They may help to explain the predominance of memory T lymphocytes (CD4R0+, CD4+) in allergic contact dermatitis and certain other skin diseases. They also confirm the results of a recent study, that showed hIL-10 to be selectively chemotactic for CD8+ T lymphocytes.

Antigens, CD↗
PubMed1990

Effects of extra- and intracellular calcium concentration on DNA replication, lateral growth, and differentiation of human epidermal cells in culture.

Variation in the extra- and intra-cellular concentration of calcium ([Ca]e and [Ca]i) affected the 3H-thymidine labeling pattern of sorted S-phase cells in human epidermal cultures. A lowering of [Ca]e resulted in retarded lateral growth but, unless [Ca]e was extremely low, caused an increase in the proportion of strongly labelled (rapidly cycling) S-phase cells. An increased desquamation of superficial cells due to a reduced cellular cohesiveness was also observed in low calcium medium. Thus, a lowering of [Ca]e might stimulate the proliferation of a pool of cycling cells destined for rapid terminal differentiation and tissue regeneration, whereas proliferation destined for lateral growth is inhibited. Attempts to decrease the [Ca]i with the calcium chelator quin-2 at low [Ca]e seemed to elevate the proportion of strongly labelled S-phase cells, whereas an increased [Ca]i obtained with the ionophore A23187 caused a dramatic decrease in the proportion of S-phase cells that showed strong 3H-thymidine incorporation. This implies that variation in both [Ca]i and [Ca]e may play a role in the regulation of proliferation and differentiation, in keratinocytes.

Aminoquinolines↗
PubMed1989

Myeloma kappa gene transcription is blocked upon fusion with fibroblasts.

Numerous studies on somatic cell hybrids have shown that expression of tissue-specific functions can be suppressed as a consequence of fusion with cells that do not express the given functions. We have further investigated this phenomenon, using as a model system the regulation of expression of kappa light chain genes in intraspecific hybrids between mouse myeloma cells and mouse fibroblasts. Hybrids containing only one genome equivalent from each parent cell (1s:1s) were isolated by fluorescence-activated cell sorting from within 10 h after fusion, and they were grown for no more than 16 days thereafter in order to ensure maximum integrity of the genomic constitution. Here we report that in hybrid cells, kappa gene transcription was specifically turned off as demonstrated by nuclear run-on assays performed on 16-day-old proliferating hybrids. Furthermore, a mechanism affecting mRNA stability may also contribute, at least initially, to the rapid depletion of cytoplasmic kappa transcripts, observed during the first few hours after fusion. Suppression was dominant and could not be overridden by increasing the relative myeloma ploidy at either the heterokaryon or the synkaryon stage. Nor could suppression be relieved by treating hybrids with cycloheximide.

Animals↗
PubMed1988

Both immunoglobulin promoter and enhancer sequences are targets for suppression in myeloma-fibroblast hybrid cells.

When immunoglobulin (Ig)-producing B cells are fused with fibroblastic cells, expression of Igs is suppressed by a mechanism that selectively abolishes transcription of Ig genes. The suppression is also maintained in proliferating hybrids. We have used gene transfer followed by cell fusion to study this phenomenon further. Here we report that expression of a rearranged Ig heavy chain gene, stably integrated into a myeloma genome, is completely suppressed upon fusion with fibroblasts by a mechanism that is equally active on the endogenous myeloma lambda light chain gene. To define regulatory sequences within the Ig transcriptional unit that are involved in this down-regulation, we examined the transcriptional contributions of the IgH chain gene enhancer and the kappa light chain gene promoter individually by linking them to a heterologous reporter gene. Mouse myeloma cells were stably transformed with such test constructs and subsequently fused with mouse fibroblasts. To avoid any significant loss of chromosomes, hybrid cells were isolated shortly after fusion by fluorescence-activated cell sorting, and proliferating hybrids were harvested within 2-3 weeks. On the basis of RNase protection mapping of cytoplasmic RNA, and of nuclear run-on assays we showed that both the kappa light chain promoter and the IgH chain enhancer contain regulatory information that is made redundant or is suppressed in the hybrid environment.

Animals↗
PubMed1988

Epidermal lymphocyte chemotactic factor specifically attracts OKT4-positive lymphocytes.

Epidermal lymphocyte chemotactic factor (ELCF) from skin overlying a positive tuberculin reaction was compared with the chemoattractants leukotriene B4 (LTB4), N-formyl-methionyl-leukyl-phenylalanine (FMLP), and complement split product C5a (C5a). The chemotactic assay used is a modified Boyden chamber technique. The lymphocytes were subsets of T lymphocytes from healthy young individuals first separated by flotation of E rosettes on Isopaque Ficoll followed by incubation of T cells with anti-CD4 and anti-CD8 monoclonal antibodies and further separation using fluorescence-activated cell sorting. ELCF specifically attracted OKT4+ lymphocytes, while LTB4, FMLP, and C5a induced significant migration in both OKT4+ and OKT8+ lymphocytes without any clear difference between the various chemoattractants or cell populations. We found no blocking of the chemotactic capacity of ELCF when we added antibodies towards IL-1 alpha and IL-1 beta to the chemotactic assay. Further recombinant IL-1 alpha and Il-1 beta did not induce any chemotactic response. Our observation may be of significance in explaining the predominance of OKT4+ cells in allergic contact dermatitis and certain other skin diseases.

CD4-Positive T-Lymphocytes↗
PubMed1986

Effects of pregnancy hormones on pancreatic islets in organ culture.

Pregnancy is associated with hyperinsulinaemia and decreased glucose tolerance which may lead to gestational diabetes. Sera from pregnant women in the last trimester were found to stimulate insulin secretion of mouse pancreatic islets maintained in organ culture suggesting a direct effect of pregnancy hormones and/or metabolites on the endocrine pancreas. Previous studies have shown that progesterone and human placental lactogen (hPL) had direct effects on isolated islets and in the present study the effects of combined addition of these hormones to the cultured islets were evaluated. hPL, 1 microgram/ml was found to prevent the decrease in the islet insulin content due to progesterone 100 ng/ml, and hPL induced an increase in the DNA-content compared with the progesterone treated islets. The effect of the hormones on the DNA synthesis was evaluated by incorporation of [3H]thymidine into newborn rat islets. Progesterone in a high concentration, 1 microgram/ml, inhibited while 0.1 and 1 microgram/ml hPL stimulated the [3H]thymidine incorporation. These results indicate that progesterone may contribute to the increased glucose-induced insulin release during pregnancy and hPL to the beta-cell hyperplasia. Since progesterone and hPL counteracted each other with regard to both insulin content and DNA-synthesis in the islets, the direct interaction between these two hormones on the beta-cell may be of importance for the changes in glucose metabolism during pregnancy.

Animals↗
PubMed1985

Percutaneous balloon catheter nephrostomy guided by ultrasound. Results of a new technique.

A new method for percutaneous nephrostomy is described. The renal pelvis is punctured guided by ultrasound and after dilatation of the tract a 10F silicone balloon catheter is inserted. The procedure was successful in 51 of 52 attempts (98%). The catheters were maintained from two to 150 days. Eight catheters slipped out unintended (16%) 27 to 86 days after the insertion. No serious complications were seen.

Catheterization↗
PubMed1980

Biosilon a new microcarrier.

A new microcarrier Biosilon has been developed on the basis of experience with polystyrene as substratum for cell cultures. Biosilon consists of polystyrene beads, which have a diameter of 160-300 micrometers, a density of 1.05 g/cm3 and carry a negative surface charge. Methods used in testing medical plastic and biomaterials can be used as quality standards for microcarriers.

Cells, Cultured↗
PubMed1980

Biosilon optimal culture conditions and various research scale culture techniques.

Research scale cultivations of L-929, MRC-5 and primary C.E. cells on Biosilon have shown that the most uniform cell attachment is obtained in systems, where the relative velocity between cells and beads is close to zero. In microcarrier cultures the attachment yield is lower compared with stationary systems, but the multiplication rate is the same or higher. The Tecam ACA-system and a rolling bottle system have proved to be promising systems for research scale.

Animals↗