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S Junker

Publications and source records attributed to S Junker.

At least 19 recordsLinked to original sources
PubMed2006

Modulation of chemokine gene expression in CD133+ cord blood-derived human mast cells by cyclosporin A and dexamethasone.

We have recently developed a protocol for generating huge numbers of mature and functional mast cells from in vitro differentiated umbilical cord blood cells. Using CD133 as a positive selection marker to isolate haematopoietic progenitors we routinely expand the number of recovered cells at least 150-fold, which vastly exceeds the yields of conventional protocols using CD34+ cells as a source of progenitors. Taking advantage of the large quantities of in vitro differentiated mast cells, here we assess at the levels of transcription and translation the kinetics of chemokine gene induction following receptor mediated mast cell activation or following pharmacological activation of specific signal transduction cascades that become activated upon classical FcepsilonRI receptor crosslinking. We demonstrate that chemokine genes encoding IL-8, MCP-1, MIP-1alpha, and MIP-1beta are induced with different kinetics and with different amplitudes in a receptor activation dependent manner, and that these events can be mimicked using pharmacological agents which activate distinct signal transduction pathways. These findings were corroborated by adding immunomodulators such as cyclosporin A and dexamethasone prior to mast cell activation. Finally, we demonstrate that the same modulators added after mast cell activation can differentially quench ongoing chemokine gene induction. Thus, considering the vast yields of mast cells, our protocol is valuable not only for studying regulation of gene expression in mast cells in general, but also as an experimental tool to develop better and more balanced treatments of mast cell related disorders.

AC133 Antigen↗
PubMed2000

Molecular mechanisms of extinction: old findings and new ideas.

Fusion experiments between somatic cells have been used for a long time as a means to understand the regulation of gene expression. In hybrids between differentiated cells such as hepatocytes or lymphocytes and undifferentiated cells such as fibroblasts a phenomenon called extinction has been described. In such hybrids expression of cell-specific genes derived from the more differentiated parental cell is selectively turned off (extinguished), whereas genes expressed from both cells like housekeeping genes remain active after fusion. Study of the molecular basis of extinction of the liver-specifically expressed tyrosine aminotransferase gene and of the B-cell-specifically expressed immunoglobulin genes has revealed that in hybrids the transcriptional program of the differentiated cells is reset. This is accompanied by a loss of expression or activity of many of the regulatory molecules that were operating in the differentiated cells. In the light of new insights in eukaryotic gene regulation we speculate that molecular mechanisms such as chromatin remodelling, recruitment to heterochromatin or subnuclear localization could underly the extinction process.

Animals↗
PubMed1997

Concomitant downregulation of IgH 3' enhancer activity and c-myc expression in a plasmacytoma x fibroblast environment: implications for dysregulation of translocated c-myc.

Regulation of immunoglobulin heavy chain (IgH) gene expression is controlled by a B cell-specific promoter, intronic enhancer and additional B cell-specific enhancer elements identified recently in the 3' end of the IgH locus. One of the latter elements, the IgH 3' enhancer, is of particular interest: (1) it is B cell-specific and active only in late B cell development; (2) in rodent plasmacytomas and in some human Burkitt's lymphomas it is part of a locus control region (LCR) that is involved in deregulation of the c-myc oncogene as a result of translocation into the IgH locus; and (3) it has been implicated in the mechanisms that control Ig gene class switch recombination. We have used a somatic cell hybridization approach to genetically analyse regulation of the activity of the IgH 3' enhancer. When mouse MPC11 plasmacytoma cells, in which the IgH 3' enhancer is active, are fused with fibroblasts, Ig expression is extinguished at the level of transcription. Here we show that in a MPC11 plasmacytoma x fibroblast environment, the IgH 3' enhancer is transcriptionally inactive. Furthermore, we demonstrate that binding of several B cell-specific transcription factors, essential for IgH 3' enhancer activity, is lacking, which may explain 3' enhancer inactivity, although the binding of repressors cannot be excluded. Moreover, the high expression level of c-myc, characteristic of the parental MPC11 cells carrying the t(12;15) translocation, is down-regulated in the hybrids to that in unfused fibroblasts. Therefore, inactivation of the IgH 3' enhancer is a multifactorial process affecting several transcription factors that control the cell-specific and developmental activity of the enhancer.

Animals↗
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↗
PubMed1994

Pre- and posttranslational upregulation of muscle-specific glycogen synthase in athletes.

Expression of muscle-specific glycogen synthase (GS) and phosphofructokinase (PFK) was analyzed in seven athletes and eight control subjects who were characterized using the euglycemic, hyperinsulinemic (2 mU.kg-1.min-1) clamp technique in combination with indirect calorimetry and biopsy sampling of vastus lateralis muscle. In the basal state, total GS activity and half-maximal GS activation by glucose 6-phosphate (G-6-P) were respectively 34% (P < 0.03) and 50% (P < 0.005) higher in athletes than in control subjects. In parallel, GS mRNA/microgram total RNA in athletes was 40% (P < 0.005) higher. No difference in GS immunoreactive protein abundance was found between the groups. PFK activity and protein levels were respectively 15% (P < 0.05) and 20% (P < 0.02) lower in athletes, whereas no differences was found in the level of PFK mRNA. After 4 h of hyperinsulinemia, total glucose disposal rate (P < 0.005) and both nonoxidative (P < 0.02) and oxidative (P < 0.03) glucose metabolism were significantly higher in athletes. In parallel, after hyperinsulinemia, the relative activation of GS by G-6-P was significantly higher in athletes, whereas total activity and gene expression of both GS and PFK were unaffected by insulin. We conclude that athletes have increased whole body insulin-stimulated nonoxidative glucose metabolism associated with both pretranslational (mRNA) and posttranslational (enzyme activity) upregulation of GS. However, the immunoreactive mass of GS is normal, emphasizing that posttranslational regulation of the GS protein activity is important for the increased glycogen synthesis rate of muscle in endurance-trained individuals.

Adult↗
PubMed1993

The effect of intensive insulin therapy on the insulin-regulatable glucose transporter (GLUT4) expression in skeletal muscle in type 1 diabetes.

Studies in normal man and rodents have demonstrated that the expression of the dominant glucose transporter in skeletal muscle, GLUT4, is regulated by insulin at supraphysiological circulating levels. The present study was designed to determine whether intensified insulin replacement therapy for 24 h given to patients with Type 1 diabetes in poor metabolic control was associated with an adaptive regulation of GLUT4 mRNA and protein levels in vastus lateralis muscle. Nine Type 1 diabetic patients with a mean HbA1c of 10.3% were included in the protocol. After intensified treatment with soluble insulin for 24 h the fasting plasma glucose concentration decreased from 20.8 +/- 2.3 (SD) to 8.7 +/- 2.3 mmol 1-1, whereas the fasting serum insulin level increased from 0.06 +/- 0.02 to 0.17 +/- 0.09 nmol 1-1. However, despite a 2.8-fold increase in serum insulin levels and more than a halving of the plasma glucose concentration for at least 15 h no significant alterations occurred in the amount of GLUT4 protein (0.138 +/- 0.056, poor control vs 0.113 +/- 0.026 arb. units, improved control, p = 0.16) or GLUT4 mRNA (96432 +/- 44985, poor control vs 81395 +/- 25461 arb. units, improved control, p = 0.54). These results suggest, that in spite of evidence that high insulin levels affect GLUT4 expression in muscle, changes in serum insulin within the physiological range do not play a major role in the short-term regulation of GLUT4 expression in Type 1 diabetic patients.

Adult↗
PubMed1993

Increased insulin-stimulated glucose uptake in athletes: the importance of GLUT4 mRNA, GLUT4 protein and fibre type composition of skeletal muscle.

In the present study the expression of GLUT4 and fibre type composition were examined in biopsies from skeletal muscle in seven male athletes and eight male sedentary subjects. Estimated maximal oxygen uptake was increased in the trained group when compared with the sedentary group (74.0 +/- 3.9 vs. 42.9 +/- 5.1 ml kg-1 min-1; P < 0.01). A biopsy of vastus lateralis muscle was taken in the fasting state, 36 h after the last bout of exercise. A second muscle biopsy was obtained following 4 h of a hyperinsulinaemic (2 mU kg-1 min-1), euglycaemic clamp. The rate of insulin-stimulated glucose uptake was increased in the trained subjects (17.34 +/- 0.53 vs. 13.53 +/- 0.79 mg kg-1 min-1, P < 0.01). In parallel, the steady state levels of GLUT4 protein and mRNA per DNA were higher in muscle biopsies obtained in the basal state from athletes than in sedentary controls, 21 and 71% respectively (P < 0.05). In the total group of participants, GLUT4 protein per DNA in the basal state and insulin-stimulated glucose uptake rate correlated positively, (r = 0.51, P = 0.05). In the insulin-stimulated state we did not find any significant correlation between GLUT4 protein per DNA and glucose uptake rate (r = 0.13, n.s.). No significant relationships between GLUT4 protein abundance per DNA and muscle fibre type distribution were observed. A significantly negative correlation was found between type 2B fibre area and insulin-stimulated glucose uptake (r = -0.63, P < 0.05). In conclusion, the abundance of GLUT4 protein and mRNA, respectively, is increased in skeletal muscle from endurance trained subjects compared to sedentary subjects. However, factors other than GLUT4 immunoreactive protein abundance seem to be determinant for the increased insulin-stimulated whole body glucose uptake in endurance trained subjects.

Adult↗
PubMed1993

Expression of the major insulin regulatable glucose transporter (GLUT4) in skeletal muscle of noninsulin-dependent diabetic patients and healthy subjects before and after insulin infusion.

In a cross-sectional study we have examined the regulatory effect of insulin in vivo on the major insulin regulatable glucose transporter (GLUT4) in vastus lateralis muscle from 12 noninsulin-dependent diabetes mellitus (NIDDM) patients and 8 healthy control subjects. Insulin-stimulated glucose uptake rate in peripheral tissue was decreased by 41% (P < 0.01) in NIDDM patients compared to healthy subjects, whereas no significant differences could be shown in the abundance of total GLUT4 protein per DNA or GLUT4 messenger RNA (mRNA) per DNA among the 2 groups in muscle biopsies obtained in the basal state. In healthy subjects, 4 h of insulin infusion (2 mU/kg/min) induced a 31% reduction (P < 0.05) in the total GLUT4 protein content per DNA and a 35% increase (P < 0.05) in GLUT4 mRNA per DNA, whereas the GLUT4 mRNA and protein responses to insulin were heterogenous and statistically unaltered in the NIDDM patients. The GLUT4 protein per DNA of muscle obtained in the basal state correlated positively with the in vivo insulin-stimulated glucose uptake rate in the control group (r = 0.82, P < 0.05), whereas there was no comparable correlation in the NIDDM group (r = 0.05, P = 0.88). Furthermore, GLUT4 protein content in skeletal muscle after 4 h of insulin infusion did not correlate with insulin-stimulated glucose uptake in any of the groups. In conclusion, 4 h of insulin infusion causing supraphysiological serum insulin levels modulates the expression of GLUT4 in skeletal muscle from healthy subjects, with divergent effects at protein and mRNA levels. The physiological significance of these observations will have to be elucidated in future studies. Factors other than total GLUT4 protein content of muscle play a role in determining insulin-stimulated glucose uptake in human skeletal muscle.

Actins↗
PubMed1991

Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS).

A new method for the detection of RNA in situ is presented. It is based on sequence-dependent annealing of unlabeled specific oligonucleotide primers to intracellular RNA and subsequent chain elongation catalyzed by reverse transcriptase. Under the conditions described, biotin-labeled nucleotides can be incorporated and the cDNA synthesized in situ can thus be detected using fluorescein-conjugated avidin. Compared to traditional in situ hybridization the use of short oligonucleotide primers has the potential advantage of being better to discriminate between closely related RNA transcripts. Compared to in situ transcription with radioactive precursors we find it more attractive to use fluorescein-conjugated avidin as detection system because it allows a more detailed study of cell and signal simultaneously.

Animals↗
PubMed1990

Extinction of an immunoglobulin kappa promoter in cell hybrids is mediated by the octamer motif and correlates with suppression of Oct-2 expression.

When immunoglobulin-expressing B cells are fused with fibroblasts, immunoglobulin expression is rapidly and selectively suppressed. here we demonstrate that the conserved octamer motif of a kappa light chain gene promoter plays a crucial role in mediating this "extinction" phenomenon. Replacement of this octamer site by an Sp1 or NF1 binding site is sufficient to bypass extinction. Furthermore, in early cell hybrids, immunoglobulin suppression is correlated with absence of the cell-specific transcription factor Oct-2 and its transcripts. Such hybrids cannot support transcription of a transiently introduced reporter plasmid, driven by an octamer-containing promoter, unless an expression vector encoding Oct-2 is cotransfected. Transfection of the same Oct-2 expression vector into hybrid cells is also sufficient to "reactivate" an integrated kappa promoter construct. Thus, our data further establish the role of Oct-2 for immunoglobulin transcription and show that in B cell x fibroblast hybrids, the lack of a necessary cell-specific transcription factor is involved in the extinction of immunoglobulin expression.

Animals↗
PubMed1990

[Comparative effect of oral fat loads with saturated, omega-6 and omega-3 fatty acids before and after fish oil capsule therapy in healthy probands].

To evaluate digestion, absorption and postprandial lipemia of diverse fats before and after fish oil capsule therapy (with ethylester) 20 healthy volunteers were recruited. A significant increase (p less than 0.001) in fecal fat excretion after fish oil capsule therapy, as well as serious diarrhoea after fat loads with 100 g of fish oil was noted. It was found, that under fish oil fat loads the total triglyceride content of the mucosa was significantly (p less than 0.001) lower compared to butter fat loads. Decrease digestion and absorption of omega-3 fatty acids was postulated.

Adult↗
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↗
PubMed1985

Time course of arrest of immunoglobulin expression in heterokaryons and early hybrids of human lymphoma cells and mouse fibroblasts. A study of transcriptional and translational events.

Early events in arrest of immunoglobulin expression were investigated at the levels of both translation and transcription in heterokaryons and early hybrids between human Daudi lymphoma cells and mouse cl. 1D cells. Large populations of 1s: 1s hybrids, isolated by fluorescence-activated cell sorting (FACS) a few hours after fusion, were grown for up to 5 days. A survey at the light-microscopical level of peroxidase-antiperoxidase-immunostained cell populations showed that arrest of expression of IgM heavy chain (mu) occurred in up to 98% of the cells. Furthermore, quantitation of mu chain contents, by using an ELISA technique, suggested that synthesis of IgM was blocked shortly after fusion. The levels of cytoplasmic mRNA specific for mu and kappa chains, respectively, decreased at rates similar to those induced in unfused Daudi cells by treatment with actinomycin D. It is concluded that arrest of immunoglobulin expression in these hybrids occurs immediately or very shortly after fusion by mechanisms that affect the levels of their cytoplasmic mRNAs.

Animals↗