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M Carlberg

Publications and source records attributed to M Carlberg.

At least 37 records · Page 2Linked to original sources
PubMed1997

Inhibition of N-linked glycosylation using tunicamycin causes cell death in malignant cells: role of down-regulation of the insulin-like growth factor 1 receptor in induction of apoptosis.

Recent studies have shown that inhibition of N-linked glycosylation using tunicamycin (TM) induces cell death in cultured cells (O. Larsson et al., J. Cell Sci., 106: 299-307, 1993; J. Y. Chang and V. Korolev, Exp. Neurol., 137: 201-211, 1996). The mechanisms underlying TM-induced cell death seem, however, to be complex in nature. In SV40-transformed cells, TM triggers within a few minutes mechanisms subsequently leading to apoptosis. These mechanisms, although still not fully understood, involve an elevation of [Ca2+]i (M. Carlberg et al., Carcinogenesis, 17: 2589-2596, 1996). In contrast, in melanoma cells, TM has to be present continuously for 24-48 h to elicit apoptosis. Before the appearance of apoptotic melanoma cells, assayed by gel-electrophoretic detection of oligonucleosomally fragmented DNA, the binding of insulin-like growth factor I (IGF-I) to the cells had become drastically decreased, which in turn was found to be due to down-regulation of IGF-I receptor proteins at the cell surface. Incubation of the cells with an antibody (alpha IR-3) against the IGF-I-binding site of the receptor resulted also in apoptosis, the kinetics of which were almost identical to those following treatment with TM. Furthermore, coincubation of a high concentration of IGF-I (50 ng/ml) with TM totally rescued the melanoma cells from apoptotic cell death during the first 48 h after addition of the drugs. This effect was shown to be abolished fully by alpha IR-3. Taken together, our data suggest strongly that N-linked glycosylation plays an important role in maintenance of viability of melanoma cells through regulating the translocation of IGF-I receptor to the cell surface.

Apoptosis↗
PubMed1997

Mevalonate-regulated mechanisms in cell growth control: role of dolichyl phosphate in expression of the insulin-like growth factor-1 receptor (IGF-1R) in comparison to Ras prenylation and expression of c-myc.

One or more mevalonate derivatives of non-sterol type have been proposed to be of indispensable importance for cell growth. Conceivable mevalonate-dependent mechanisms involved in growth control are farnesylation of Ras proteins, regulation of c-myc expression, and N-linked glycosylation of the IGF-1 receptor. The latter mechanism might be rate-limited by dolichyl phosphate, which acts as a donor of oligosaccharides in glycoprotein synthesis in the endoplasmic reticulum. In order to study the significance for cell proliferation of the three aforementioned mevalonate-dependent processings, their inhibitory response due to mevalonate deprivation was explored and compared with the effect on DNA synthesis in the malignant melanoma cell line SK-MEL-2. We found that mevalonate depletion due to treatment with 3 microM lovastatin for 24 h, which efficiently growth-arrested the cells, hardly at all affected the expression of c-myc, and although Ras prenylation was inhibited by 50%, the most pronounced effect of lovastatin was seen on N-linked glycosylation of IGF-1 receptors, which was inhibited by more than 95%. The order and magnitude of the decreased IGF-1 receptor glycosylation, which was followed by a decreased expression of IGF-1 receptors at the cell membrane, correlated well with the inhibition of DNA synthesis. We investigated whether dolichol, and in particular dolichyl phosphate, through its participation in N-linked glycosylation, act as regulators of IGF-1 receptor expression. First, we could confirm that exogenous dolichol became phosphorylated and in this form took part in the glycosylation processing. Secondly, we showed that dolichyl phosphate, in a dose-dependent manner, could increase the number of IGF-1 receptors at the cell membrane, simultaneously as DNA synthesis was stimulated. Taken together, our results provide direct evidence for an important role of dolichyl phosphate as a regulator of cell growth through limiting N-linked glycosylation of the IGF-1 receptor.

Cell Division↗
PubMed1996

Doxorubicin- and daunorubicin-induced energy deprivation and nucleotide degradation in isolated cardiomyocytes.

Cytotoxic mechanisms of the antitumor agents daunorubicin and doxorubicin were elucidated in isolated cardiomyocytes from adult rats. Incubation with daunorubicin resulted in a concentration-dependent loss of cell viability and changes of the cell structure. Only the highest concentration of doxorubicin (1 mM) caused similar effects. Doxorubicin was found to stimulate oxygen consumption by cardiomyocytes (about 20%), while the opposite effect was observed after daunorubicin treatment. A rapid decrease of the mitochondrial ATP content (more than 40%) and elevation of the cytosolic ADP level (doxorubicin 2-fold and daunorubicin 6-fold) was followed by increased release of adenosine and inosine to the surrounding medium. When myocytes were exposed to an anthracycline concentration lower than plasma levels measured clinically (0.15 microM), doxorubicin and daunorubicin significantly decreased the intracellular ADP and NAD levels. Isolated cardiomyocytes were found to be able to form daunorubicinol from daunorubicin. In contrast, no conversion of doxorubicin was detected in our experiments. In conclusion, our data demonstrate that decreased ATP production and increased nucleoside formation are major events in the toxicity induced by daunorubicin and doxorubicin in isolated cardiomyocytes. The results also suggest that the toxic effects may be caused by separate mechanisms.

Adenosine Diphosphate↗
PubMed1996

Mevalonic acid is limiting for N-linked glycosylation and translocation of the insulin-like growth factor-1 receptor to the cell surface. Evidence for a new link between 3-hydroxy-3-methylglutaryl-coenzyme a reductase and cell growth.

Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth.

Base Sequence↗
PubMed1996

Stimulatory effect of PDGF on HMG-CoA reductase activity and N-linked glycosylation contributes to increased expression of IGF-1 receptors in human fibroblasts.

In the present paper, we show that the prereplicative period in platelet-derived growth factor (PDGF)-stimulated human diploid fibroblasts (HDF) includes an early increase in 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity followed by an increased N-linked glycosylation. HMG-CoA reductase inhibitors abolished these events and also prevented progression of cells into S-phase, indicating that mevalonate (MVA)-dependent glycosylation might be required in PDGF-mediated cell growth. Furthermore, PDGF was demonstrated to increase the number of insulin-like growth factor 1 (IGF-1) binding sites in HDF. This event, which was dependent on MVA, took place late in the prereplicative period and was corrected to a significant increase in the expression of de novo synthesized IGF-1 receptor (IGF-1R) proteins at the cell membrane. The PDGF-induced IGF-1R expression was suppressed in the presence of tunicamycin, an inhibitor of N-linked glycosylation. Taken together, our findings suggest an important role of MVA and N-linked glycosylation in PDGF-mediated growth activation of HDF.

Cell Division↗
PubMed1996

Short exposures to tunicamycin induce apoptosis in SV40-transformed but not in normal human fibroblasts.

When SV40-transformed fibroblasts (line 90VAVI) were exposed to tunicamycin, an inhibitor of N-linked glycosylation, an extensive cell death occured compared with untransformed fibroblasts. A considerable cell loss was obtained within 24 h after tunicamycin addition, and after 72 h there were hardly any virus-transformed cells alive. A 2-h pulse treatment with tunicamycin was found to be almost as effective as a continuous 48-h treatment in killing the cells. Even such a short exposure as 7 min resulted in a drastically decreased cell viability (54%). The morphology of the dying tunicamycin-treated 90VAVI cells suggested that they were undergoing apoptosis. This was also supported by the appearance of nuclear condensation, as assayed by propidium iodide uptake, which was detectable within 2 h after tunicamycin addition. Furthermore, analysis of DNA from tunicamycin-treated 90VAVI cells by field inversion gel electrophoresis revealed DNA degradation into 50 kbp fragments within 2 h, and conventional agarose gel electrophoresis showed 'DNA laddering', indicating internucleosomal DNA cleavage, detectable after 36 h. Together with the finding that tunicamycin within seconds caused an elevation of [Ca2+]i, a well documented early feature of apoptosis in many experimental systems, these results strongly suggest that tunicamycin-induced cell death in 90VAVI is due to apoptosis. The short tunicamycin exposure required to trigger cell death in 90VAVI indicates that the apoptotic process is irreversibly induced soon after its addition. It seems unlikely that the pool of one or several specific N-linked glycoproteins could be depleted during such a short period. Instead the overall accumulation of unglycosylated proteins in ER might contribute to the apoptotic response in 90VAVI. Tunicamycin also killed and induced DNA degradation in the breast cancer cell line MDA-231.

Apoptosis↗
PubMed1996

Functional domains in the Mig1 repressor.

Mig1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. It is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 proteins and binds to a GC-rich motif that resembles the GC boxes recognized by these proteins. We have performed deletion mapping in order to identify functional domains in Mig1. We found that a small C-terminal domain comprising the last 24 amino acids mediates Mig1-dependent repression of a reporter gene. This effector domain contains several leucine-proline dipeptide repeats. We further found that inhibition of Mig1 activity in the absence of glucose is mediated by two internal elements in the Mig1 protein. A Mig1-VP16 hybrid activator was used to further investigate how Mig1 is regulated. Mig1-VP16 can activate transcription from promoters containing Mig1-binding sites and suppresses the inability of Snf1-deficient cells to grow on certain carbon sources. We found that a deletion of the SNF1 gene increases the activity of Mig1-VP16 fivefold under derepressing conditions but not in the presence of glucose. This shows that the hybrid activator is under negative control by the Snf1 protein kinase. Deletion mapping within Mig1-VP16 revealed that regulation of its activity by Snf1 is conferred by the same internal elements in the Mig1 sequence that mediate inhibition of Mig1 activity in the absence of glucose.

Amino Acid Sequence↗
PubMed1996

Islet constitutive nitric oxide synthase: biochemical determination and regulatory function.

Recent immunohistochemical findings suggested that a constitutive nitric oxide synthase (cNOS) resides in endocrine pancreas. Here we provide direct biochemical evidence for the presence of cNOS activity in isolated islets. The regulating influence of this nitric oxide synthase (NOS) activity for islet hormone release was also investigated. We observed that cNOS activity could be quantitated in islet homogenates by monitoring the formation of L-citrulline from L-arginine using an Amprep CBA cation-exhange minicolumn before derivatization with o-phthaldialdehyde and subsequent high-performance liquid chromatography analysis. The islet NOS was dependent on both Ca2+ and calmodulin and suppressed by the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). This effect was enantiomerically specific. Islet insulin release induced by a mixture of L-arginine and glucose was enhanced by L-NAME, whereas L-arginine-induced glucagon release was inhibited. The effect of L-NAME on insulin release was dose dependently potentiated by increasing glucose concentrations, suggesting that glucose is an important regulator of islet NO production. Complementary in vivo studies showed similar results, i.e., the insulin secretory response to a mixture of glucose and L-arginine was extremely enhanced by pretreatment with L-NAME, whereas L-arginine-stimulated glucagon response was suppressed. Finally, in isolated islets, the intracellular nitric oxide (NO) donor hydroxylamine suppressed insulin release and increased glucagon release. In summary, the islets of Langerhans contain a constitutive, Ca2+/calmodulin-dependent isoform of NOS. Islet NO suppressed insulin but enhanced glucagon secretion. The data also suggest a negative feedback by NO on glucose-induced insulin release. The islet NO system is a novel and important regulatory factor in insulin and glucagon secretion.

Animals↗
PubMed1995

Anatomical basis for interactions of enkephalins with other transmitters in the CNS of a snail.

Immunocytochemical techniques for double staining were employed to investigate the morphological basis for interactions between enkephalins and other neuroactive compounds in the behavior of the gastropod mollusc Cepaea nemoralis. Coexistence of each of the two enkephalins with FMRFamide, serotonin or GABA-like immunoreactivity was found in certain neurons in cerebral, parietal, and pedal ganglia. Tyrosine hydroxylase-immunoreactive neurons were occasionally seen in close apposition to, but never colocalized with, the enkephalins. A comparison between these anatomical observations and previous behavioral studies suggests that in gastropod molluscs cotransmission of enkephalins with classical transmitters may, at least partly, reflect synergism of these substances in the control of definite behavioral programs.

Animals↗
PubMed1995

Complex avoidance behaviour and its neurochemical regulation in the land snail Cepaea nemoralis.

1. In hot plate experiments, the pulmonate land snail Cepaea nemoralis displays a biphasic passive/active avoidance behaviour composed of retraction and subsequent searching mediated by antagonistic muscular systems. The switch, between the behaviours, is under neuronal control. 2. Leu- and met-enkephalin, as well as FMRFamide-antiserum, attenuated the retraction response and potentiated the searching behaviour. Opposite effects were achieved by injection of antisera to the enkephalins. 3. Both retraction and searching behaviours were potentiated by 5-HT. Methysergide antagonized the effects of the enkephalins on the searching behaviour. 4. We conclude that endogenous opioids act antagonistic to FMRFamide in the neuronally controlled switch between passive and active avoidance behaviour.

Animals↗
PubMed1995

NO-synthase: what can research on invertebrates add to what is already known?

The present study attempts to review presently known data regarding the distribution of nitric oxide (NO) synthase and the function of NO in invertebrate species. NO is synthesized from L-arginine by the enzyme NO-synthase, and activates guanylate cyclase which in turn leads to an increase in levels of cGMP in target cells. Major contributions to the knowledge of NO as a messenger molecule in invertebrates have been made by NADPH-diaphorase histochemistry and biochemical assays. These techniques suggest the presence of a L-arginine/NO pathway in a variety of tissues, thus implicating multiple roles for NO in invertebrates.

Animals↗
PubMed1995

Perioperative autotransfusion and functional coagulation analysis in total hip replacement.

Functional coagulation analyses like Sonoclot and thromboelastography have not been evaluated during perioperative autotransfusion. We have prospectively studied three different transfusion regimes in 45 patients undergoing total hip arthroplasty. Blood losses were replaced either with heterologous erythrocyte concentrate (group I), intra- and postoperative autotransfusion of blood salvaged with cellsaver technique (group II) or predonated autologous erythrocyte concentrates together with salvaged blood (group III). Routine and functional coagulation analyses with a Sonoclot were performed preoperatively, 6 hours postoperatively (6 h), day 1-5 and 10. An early postoperative hypo- and late postoperative hypercoagulative phase could be detected with Sonoclot signs of platelet function and fibrin deposition in all groups. Sonoclot coagulation analyses better correlated to both blood loss and dextran dosage than APTT and platelet count in the routine coagulation analyses. Functional coagulation analysis has a potential use in individualizing plasmasubstitution and thromboprophylaxis regimes during autotransfusion in THR.

Blood Coagulation↗
PubMed1994

NADPH-diaphorase histochemistry and nitric oxide synthase activity in deutocerebrum of the crayfish, Pacifastacus leniusculus (Crustacea, Decapoda).

The activity of an nitric oxide synthase in the deutocerebrum of the crayfish Pacifastacus leniusculus was investigated with histochemical and biochemical methods. By using the NADPH-diaphorase histochemical reaction, known as a selective marker for NO synthase in mammals, it was possible to localize specific neuronal elements in the crayfish. Pronounced diaphorase-staining was observed in peripheral olfactory sensory cells and in the neuropil of the olfactory lobes. Less intense diaphorase-staining also occurred in other deutocerebral neuropils, such as the accessory lobes, the lateral antennular neuropil and in the deutocerebral commissure neuropil. The biochemical assay revealed a calcium/calmodulin-dependent formation of citrulline from L-arginine in brain homogenate. It was also possible to show that the selective NO synthase inhibitor L-NOARG decreased the formation of citrulline. These data indicate a role for NO as an intercellular messenger in the crayfish.

Amino Acid Oxidoreductases↗
PubMed1994

Mevalonate is essential for growth activation of human fibroblasts: evidence for a critical role of protein glycosylation in the prereplicative period.

Human diploid fibroblasts, arrested following serum or mevalonate depletion, were restimulated to a maximal rate of DNA synthesis within 24 h after the addition of serum or mevalonate, respectively. In both cases the initiation of DNA synthesis was preceded by a 12-h prereplicative phase. Upon the stimulation with serum there was a rapid increase in HMG-CoA reductase activity, reflecting an elevated formation of mevalonate, which reached its maximal value 4 h after serum replenishment. If this serum-induced increase in mevalonate synthesis was inhibited, the subsequent initiation of DNA synthesis was prevented. Serum stimulation also increased the level of N-linked glycosylation, an event that was dependent on the increase in HMG-CoA reductase activity. After treatment of the cells with tunicamycin, an inhibitor of N-linked glycosylation, they failed to enter the S-phase. However, an increased level of N-linked glycosylation was not required during the whole of the period after serum stimulation. Instead, it seemed to be of critical importance only during the mid stage of the prereplicative phase (i.e., 4-8 h after stimulation). Our data suggest that the N-linked glycosylation required for initiation of DNA synthesis is of high-molecular-weight (90-240 kDa) proteins. These high-molecular-weight glycoproteins may include growth factor receptors. Indirect evidence raises the possibility that the expression of growth factor receptors may play a regulatory role in the mevalonate-dependent growth activation of human fibroblasts.

Cell Division↗
PubMed1994

Assay of neuronal nitric oxide synthase by HPLC determination of citrulline.

Biological membranes from different tissue sources were incubated for nitric oxide synthase (NOS) activity under standard conditions at 20 degrees C and compared with rat cerebellar cytosol. NOS activity was monitored as the formation of L-citrulline from L-arginine. Samples were purified on Amprep CBA cation-exchange minicolumns prior to derivatization with o-phthaldialdehyde (OPA) and HPLC analysis. The OPA derivatives of L-citrulline and L-arginine eluted well separated within 15 min during isocratic elution at room temperature. A linear relation between peak height and quantity of L-citrulline was seen down to the detection limit at 0.1 pmol L-citrulline. Formation of L-citrulline was measurable in rat cerebellar cytosol as well as in preparations not previously assayed for NOS activity, including rat colon, cat oesophagus and crayfish (Pacifastacus leniusculus) brain. The method provides a sensitive and non-radioactive method for assaying NOS activity in small tissue samples and in tissues with low to moderate levels of NOS activity.

Amino Acid Oxidoreductases↗
PubMed1993

Isoprenoid regulation of cell growth: identification of mevalonate-labelled compounds inducing DNA synthesis in human breast cancer cells depleted of serum and mevalonate.

Growth arrest induced by serum depletion and/or treatment with mevinolin (an inhibitor of mevalonate synthesis) in the human breast cancer cell line Hs578T was overcome by exogenous mevalonate, indicating that some product or metabolite of mevalonate may be involved in the mediation of serum-regulated growth of these cells. In the search for such compounds we first tested a variety of known end products of mevalonate with respect to their ability to counteract the inhibition of DNA synthesis caused by serum-free medium and mevinolin. Thereby high doses (10 micrograms/ml) of dolichol-20 were found to cause a partial counteraction. After straight-phase HPLC purification of endogenous lipids, isolated from 3H- or 14C-mevalonate-labelled Hs578T cultures, we found that non-sterol lipids co-eluting with dolichols efficiently induced DNA synthesis. After further purification with reverse-phase HPLC it was confirmed that virtually all of this effect was achieved by compounds(s) (seen as a single UV and radioactive peak) co-eluting with dolichol-20. Nanogram doses, at most, of this (these) compound(s) elicited a substantial stimulation of DNA synthesis. The lipid(s) also counteracted the inhibition by mevinolin of N-linked glycosylation, indicating that it (they) also interfere(s) with this processing. Since treatment with tunicamycin (an inhibitor of N-linked glycosylation) abolished this growth-stimulative effect, N-linked glycosylation seems to be a necessary event in the processes leading to lipid-induced initiation of DNA synthesis.

Blood↗
PubMed1993

Biogenic amines in coelenterates.

1. This mini review highlights the most important findings during three decades of research on biogenic amines in coelenterates. 2. Histochemical, analytic chemical and physiological evidences clearly indicate that dopamine is used as an intercellular messenger in hydrozoans. 3. The colonian anthozoan Renilla, has beta-adrenergic mechanisms in monitoring bioluminescence and serotoninergic mechanisms in rhythmic contractions.

Animals↗