Biomedical subjects

J E Eriksson

Publications and source records attributed to J E Eriksson.

At least 19 recordsLinked to original sources

PubMed2001

Human inhibitor of apoptosis protein (IAP) survivin participates in regulation of chromosome segregation and mitotic exit.

A signaling cascade termed the "spindle checkpoint" monitors interactions between the kinetochores of chromosomes and spindle microtubules to prevent precocious separation of sister chromatids. We have investigated the role of human inhibitor of apoptosis protein (IAP) surviving in regulation of cell division. We demonstrate that HeLa and PtK1 cells transfected or microinjected with surviving anti-sense oligonucleotides produce significantly more polyploid and micronucleated progeny cells and show abortive mitosis when treated with spindle poisons. Furthermore, perturbation of surviving function in HeLa and PtK1 cells with anti-surviving antibodies at the beginning of mitosis affects the normal timing of separation of sister chromatids and disturbs the 3F3/2 phosphoepitope-recognized tension sensing mechanism of the spindle checkpoint. This leads to premature separation of sister chromatids, which results in an uneven distribution of chromosomes between the newly formed progeny cells-an event associated with tumor formation in many cell types. Finally, cells injected with anti-surviving antibody exit mitotic block induced with microtubule drugs. Our data suggest that surviving protein may function within the spindle checkpoint pathway.

Antibodies↗

PubMed2001

Activation of the MKK4-JNK pathway during erythroid differentiation of K562 cells is inhibited by the heat shock factor 2-beta isoform.

In this study we report the activation of c-Jun N-terminal kinase (JNK) in human K562 erythroleukemia cells undergoing hemin-mediated erythroid differentiation, which occurs concomitantly with activation of heat shock factor 2 (HSF2) and leads to a simultaneous in vivo phosphorylation of c-Jun. The activation of JNK occurs through activation of mitogen-activated protein kinase kinase (MKK) 4 and not by activation of MKK7 or inhibition of JNK-directed phosphatases. We have previously shown that overexpression of the HSF2-beta isoform inhibits the activation of HSF2 upon hemin-induced erythroid differentiation. Here we demonstrate that HSF2-beta overexpression blocks the hemin-induced activation of the MKK4-JNK pathway, suggesting an erythroid lineage-specific JNK activation likely to be regulated by HSF2.

Anisomycin↗

PubMed2001

Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1.

Heat shock factor 1 (HSF1) is a serine-rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA-binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild-type HSF1 when expressed in hsf1(-/-) cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.

Antibodies↗

PubMed2001

Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase.

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

Amino Acid Sequence↗

PubMed2001

MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors.

The tumor necrosis factor (TNF), Fas, and TNF-related apoptosis-inducing ligand (TRAIL) receptors (R) are highly specific physiological mediators of apoptotic signaling. We observed earlier that a number of FasR-insensitive cell lines could redirect the proapoptotic signal to an anti-apoptotic ERK1/2 signal resulting in inhibition of caspase activation. Here we determine that similar mechanisms are operational in regulating the apoptotic signaling of other death receptors. Activation of the FasR, TNF-R1, and TRAIL-R, respectively, rapidly induced subsequent ERK1/2 activation, an event independent from caspase activity. Whereas inhibition of the death receptor-mediated ERK1/2 activation was sufficient to sensitize the cells to apoptotic signaling from FasR and TRAIL-R, cells were still protected from apoptotic TNF-R1 signaling. The latter seemed to be due to the strong activation of the anti-apoptotic factor NF-kappaB, which remained inactive in FasR or TRAIL-R signaling. However, when the cells were sensitized with cycloheximide, which is sufficient to sensitize the cells also to apoptosis by TNF-R1 stimulation, we noticed that adenovirus-mediated expression of constitutively active MKK1 could rescue the cells from apoptosis induced by the respective receptors by preventing caspase-8 activation. Taken together, our results show that ERK1/2 has a dominant protecting effect over apoptotic signaling from the death receptors. This protection, which is independent of newly synthesized proteins, acts in all cases by suppressing activation of the caspase effector machinery.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Production and specificity of mono and polyclonal antibodies against microcystins conjugated through N-methyldehydroalanine.

Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae). Their toxicity is associated with specific inhibition of intracellular protein phosphatases type-1 and type-2A (PP1 and PP2A, respectively). We have developed a battery of antibodies to microcystins using chemical modification (aminoethylation) of one of its core amino acids, N-methyl-dehydroalanine. The developed antibodies displayed different reactivities to closely related MCs. Selected monoclonal antibodies were used for quantitative competitive ELISA assays. The analytical sensitivity of these assays was up to 1 ng/ml. Comparison of the developed ELISA tests with HPLC-based measurements of MCs in laboratory and field samples showed a good correspondence between the results yielded by these two methods. The antibodies developed by this technique provide the means for developing extremely sensitive and specific analytical assays for direct measurement of toxins in cyanobacterial or water samples.

Alanine↗

PubMed2001

A time-resolved fluoroimmunometric assay for the detection of microcystins, cyanobacterial peptide hepatotoxins.

An immunoassay based on the time-resolved fluorometry (TR-FIA) was developed for microcystins, cyanobacterial peptide hepatotoxins. The assay was performed in a competitive mode and it utilised the monoclonal antibodies raised against microcystin-LR, and a europium chelate of microcystin-LR as a competitive antigen. The sensitivity of the assay was 0.1microg/l. The detection method of TR-FIA was compared to a commercially available kit based on the enzyme-linked immunosorbent assay (ELISA). The same level of sensitivity could be obtained with TR-FIA (in a non-optimised system). The simplified method of TR-FIA leads to a shorter analysis time.

Antibodies, Monoclonal↗

PubMed2001

Production and specificity of monoclonal antibodies against nodularin conjugated through N-methyldehydrobutyrine.

Nodularin (Nod) is a cyclic pentapeptide hepatotoxin produced by the cyanobacterial genus Nodularia living in brackish waters and coastal lagoons. The toxicity of Nod is due to specific inhibition of the type-1 and type-2A intracellular protein phosphatases (PP1 and PP2A, respectively). We have developed a monoclonal antibody against Nod using chemical modification (aminoethylation) of one of its core amino acids, N-methyldehydrobutyrine. The developed antibody is highly specific for Nod, with negligible reactivity to the closely related cyanobacterial toxin microcystin (MC). The monoclonal antibody was employed for quantitative competitive ELISA assay. The analytical sensitivity of the assay was up to 0.2 ng/ml. Comparison of the developed ELISA test with HPLC-based measurements of Nod, with both laboratory and field samples, showed a good correspondence between the results yielded by these two methods. The antibodies developed by this technique provide means for developing extremely sensitive and specific analytical assays for direct measurement of nodularin and related toxins in cyanobacterial or water samples.

Alanine↗

PubMed2001

Disturbances in hepatic cell-cycle regulation in mice with assembly-deficient keratins 8/18.

Simple epithelial tissues such as liver and pancreas express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. K8 and K18 null mice and transgenic mice that express mutant K18 (K18C) manifest several hepatocyte abnormalities and demonstrate that K8/18 are important in maintaining liver tissue and cell integrity, although other potential functions remain uncharacterized. Here, we report an additional abnormal liver phenotype, which is similar in K8 null, K18 null, and K18C mouse models. Liver histologic examination showed large polynuclear areas that lacked cell membranes, desmosomal structures, and filamentous actin. Similar, but less prominent, areas were observed in the pancreas. The parenchyma outside the polynuclear areas displayed irregular sinusoidal structures and markedly enlarged nuclei. Most K8 null hepatocytes were positive for the proliferating cell nuclear antigen (PCNA) with a doubled DNA content in comparison with the predominantly PCNA-negative wild-type hepatocytes. The distribution of the 14-3-3zeta protein was also altered in K8 null mice. Taken together, our results indicate that absence of keratin filaments causes disturbances in cell-cycle regulation, driving cells into the S-G2 phase and causing aberrant cytokinesis. These effects could stem from disturbed functions of K8/18-dependent cell-cycle regulators, such as the signaling integrator, 14-3-3.

Actins↗

PubMed2001

Primary chondrocytes resist hydrostatic pressure-induced stress while primary synovial cells and fibroblasts show modified Hsp70 response.

OBJECTIVE: During joint loading, chondrocytes in the articular cartilage are subjected to gradients of high compressive hydrostatic pressure (HP). In response to diverse chemical or physical stresses, heat shock genes are induced to express heat shock proteins (Hsps). This study sought to examine the role of Hsps in baroresistance in primary bovine chondrocytes and synovial cells, as well as in primary human fibroblasts. METHODS: Northern blotting was used to analyze the steady-state levels of hsp70 mRNA in the primary cells exposed to HP or heat stress. Hsp70 protein accumulation was analyzed by Western blotting, and the DNA-binding activity was examined by gel mobility shift assay. RESULTS: Primary bovine chondrocytes which have been adapted to live under pressurized conditions showed negligible Hsp70 response upon HP loading, whereas primary bovine synovial cells and human fibroblasts accumulated hsp70 mRNA and protein when subjected to HP. The response was initiated without activation of the heat shock transcription factor 1. Interestingly, pre-conditioning of the barosensitive fibroblasts with HP or heat shock reduced the Hsp70 response, indicating induction of baroresistance. CONCLUSION: This study suggests that Hsp70 can play an important role in the early stages of adaptation of cells to HP. Thus, the Hsp70 gene expression upon HP loading may serve as one indicator of the chondrocytic phenotype of the cells. This can be of use in the treatment of cartilage lesions.

Animals↗

PubMed2001

The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle.

Intermediate filament (IF) proteins show specific spatial and temporal expression during development of skeletal muscle. Nestin, the least known muscle IF, has an important role in neuronal regeneration. Therefore, we analyzed the expression pattern of nestin as related to that of vimentin and desmin during skeletal muscle regeneration. Nestin and vimentin appear at 6 h post-injury in myoblasts, with maximum expression around day 3-5 post-injury. Thereafter, vimentin expression ceases completely, whereas that of nestin is downregulated to remain only in the sarcoplasm next to neuromuscular and myotendinous junctions. Desmin appears at 6-12 h post-injury and becomes the predominant IF in myofibers simultaneously with the appearance of cross-striations. The expression pattern and colocalization of nestin and vimentin, known to form heteropolymers, suggests that they are essential during the early dynamic phase of the myofiber regeneration when migration, fusion, and structural modeling of myogenic cells occurs, whereas desmin is responsible for keeping myofibrils in register in mature myofibers. In conclusion, the expression of nestin is dynamically orchestrated with that of vimentin and desmin during skeletal muscle regeneration and recapitulates that seen during myogenesis, i.e. these IFs have key functional roles in the construction and restoration of skeletal myofibers.

Animals↗

PubMed2000

MAPK/ERK signaling in activated T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly.

When T cells are activated, the expression of the CD95 ligand is elevated, with the purpose of inducing apoptosis in target cells and to later eliminate the activated T cells. We have shown previously that mitogen-activated protein kinase (MAPK or ERK) signaling suppresses CD95-mediated apoptosis in different cellular systems. In this study we examined whether MAPK signaling controls the persistence and CD95-mediated termination of an immune response in activated T cells. Our results show that activation of Jurkat T cells through the T cell receptor immediately suppresses CD95-mediated apoptosis, and that this suppression is mediated by MAPK activation. During the phase of elevated MAPK activity, the activation of caspase-8 and Bid is inhibited, whereas the assembly of a functional death-inducing signaling complex (DISC) is not affected. These results explain the resistance to CD95 responses observed during the early phase of T cell activation and suggest that MAPK-activation deflects DISC signaling from activating caspase-8 and Bid. The physiological relevance of the results was confirmed in activated primary peripheral T cells, in which inhibition of MAPK signaling markedly sensitized the cells to CD95-mediated apoptosis.

Apoptosis↗

PubMed2000

Differences in spindle association of the mitotic checkpoint protein Mad2 in mammalian spermatogenesis and oogenesis.

We have investigated expression and subcellular localization of the spindle checkpoint protein Mad2 during rat and mouse spermatogenesis and in superovulated mouse oocytes. Our immunofluorescence studies demonstrate substantial differences in the localization patterns of kinetochore-associated Mad2 in these meiotic systems compared with previous studies of mitosis. In addition, the association of Mad2 with second-division-metaphase kinetochores differed significantly in male versus female meiosis. In spermatogenesis, Mad2 remained at most kinetochores throughout the entire first meiotic division and was lost only at metaphase of the second meiotic division. This result indicates that loss of kinetochore-associated Mad2 is not essential for the metaphase-to-anaphase transition during the first meiotic division. Disruption of the male meiotic spindles with the microtubule depolymerizing agent nocodazole resulted in the appearance of Mad2 at nearly all kinetochores. In contrast, the microtubule stabilizer taxol induced the loss of Mad2 from the majority of the first-division-metaphase kinetochores in which it was normally present in untreated cells. In contrast to the situation in spermatogenesis, Mad2 persisted at the kinetochores of normal, second-division oocytes at metaphase. These findings suggest that the role of the kinetochore in signaling in the spindle checkpoint may differ markedly between mammalian mitosis and meiosis, between the two meiotic divisions, and between male and female meiosis.

Animals↗

PubMed2000

Protein synthesis is required for stabilization of hsp70 mRNA upon exposure to both hydrostatic pressurization and elevated temperature.

We have recently described that in chondrocytic cells high hydrostatic pressure (HP) causes a heat shock response via mRNA stabilization without a transcriptional activation of the hsp70 gene. In this study, we investigated whether this exceptional regulatory mechanism occurs more generally in different types of cells. Indeed, hsp70 mRNA and protein accumulated in HeLa, HaCat and MG-63 cells under 30 MPa HP, without DNA-binding of heat shock transcription factor 1 (HSF1) to the heat shock element of the hsp70 gene or formation of nuclear HSF1 granules, revealing a lack of transcriptional activation. Moreover, we observed that protein synthesis is needed for mRNA stabilization. Thus, high HP offers a model to study the mechanisms of hsp70 mRNA stabilization without HSF1-mediated induction of the heat shock gene response.

HSP70 Heat-Shock Proteins↗

PubMed2000

Microcystin-LR toxicodynamics, induced pathology, and immunohistochemical localization in livers of blue-green algae exposed rainbow trout (oncorhynchus mykiss).

With this retrospective study, we investigated the temporal pattern of toxin exposure and pathology, as well as the topical relationship between hepatotoxic injury and localization of microcystin-LR, a potent hepatotoxin, tumor promoter, and inhibitor of protein phosphatases-1 and -2A (PP), in livers of MC-gavaged rainbow trout (Oncorhynchus mykiss) yearlings, using an immunohistochemical detection method and MC-specific antibodies. H&E stains of liver sections were used to determine pathological changes. Nuclear morphology of hepatocytes and ISEL analysis were employed as endpoints to detect the advent of apoptotic cell death in hepatocytes. Trout had been gavaged with lyophilized cyanobacteria (Microcystis aeruginosa, strain PCC 7806) at acutely toxic doses of 5700 microg microcystin (MC) per kg of body weight (bw), as described previously (Tencalla and Dietrich, 1997). Briefly, 3 control and 3 test animal were killed 1, 3, 12, 24, 48, and 72 h after bolus dosing, and livers were fixed and paraffin embedded for histological analysis and later retrospective histochemical analyses. The results of the immunohistochemistry reported here revealed a time dependent, discernible increase in MC-positive staining intensity throughout the liver, clearly not concurring with the kinetics of hepatic PP inhibition observed in the same fish and reported in an earlier publication by Tencalla and Dietrich (1997). After 3 h, marked and increasing MC-immunopositivity was observed in the cytoplasm, as well as the nuclei of hepatocytes. Apoptotic cell death could be detected after 48 h, at the very earliest. These data suggest that accumulation of MC and subsequent changes in cellular morphology, PP inhibition, and hepatocyte necrosis represent the primary events in microcystin induced hepatotoxicity and appear to be associated with the reversible interaction of MC with the PP. In contrast, apoptotic cell death, as demonstrated here, seems to be of only secondary nature and presumably results from the covalent interaction of MC with cellular and nuclear PP as well as other thiol containing cellular proteins.

Animals↗

PubMed2000

Effector caspases are dispensable for the early nuclear morphological changes during chemical-induced apoptosis.

Nuclear morphological changes during apoptosis are very distinct and effector caspases have been implicated to play a central role in these processes. To investigate this in greater detail we examined the effect of blocking caspase activity and its activation on the nuclear morphological change in Jurkat T cells undergoing apoptosis after staurosporine treatment. In the presence of caspase inhibitors, like benzyloxycarbonyl-Val-Ala-Asp fluoro-methylketone (z-VAD-FMK), N-acetyl Tyr-Val-Ala-Asp chloromethylketone (Ac-YVAD-CMK) and benzyloxy-carbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-FMK), staurosporine-treated Jurkat cells displayed a nuclear morphological change distinct from that of normal and apoptotic cells. This nuclear morphological change is an early event, characterised by convoluted nuclei with cavitations, and clumps of chromatin abutting to inner regions of the nuclear envelope between the nuclear pores. Both the nuclear envelope and endoplasmic reticulum were grossly dilated. This pre-apoptotic nuclear change precedes the externalisation of phosphatidylserine, chromatin condensation and DNA laddering, and can be dissociated from the formation of high molecular weight DNA fragments and cell shrinkage. Although cytochrome c efflux from the mitochondria and the processing of caspase-3 were observed in Jurkat cells with pre-apoptotic nuclear morphology, caspase-2, -6, -7 and -8 were not activated. In the presence of z-DEVD-FMK or Ac-YVAD-CMK, caspase-3 was processed to both the p17 and p20 fragments in staurosporine-treated cells, but only to p20 fragment in the presence of z-VAD-FMK. However, the caspase-3 substrate, poly(ADP ribose) polymerase was not cleaved in the presence of z-VAD-FMK, despite >70% of the cells have pre-apoptotic nuclei. In addition, caspase-3 null MCF-7 cells also undergo pre-apoptotic nuclear change when treated with staurosporine in the presence of caspase inhibitors, indicating that caspase-3 is not required for the early nuclear morphological change in cells undergoing apoptosis. Although cell death in staurosporine-treated Jurkat cells was markedly delayed, they eventually die without discernible downstream apoptotic features. Other apoptotic stimuli like etoposide and the heavy metal chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine also induced this nuclear morphological change in Jurkat cells in the presence of z-VAD-FMK. In summary, the effector caspases are not involved in early nuclear morphological change, which precedes the conventional hallmark morphological changes associated with chemical-induced apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Phosphorylation-Based signaling in Fas receptor-mediated apoptosis.

Apoptosis or programmed cell death plays an essential role during development of the immune system, in immune responses, and in the control of tissue homeostasis in the adult. An important physiological mediator of apoptosis is the Fas/APO-1/CD95 receptor (FasR), a surface receptor belonging to the tumor necrosis factor receptor family. Apoptosis consists of a series of characteristic features that occur following activation of caspases, a collective term for apoptosis-specific proteases. The focus in FasR research has been on determining the mechanisms resulting in caspase activation. However, the role of phosphorylation-based signaling has received increasing attention both as an outcome of FasR activation and as a factor regulating FasR responses. Tyrosine-directed phosphorylation has been implicated to be induced and required during FasR stimulation. The FasR also activates all major signaling pathways that belong to the family of mitogen-activated protein kinase (MAPK) pathways, by either caspase-independent or -dependent mechanisms. Furthermore, phosphorylation-based signaling serves as a potent modifier of FasR responses. In this respect, especially the extracellular signal-regulated kinase and the phosphoinositide 3-kinase signaling pathways have been established as important regulators. This type of control seems to be directly phosphorylation-mediated without the requirement of newly synthesized proteins. Signaling through phosphorylation also regulates the expression of the Fas ligand (FasL), the FasR, as well as various other proteins that affect the outcome of receptor stimulation. While the involvement of phosphorylation has been established in FasR responses, the targets, molecular mechanisms, and biological significance of this aspect of the FasR signaling machinery still require further elucidation.

Apoptosis↗

PubMed1999

Intermediate filament protein partnership in astrocytes.

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.

Animals↗