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Bodil Norrild

Publications and source records attributed to Bodil Norrild.

12 recordsLinked to original sources
PubMed2005

Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping.

Decapping is a key step in mRNA turnover. However, the composition and regulation of the human decapping complex is poorly understood. Here, we identify three proteins that exist in complex with the decapping enzyme subunits hDcp2 and hDcp1: hEdc3, Rck/p54, and a protein in decapping we name Hedls. Hedls is important in decapping because it enhances the activity of the catalytic hDcp2 subunit and promotes complex formation between hDcp2 and hDcp1. Specific decapping factors interact with the mRNA decay activators hUpf1 and TTP, and TTP enhances decapping of a target AU-rich element (ARE) RNA in vitro. Each decapping protein localizes in cytoplasmic processing bodies (PBs), and overexpression of Hedls produces aberrant PBs and concomitant accumulation of a deadenylated ARE-mediated mRNA decay intermediate. These observations suggest that multiple proteins involved in human decapping are important subunits of PBs and are activated on ARE-mRNAs by the protein TTP.

Cell Line↗
PubMed2005

Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation.

Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1-tandelta0/tandeltaN] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.

Cell Membrane↗
PubMed2005

The 3' region of human papillomavirus type 16 early mRNAs decrease expression.

BACKGROUND: High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome. METHODS: We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome was inserted into the 3' UTR of the reporter mRNA. Pools containing thousands of independent integrations were tested for the steady state levels of the reporter mRNA by Real Time PCR and reporter protein by a GUS enzymatic activity assays. In addition, we tested the cellular distribution and half lives of the reporter mRNAs. The integrity of the reporter mRNAs were tested by northern blotting. RESULTS: We show that the 3' region of the HPV-16 early mRNAs (HPV-16 nucleotide (nt.) 2582-4214) act in cis to decrease both mRNA and protein levels. This region seems to affect transcription from the exogenous minimal CMV promoter or processing of the reporter mRNA. The observed repression was most pronounced at the protein level, suggesting that this sequence may also affect translation. For the HPV types: 2, 6, 11, 13, 18, 30, 31, and 35 we have investigated the regulatory effect of the regions corresponding to the HPV-16 nt. 3358-4214. For all types, except HPV-18, the region was found to repress expression by posttranscriptional mechanisms. CONCLUSION: We find that the 3' region of HPV-16 early mRNAs interfere with gene expression. It is therefore possible that the deletion of the 3' part of early HPV-16 mRNAs occurring during cervical oncogenesis could contribute to transformation of cells through deregulation of the viral oncogene synthesis. Moreover, we find that the corresponding region from several other HPV types also repress expression, suggesting that the repression by this region may be a general feature of the HPV life cycle.

3' Untranslated Regions↗
PubMed2005

Herpes simplex virus-cell interactions studied by low-fading contrasted immunofluorescence.

The low-fading immunofluorescence with propidium iodide contrast described here is recommended for light and confocal viral antigen identification and other cell biology studies because: (1) it is a simple, rapid, sensitive, and reproducible technique; (2) phase-contrast microscopy is unnecessary; (3) contrast is optimal without blurring the fluorescent labeling; (4) autofluorescence is minimal, even in fixed cells; (5) background staining is minimal; (6) fading is invisible for at least 5-min exposures, even in preparations with weak antigen presentation; (7) fluorescence is stable after storage in the dark at -20 degrees C; (8) fluorochromes are small-sized markers without steric hindrance; and (9) there is no need for silver enhancement or substrate solutions, which increase the risk of diffusion and other artifacts.

Clinical Laboratory Techniques↗
PubMed2005

Herpes simplex virus-cell interactions studied by immunogold cryosection electron microscopy.

A technique is presented for high-resolution postembedding immunolocalization of one or two (or several) antigens in the same ultrathin cryosection using primary monoclonal antibodies from the same species. The optimized three-layer indirect immunogold-labeled cryosection electron microscopy described is recommended for studies of virus-cell interactions, because: (1) it is a simple and reproducible method; (2) colloidal gold markers are electron-dense, stable, and easy to recognize; (3) the membraneous ultrastructure and immunolabeling are well preserved; (4) immunolabeling is less in the two-layer method; (5) silver-enhanced gold particles vary in size and shape; (6) it is possible to demonstrate herpes simplex virus type 1 glycoproteins gC-1 and gD-1 in the nuclear membranes and gC-1- and gD-1-labeled viral particles in the perinuclear space and to observe virions in the endoplasmic reticulum and Golgi area. The use of buffered 3% paraformaldehyde plus 2% glutaraldehyde for 2 h at room temperature effectively destroys free anti-IgG binding sites on the secondary antibodies in double-labeling immunogold cryosection electron microscopy and is recommended because: (1) inactivation is obtained through buffered primary fixative; (2) the method is simple and reproducible; (3) cross-labeling is effectively avoided; (4) silver-intensification, high temperature, and methyl cellulose cover of ultrathin cryosections are avoided between the staining sequences; and (5) ultrastructure and antigenicity are well preserved.

Epithelial Cells↗
PubMed2004

Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation.

Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induction of cell death. We have used the osteosarcoma cell line U2OS cells provided with E7 and the cdk2 inhibitor p21 (cip1/waf1) under inducible control, as a model system for the analysis of E7-mediated apoptosis. Our data shows that simultaneous expression of E7 and p21 proteins induces cell death, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge, this is the first time a role for cathepsin B is reported in HPV-induced apoptotic signalling.

Apoptosis↗
PubMed2003

A promoter within the E6 ORF of human papillomavirus type 16 contributes to the expression of the E7 oncoprotein from a monocistronic mRNA.

Human papillomavirus type 16 (HPV-16) has the capacity to transform human primary keratinocytes. Maintenance of the transformed phenotype requires constitutive expression of the oncoproteins E6 and E7. The low-risk HPV types express E7 from monocistronic mRNA, but for the high-risk types, no mRNA that encodes E7 as the first open reading frame (ORF) has been identified. We recently identified a transcription initiation site within the E6 ORF of HPV-16 at nt 542. In the present study we have characterized the P542 promoter, which putatively controls monocistronic expression of E7. The monocistronic mRNA is not very abundant, but we have shown that an E7-luciferase fusion protein can be expressed in SiHa cells from a monocistronic HPV-16 transcript initiated at nt 542. The monocistronic mRNA expresses E7-luciferase more efficiently than the most abundant in vivo-like mRNA E6*IE7, initiated by P97 and spliced from nt 226 to 409. Furthermore, the translation initiation of E7 is most abundant from the monocistronic mRNA. We have also shown that the P542 promoter is downregulated by the transcription factor activator protein 4 (AP-4) and the differentiation-dependent factor hSkn-1a, both binding downstream of the transcription initiation site. In conclusion, we have found that P542 is a relatively weak promoter compared with P97 and may be downregulated in differentiated epithelial cells.

Animals↗
PubMed2003

Identification and characterization of a cluster of transcription start sites located in the E6 ORF of human papillomavirus type 16.

Human papillomavirus type 16 (HPV-16) is the prototype strain among the malignant types of HPV in the western world. The main promoter, P97, located in front of the E6 ORF, has been shown to control expression of the oncogenes E6 and E7. These oncogenes are expressed continuously in HPV-16-transformed cells. In contrast to malignant HPV types, non-malignant HPV types have separate promoters driving the expression of E6 and E7. Experiments have shown that the translation of E7 is more efficient from monocistronic than bicistronic transcripts encoding both E6 and E7. Here, identification of a cluster of transcription start sites located in the E6 ORF of HPV-16 is presented. Transcripts from this region contain the E7 ORF as the first reading frame. The cluster consists of multiple transcription start sites located around nt 441. Additional transcription start sites were identified in a cluster around nt 480. A transcription start site has been identified previously at nt 480 but has never been characterized further. The region responsible for transcription activity was mapped to nt 272-448. Mutational analysis showed that initiation of transcription is independent of a TATA-box element, which is consistent with the finding of multiple transcription start sites. Furthermore, it is shown that proteins from HeLa and SiHa nuclear cell extracts bind to the two regions at nt 291-314 and 388-411, and that these two regions influence transcription activity in a cell type-dependent manner.

Cell Line, Tumor↗
PubMed2003

The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells.

Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus-cell interactions. The properties of uninfected and HSV-1-infected L fibroblasts and gro29 cells investigated by protein assay, immunoblot, titration assay, immunofluorescence light microscopy and immunogold cryosection electron microscopy are reported. The ultrastructure of both HSV-1-infected L and gro29 cells confirmed primary envelopment of virions at the nuclear membranes followed by maturing multiple de-envelopments and re-envelopments in the endoplasmic reticulum and in the Golgi complex. The gro29 cells presented changed cytoskeleton, abolished egress of virions, and were defective in the trafficking of glycoproteins, giving rise to accumulation of viral particles and glycoproteins in the endoplasmic reticulum and the Golgi complex. The results suggest that gro29 cells harbour a causal underlying defect of the cytoskeleton in addition to the HSV-1-induced cytoskeletal changes.

Actins↗
PubMed2002

Morphologic, immunohistochemical, immunologic, ultrastructural, and time-related study of herpes simplex virus type 1-infected cultured human fibroblasts.

Membrane glycoproteins of enveloped animal viruses are synthesized, processed, and transported inside infected cells. Expression of viral glycoproteins on the surface of viral particles and host cells are essential for many biologic functions. In the case of herpes simplex virus, the glycoprotein molecules may act as nucleation points for virus assembly and budding at the nuclear membrane. The temporal distribution of herpes simplex virus type 1 particles and glycoproteins in cultured human fibroblasts was studied by titration plaque assay, immunoblots, immunofluorescence light microscopy, and immunogold cryosection electron microscopy to describe the virus-cell interactions. These concordant analyses revealed significant release of infectious viral particles to the medium at 6 hours postinfection, that the capacity of the host cells to make infectious viral particles was complete at 18 hours postinfection, and that the infection brought time-related modifications of tubulin, cell morphology, and viral glycoproteins. The data presented is in accord with the theory of envelopment at the nuclear membranes containing immature glycoproteins followed by multiple deenvelopments and reenvelopments of the virus particles during the transport and maturation in the endoplasmic reticulum and the Golgi complex.

Cells, Cultured↗
PubMed2002

Temporal morphogenesis of herpes simplex virus type 1-infected and brefeldin A-treated human fibroblasts.

BACKGROUND: Insights in the herpesvirus-cell interactions are of general cell biology interest, especially to studies of intracellular transport, and of considerable significance in the efforts to generate drugs, vaccines, and gene therapy. However, the pathway of virus particle egress and maturation is a contentious issue. MATERIALS AND METHODS: The intracellular transport was inhibited in cultured herpes simplex virus type 1 (HSV-1) infected human fibroblasts by brefeldin A (BFA). The virus-cell interactions including the viral envelopment, transport of HSV-1 virions, and transport of viral glycoprotein D (gD-1) and glycoprotein C (gC-1) were studied by titration assay, immunoblot, immunofluorescence light microscopy, and immunogold electron microscopy of cryosections. RESULTS: gD-1 and gC-1 were synthesized and normally transported to the plasma membranes of untreated HSV-1 infected host cells. BFA (1 microg/ml medium) effectively blocked the transport of the glycoproteins to the plasma membranes and affected the tubulin and vimentin of the cytoskeleton. Viral particles and glycoproteins accumulated in the perinuclear space and the endoplasmic reticulum of BFA treated cells. Withdrawal of BFA influence up to 9 hr resulted in restored tubulin and vimentin, transport of glycoproteins to the plasma membranes, and steady release of infectious viral particles to the extracellular space superior to the cellular assembly of new virions. The ultrastructural data presented support that the primary envelopment of viral particles occur at the nuclear membranes containing immature glycoproteins followed by multiple de-envelopments and re-envelopments of the virions during the transport and maturation in the endoplasmic reticulum and the Golgi complex. CONCLUSIONS: BFA-induced changes include the cytoskeleton with significant effect on HSV-1 maturation and egress. The data support a multiple-step envelopment of HSV-1 in a common pathway of glycoprotein synthesis and virion egress.

Antibodies, Monoclonal↗