| 1. |
- Deshpande, J, et al.
(författare)
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Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death.
- 1992
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Ingår i: Experimental brain research. Experimentelle Hirnforschung. Expérimentation cérébrale. - 0014-4819. ; 88:1, s. 91-105
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Tidskriftsartikel (refereegranskat)abstract
- The ultrastructural changes in the pyramidal neurons of the CA1 region of the hippocampus were studied 6 h, 24 h, 48 h, and 72 h following a transient 10 min period of cerebral ischemia induced by common carotid occlusion combined with hypotension. The pyramidal neurons showed delayed neuronal death (DND), i.e. at 24 h and 48 h postischemia few structural alterations were noted in the light microscope, while at 72 h extensive neuronal degeneration was apparent. The most prominent early ultrastructural changes were polysome disaggregation, and the appearance of electron-dense fluffy dark material associated with tubular saccules. Mitochondria and nuclear elements appeared intact until frank neuronal degeneration. The dark material accumulated with extended periods of recirculation in soma and in the main trunks of proximal dendrites, often beneath the plasma membrane, less frequently in the distal dendrites and seldom in spines. Protein synthesis inhibitors (anisomycin, cycloheximide) and an RNA synthesis inhibitor (actinomycin D), administered by intrahippocampal injections or subcutaneously, did not mitigate neuronal damage. Therefore, DND is probably not apoptosis or a form of programmed cell death. We propose that the dark material accumulating in the postischemic period represents protein complexes, possibly aggregates of proteins or internalized plasma membrane fragments, which may disrupt vital cellular structure and functions, leading to cell death.
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| 2. |
- Källberg, Eva, et al.
(författare)
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Analysis of somatic mutation activity in multiple V kappa genes involved in the response to 2-phenyl-5-oxazolone
- 1993
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Ingår i: International Immunology. - : Oxford University Press (OUP). - 0953-8178 .- 1460-2377. ; 5:6, s. 81-573
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Tidskriftsartikel (refereegranskat)abstract
- We have studied somatic mutation activity early in a response to 2-phenyl-5-oxazolone coupled to ovalbumin (phOx-OVA). Although the V kappa Ox1 gene rearranged to J kappa 5 is known to predominate in this response, other closely related V kappa genes are involved. We compared the introduction of point mutations into V kappa Ox1 genes and into a set of related V kappa genes rearranged to the same J kappa segment at two time points after primary immunization. The result showed that quantitation of mutations in a single rearrangement substrate leads to an underestimation of the total mutational activity. There is pronounced somatic mutation activity early within genes that may be absent later in the response. We also show that multiple somatic mutations can be detected in B cells from draining lymph nodes after foot-pad injection with phOx-OVA already at day 7 after immunization. The data suggest a system in which mutation acts early in the response on a wide range of substrates and that selection and expansion of high affinity paratopes occurs later.
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| 3. |
- Schwab, U E, et al.
(författare)
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Increased adherence of Staphylococcus aureus from cystic fibrosis lungs to airway epithelial cells.
- 1993
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Ingår i: The American review of respiratory disease. - 0003-0805. ; 148:2, s. 365-9
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Tidskriftsartikel (refereegranskat)abstract
- Airway colonization by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF). To assess the pathogenesis of selective colonization with this organism, we compared the capacity of S. aureus isolated from the respiratory tract of CF and non-CF patients to adhere to epithelial cells from the upper and lower airways of CF and control subjects. Bacterial adherence to bronchial epithelial cell lines was significantly greater for CF than for non-CF isolates (p < 0.001). Of 17 CF S. aureus isolates 12 adhered at a level > 1 bacterium per cell; this was true for only 1 of 14 non-CF isolates. CF S. aureus isolates also bound more avidly than non-CF isolates to ciliated (p < 0.05) and squamous nasal cells (p < 0.02) and buccal epithelial cells (p < 0.005) freshly harvested by scraping. Each S. aureus isolate bound with equal avidity to epithelial cells from CF patients and healthy individuals. Adherence was not related to sex, age, severity of pulmonary disease, presence of other microorganisms in the airways, or genotype of the CF hosts. Binding of S. aureus was blocked by proteinase treatment of organisms, suggesting that adherence is mediated by one or more peptide adhesins. We propose that the high prevalence of adherent S. aureus is due either to selection of adherent strains by CF airways or to induction of an adherent phenotype by factors residing at the CF airways surface.
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| 4. |
- Hillarp, Andreas, et al.
(författare)
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Cloning of cDNA coding for the beta chain of human complement component C4b-binding protein : sequence homology with the alpha chain
- 1990
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 87:3, s. 1183-1187
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Tidskriftsartikel (refereegranskat)abstract
- The major form of complement component C4b-binding protein, a regulator of the complement system, is composed of seven identical 70-kDa alpha chains, each containing a binding site for the complement protein C4b. We recently showed that C4b-binding protein also contains a unique 45-kDa beta chain. It is disulfide-linked to the central core and contains a binding site for the vitamin K-dependent protein S. We have now isolated and characterized full-length cDNA clones for the beta chain. In addition, 57% of the structure was determined by protein sequencing of tryptic and chymotryptic peptides. Two clones, A8 and C1, isolated from different libraries were sequenced. Except for a deleted triplet encoding Ala-3 in clone A8, the two clones were identical and coded for a leader sequence of 17 amino acids and a mature protein of 235 amino acids (including Ala-3). By N-terminal amino acid sequencing, the Ala-3 heterogeneity was confirmed and a third beta-chain species starting at Glu-4 was identified. The beta chain contains five potential N-linked glycosylation sites, and endoglycosidase digestion suggested that the beta chain contained multiple complex carbohydrate side chains. Northern blot analysis of human liver mRNA, using the beta-chain cDNA as the probe, demonstrated a major mRNA species of approximately 1.0 kilobase. From the N terminus, the beta chain contains three tandem repeat units (60 amino acids long) that are homologous to those present in the alpha chain. The C-terminal region, which was unrelated to the tandem repeats, demonstrated sequence similarity with the corresponding region of the alpha chain. In both alpha and beta chains these regions contain two cysteine residues that probably form the interchain disulfide bridges.
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| 5. |
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| 6. |
- Xu, X, et al.
(författare)
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Postreceptor events involved in the up-regulation of beta-adrenergic receptor mediated lipolysis by testosterone in rat white adipocytes.
- 1993
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Ingår i: Endocrinology. - 0013-7227. ; 132:4, s. 1651-7
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Tidskriftsartikel (refereegranskat)abstract
- In the previous studies we have shown that testosterone increases lipolytic responsiveness to catecholamines in rat white adipocytes, and that is associated with an up-regulation of beta-adrenergic receptor density. However, the postreceptor events involved in the testosterone induced enhancement of beta-adrenergic receptor activated lipolysis in these cells have not been adequately studied, and were therefore investigated in the present study. Male Sprague Dawley rats were divided into three groups: control, castrated, and castrated treated with testosterone. The beta-adrenergic receptor-mediated cAMP accumulation, measured with RIA after isoproterenol (a beta-adrenergic agonist) stimulation was decreased in castrated rats, and reversed by testosterone treatment, suggesting a testosterone effect at or proximal to adenylate cyclase. However, no differences between the groups were found in abundance of G alpha protein messenger RNAs (G alpha s, G alpha i-1, and G alpha i-2) as analyzed by Northern blot and a solution hybridization RNase protection assay, or in G protein mass measured with a quantitative enzyme-linked immunosorbent assay in fat cell membrane preparation. Lipolysis stimulated by N6-monobutyryl-cAMP was reduced in castrated rats and recovered by testosterone treatment, suggesting that components distal to the adenylate cyclase, i.e. protein kinase A (PKA) and/or hormone sensitive lipase (HSL) also are involved in testosterone regulation of lipolysis. In conclusion, these and previous results suggest that the testosterone-induced increase in lipolytic response to catecholamines in rat white adipocytes is mediated through several events including an increased beta-adrenergic receptor density, probably an increased adenylate cyclase activity and an increased protein kinase A/hormone sensitive lipase activity at the postreceptor level with apparent absence of effect on the expression of G-proteins.
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| 7. |
- Fåhraeus, R, et al.
(författare)
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Response to cAMP levels of the Epstein-Barr virus EBNA2-inducible LMP1 oncogene and EBNA2 inhibition of a PP1-like activity.
- 1994
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Ingår i: The EMBO journal. - 0261-4189. ; 13:24, s. 6041-51
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Tidskriftsartikel (refereegranskat)abstract
- The expression of the Epstein-Barr virus LMP1 oncogene is regulated by viral and non-viral factors in a tissue dependent fashion. The virus encoded transcription factor EBNA2 induces its expression in human B-cells. However, this induction also requires the contribution of cellular and/or other viral factors. In nasopharyngeal carcinoma cells and in cells from Hodgkin's lymphoma, LMP1 gene transcription is independent of viral products. Here we show that the effect of a factor binding to a cAMP responsive-like element (CRE) in the LMP1 gene transcription regulatory sequence (LRS) is essential for efficient promoter activity in the DG75 B-cell line and that elevation of cAMP levels in the cells induces LRS-derived CAT activity in a CRE dependent fashion. Incubation of two EBV-immortalized B-cell lines expressing endogenous EBNA2A with 8-Br cAMP increased the levels of the latency associated 66 kDa LMP1 within 2 h. Interestingly, LMP1 expression in DG75 cells conferred resistance to the inhibitory effect of 8-Br cAMP on cell proliferation. The protein phosphatase 1 and 2A (PP1 and PP2A, respectively) inhibitor okadaic acid also stimulated LRS-CAT activity in DG75 cells. EBNA2A from an EBV-immortalized B-cell line co-immunopurified with a PP1-like protein. An EBNA2A fragment spanning residues 324-436 fused to the GST protein specifically rescued a PP1/PP2A-like component from DG75 cell extracts. This GST-EBNA2A fusion product inhibited a PP1-like activity in nuclear extracts from these cells.
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| 8. |
- Dallman, M. J., et al.
(författare)
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Cytokine gene expression : analysis using northern blotting, polymerase chain reaction and in situ hybridization
- 1991
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Ingår i: Immunol Rev. ; 119, s. 163-79
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Tidskriftsartikel (refereegranskat)abstract
- We describe here the use of northern blotting, PCR and in situ hybridization for the analysis of cytokine gene expression. These techniques, each with their advantages and disadvantages, have been used to monitor cytokine gene expression in sites of immune reactivity and in the developing thymus. Whilst expression of a gene usually correlates well with protein production from that gene, this may not always be the case. The development of methods to analyze protein production in situ, for instance by immunohistochemistry, together with analysis of mRNA expression will allow us to begin to understand the role of cytokines within the immune system of the intact animal.
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| 9. |
- Åström, Anders, et al.
(författare)
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A simple procedure for isolation of eukaryotic mRNA polyadenylation factors
- 1991
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 202:3, s. 765-773
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Tidskriftsartikel (refereegranskat)abstract
- We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.
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| 10. |
- Åström, Jonas, et al.
(författare)
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In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:10, s. 3067-3071
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Tidskriftsartikel (refereegranskat)abstract
- We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.
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