| 1. |
- Belyaev, I Y, et al.
(författare)
-
Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis
- 1999
-
Ingår i: Biochimica et Biophysica Acta - General Subjects. - 0304-4165 .- 1872-8006. ; 1428:2-3, s. 348-356
-
Tidskriftsartikel (refereegranskat)abstract
- The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2. mg/ml, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with gamma-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation.
|
|
| 2. |
- Söderqvist, Henrik, et al.
(författare)
-
Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain
- 1997
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 250:3, s. 808-13
-
Tidskriftsartikel (refereegranskat)abstract
- The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121.
|
|
| 3. |
- Elmroth, Kerstin, 1970, et al.
(författare)
-
Radiation and hypothermia: changes in DNA supercoiling in human diploid fibroblasts
- 1999
-
Ingår i: Anticancer Res. - 0250-7005 .- 1791-7530. ; 19:6B, s. 5307-11
-
Tidskriftsartikel (refereegranskat)abstract
- The influence of hypothermia (2 degrees, 15 degrees and 28 degrees C) upon the effect of X-irradiation on chromatin from human diploid fibroblast cells (AG1518) was studied using the fluorescent halo assay. Rewinding of supercoils was inhibited in a dose-dependent manner when cells were irradiated with 4, 8 or 16 Gy. This inhibition of rewinding was reduced when cells were irradiated at subnormal temperatures compared with cells irradiated at 37 degrees C. One hour's preincubation at low temperature did not influence rewinding. When AG1518 cells were irradiated at 37 degrees C in the presence of the radical scavenger DMSO (0.5 M), the radiation-induced damage was reduced. No additional protection of DMSO in hypothermic cells (2 degrees C) was found, possibly indicating that OH-radical-mediated effects are more temperature dependent. These results are similar to those recently found for the malignant MCF-7 cell line.
|
|
| 4. |
- Bergström, R., et al.
(författare)
-
Trends in cancer of the cervix uteri in Sweden following cytological screening
- 1999
-
Ingår i: British Journal of Cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 81:1, s. 159-166
-
Tidskriftsartikel (refereegranskat)abstract
- Trends in cervical cancer incidence following the introduction of screening have mostly been studied using cross-sectional data and not analysed separately for squamous cell cancer and adenocarcinomas. Using Swedish nationwide data on incidence and mortality, we analysed trends during more than 3 decades and fitted Poisson-based age-period-cohort models, and also investigated whether screening has reduced the incidence of adenocarcinomas of the cervix. The incidence of reported cancer in situ increased rapidly during 1958-1967. Incidence rates of squamous cell cancer, fairly stable before 1968, decreased thereafter by 4-6% yearly in women aged 40-64, with a much smaller magnitude in younger and older women. An age-cohort model indicated a stable 70-75% reduction in incidence for women born 1940 and later compared with those born around 1923. The incidence of adenocarcinomas doubled during the 35-year study period. The mortality rate increased by 3.6% before 1968 and decreased by 4.0% yearly thereafter. Although a combination of organized and opportunistic screening can reduce the incidence of squamous cell cancer substantially, the incidence of adenocarcinomas appears uninfluenced by screening.
|
|
| 5. |
- Berndt, Kurt D, et al.
(författare)
-
Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water
- 1996
-
Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 24, s. 304-313
-
Tidskriftsartikel (refereegranskat)abstract
- The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using "realistic" potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.
|
|
| 6. |
- Brenden, N., et al.
(författare)
-
Differential MHC expression requirements for positive selection of separate TCR Vb families
- 1999
-
Ingår i: Immunogenetics. - : Springer Science and Business Media LLC. - 0093-7711 .- 1432-1211. ; 49:1, s. 1-6
-
Tidskriftsartikel (refereegranskat)abstract
- Positive selection has been proposed to be involved in protection from diabetes. We examined positive selection by fluorescence-activated cell sorter analyses in thymocytes of protected and susceptible E-transgenic and non-transgenic NOD mice. Three Vb families showed positive selection in E-transgenic mice. Vb6+CD4+ and Vb10+CD4+ thymocytes were found at higher frequencies in both protected NOD-Ea and susceptible NOD-DY mice. The increased frequencies of Vb13+CD8+ thymocytes were found in protected NOD-Ea mice only, and not in susceptible NOD-DY transgenic mice. These three Vb families were further examined in bone-marrow chimeras between NOD-Ea and non-transgenic NOD mice, where we could examine the contribution of E-expressing bone-marrow-derived cells in positive selection. We find that NOD-Ea→NOD-Ea chimeras have an increased positive selection of Vb13+CD8+ cells and that positive selection is more efficient when both thymic epithelium and bone-marrow-derived cells express the E molecule. This was also seen for Vb6+CD4+ cells. However, for Vb6, bone-marrow-derived cells alone were also capable of positive selection. Positive selection of Vb10+CD4+ cells was restricted to E-expressing thymic epithelium only. For Vb13+CD8+ cells, we found that positive selection is most efficient with E-expression on both thymic epithelium and bone-marrow-derived cells, although positive selection also occurs with E-positive epithelium only. For Vb6+CD4+ cells, the dominating selecting cells are bone-marrow-derived cells, and Vb10+CD4+ cells seem to be selected exclusively by the thymic epithelium. Thus, the conditions for positive selection seem to vary considerably between different Vb families.
|
|
| 7. |
- Brenden, N., et al.
(författare)
-
Disease-protected major histocompatibility complex Ea-transgenic non- obese diabetic (NOD) mice show interleukin-4 production not seen in susceptible Ea-transgenic and non-transgenic NOD mice
- 1998
-
Ingår i: Immunology. - : Wiley. - 0019-2805 .- 1365-2567. ; 95:1, s. 1-7
-
Tidskriftsartikel (refereegranskat)abstract
- The non-obese diabetic (NOD) mouse is an animal model for insulin- dependent diabetes that has many similarities to the human disease. NOD mice transgenic for the Ea gene, allowing expression of the E molecule, are protected from diabetes and rarely develop insulitis. An Ea transgene mutated in the promoter region, (ΔY) lacks E expression on most B cells, thymic medullary epithelium and primary antigen-presenting cells, and confers no protection whatsoever. We have used these transgenic NOD mice, together with non-transgenic NOD mice, to study the correlation of E expression and production of interleukin-4 (IL-4) and interferon-γ (IFN-γ). We show that protected E-transgenic NOD mice have elevated levels of IL-4 compared with non-transgenic mice, both in the thymus and in the periphery. However, susceptible ΔY-transgenic mice have elevated thymic IL-4 levels, but express almost as little IL-4 as non-transgenic NOD mice in the periphery. This drop in peripheral IL-4 production seen in ΔY-transgenic mice thus correlates with the decreased E expression in the periphery of ΔY-transgenic NOD mice. In contrast, there were no differences in IFN-γ production between the three NOD lines. We suggest that Ea-transgenic NOD mice have E-selected regulatory T cells producing IL-4, which are subsequently activated by E-expressing primary antigen-presenting cells in the periphery. This activation would then be instrumental for the E-mediated protection from disease in NOD mice. Such a process would explain the total absence of protection in ΔY-transgenic NOD mice, despite their widespread E expression.
|
|
| 8. |
- Brenden, N., et al.
(författare)
-
E expression is needed on both bone marrow derived cells and thymic epithelium to increase IL-4 production and achieve protection in NOD bone marrow chimeras
- 1999
-
Ingår i: Cytokine. - : Elsevier BV. - 1043-4666 .- 1096-0023. ; 11:10, s. 766-772
-
Tidskriftsartikel (refereegranskat)abstract
- The NOD mouse is an animal model for insulin-dependent diabetes with many similarities to the human disease. NOD mice which are transgenic for the Ea gene, allowing expression of the E molecule, are protected from diabetes and rarely develop insulitis. We have constructed bone marrow chimeras between transgenic and non-transgenic NOD mice to study the correlation of E expression on bone marrow derived cells and thymic epithelium vs the production of IL-4 and IFN-γ. We show that NOD-E→NOD-E and NOD-E→NOD chimeras have elevated levels of IL-4 compared to NOD→NOD and NOD→NOD-E chimeras in the thymus. However, in the periphery the protected NOD-E→NOD-E show much higher IL-4 levels than any of the other chimeras. This drop in peripheral IL-4 production seen in NOD-E→NOD, NOD→NOD-E and NOD→NOD chimeras correlates with the increased insulitis seen in these mice compared to NOD-E→NOD-E. In contrast, there were no differences in IFN-γ production between the chimeras. We suggest that the precommitted, regulatory T cells, selected in an E-expressing thymic environment, need continuous interaction with E-expressing primary antigen presenting cells in the periphery for optimal IL-4 production. Decrease in IL-4 production correlates with increased insulitis.
|
|
| 9. |
- Fang, Hong, et al.
(författare)
-
Identification of the metallo-beta-lactamase gene from clinical isolates of Bacteroides fragilis
- 1999
-
Ingår i: Anaerobe. - : Elsevier BV. - 1075-9964 .- 1095-8274. ; 5:3-4, s. 431-434
-
Tidskriftsartikel (refereegranskat)abstract
- Bacteroides fragilis is one of the organisms known to produce carbapenem-hydrolysing metallo-beta-lactamase, which can confer resistance to a wide variety of beta-lactams. The purpose of this study was to identify carbapenem-hydrolysing metallo-beta-lactamase-producing B. fragilis strains by means of PCR assay, nucleotide sequencing and enzyme inhibition studies. Ten beta-lactam-resistant B. fragilis isolates were investigated. Four imipenem-resistant strains among the 10 isolates gave positive reactions in the PCR assay. The nucleotide sequences of the PCR products from two imipenem-resistant strains shared >98% similarity with the metallo-beta-lactamase gene from B. fragilis TAL 3636, which was used as a control. The amino acid sequence homology between the two imipenem-resistant strains and B. fragilis TAL 3636 was 99.2%. These strains produced high amounts of Zn2+-dependent beta-lactamases which were inactivated by EDTA.
|
|
| 10. |
- Hammarström, A, et al.
(författare)
-
Solution structure of a naturally-occurring zinc-peptide complex demonstrates that the N-terminal zinc-binding module of the Lasp-1 LIM domain is an independent folding unit
- 1996
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 35:39, s. 12723-12732
-
Tidskriftsartikel (refereegranskat)abstract
- The three-dimensional solution structure of the 1:1 complex between the synthetic peptide ZF-1 and zinc was determined by H-1 NMR spectroscopy. The peptide, initially isolated from pig intestines, is identical in sequence to the 30 N-terminal amino acid residues of the human protein Lasp-1 belonging to the LIM domain protein family. The final set of 20 energy-refined NMR conformers has an average rmsd relative to the mean structure of 0.55 Angstrom for the backbone atoms of residues 3-30, Calculations without zinc atom constraints unambiguously identified Cys 5, Cys 8, His 26, and Cys 29 as the zinc-coordinating residues. LIM domains consist of two sequential zinc-binding modules and the NMR structure of the ZF-1(-)zinc complex is the first example of a structure of an isolated module. Comparison with the known structures of the N-terminal zinc-binding modules of both the second LIM domain of chicken CRP and rat GRIP with which ZF-1 shares 50% and 43% sequence identity, respectively, supports the notion that the zinc-binding modules of the LIM domain have a conserved structural motif and identifies local regions of structural diversity. The similarities include conserved zinc-coordinating residues, a rubredoxin knuckle involving Cys 5 and Cys 8, and the coordination of the zinc ion by histidine N-delta in contrast to the more usual coordination by N-epsilon observed for other zinc-finger domains, The present structure determination of the ZF-1(-)zinc complex establishes the N-terminal half of a LIM domain as an independent folding unit. The structural similarities of N- and C-terminal zinc-binding modules of the LIM domains, despite limited sequence identity, lead to the proposal of a single zinc-binding motif in LIM domains. The coordinates are available from the Brookhaven protein data bank, entry 1ZFO.
|
|
