| 1. |
- Beltran-Pardo, Eliana, et al.
(författare)
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Sequence analysis of the DNA-repair gene rad51 in the tardigrades Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi
- 2013
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Ingår i: Journal of limnology. - 1129-5767 .- 1723-8633. ; 72:s1, s. 80-91
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Tidskriftsartikel (refereegranskat)abstract
- Tardigrades are known for being resistant to extreme conditions, including tolerance to ionising and UV radiation in both the hydratedand the dehydrated state. It is known that these factors may cause damage to DNA. It has recently been shown that single and double DNAstrand breaks occur when tardigrades are maintained for a long time in the anhydrobiotic state. This may suggest that perhaps tardigrades rely on efficient DNA repair mechanisms. Among all proteins that comprise the DNA repair system, recombinases such as RecA or Rad51 have a very important function: DNA exchange activity. This enzyme is used in the homologous recombination and allows repair of thedamaged strand using homologous non-damaged strands as a template. In this study, Rad51 induction was evaluated by western blot in Milnesium cf. tardigradum, after exposure to gamma radiation. The Rad51 protein was highly induced by radiation, when compared to the control. The rad51 genes were searched in three tardigrades: Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi. The gene sequences were obtained by preparing and sequencing transcriptome libraries for H. dujardini and M. cf. harmsworthi and designing rad51 degenerate primers specific for M. cf. tardigradum. Comparison of Rad51 putative proteins from tardigrades with other organisms showed that they are highly similar to the corresponding sequence from the nematode Trichinella spiralis. A structure-based sequence alignment from tardigrades and other organisms revealed that putative Rad51 predicted proteins from tardigrades contain the expected motifs for these important recombinases. In a cladogram tree based on this alignment, tardigrades tend to cluster together suggesting that they have selective differences in these genes that make them diverge between species. Predicted Rad51 structures from tardigrades were also compared with crystalline structure of Rad51 in Saccharomyces cerevisiae. These results reveal that S. cerevisiae Rad51 structure is very similar to that of the three analysed tardigrades. On the other hand the predicted structure of Rad51 from M. cf. harmsworthi and H. dujardini are closer related to each other, than each of them to that of M. cf. tardigradum.
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| 2. |
- Jönsson, Ingemar, 1959-, et al.
(författare)
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Tolerance to gamma-irradiation in eggs of the tardigrade Richtersius coronifer depends on stage of development
- 2013
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Ingår i: Journal of limnology. - 1129-5767 .- 1723-8633. ; 72:s1, s. 73-79
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Tidskriftsartikel (refereegranskat)abstract
- Tardigrades are known as one of the most radiation tolerant animals on Earth, and several studies on tolerance in adult tardigrades have been published. In contrast, very few studies on radiation tolerance of embryonic stages have been reported. Here we report a study on tolerance to gamma irradiation in eggs of the eutardigrade Richtersius coronifer. Irradiation of eggs collected directly from a natural substrate (moss) showed a clear dose-response, with a steep decline in hatchability at doses up to 0.4 kGy followed by a relatively constant hatchability around 25% up to 2 kGy, and a decline to ca. 5% at 4 kGy above which no eggs hatched. Analysis of the time required for eggs to hatch after irradiation (residual development time) showed that hatching of eggs after exposure to high doses of gamma radiation was associated with short residual development time. Since short residual development time means that the egg was irradiated at a late developmental stage, this suggests that eggs were more tolerant to radiation late in development. This was also confirmed in another experiment in which stage of development at irradiation was controlled. No eggs irradiated at the early developmental stage hatched, and only one egg at middle stage hatched, while eggs irradiated in the late stage hatched at a rate indistinguishable from controls. This suggests that the eggs are more sensitive to radiation in the early stages of development, or that tolerance to radiation is acquired only late in development, shortly before the eggs hatch, hypotheses that are not mutually exclusive. Our study emphasizes the importance of considering specific cell cycle phases and developmental stages in studies of tolerance to radiation in tardigrades, and the potential importance of embryonic studies in revealing the mechanisms behind the radiation tolerance of tardigrades and other cryptobiotic animals.
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| 3. |
- Zorita, Saioa, et al.
(författare)
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Steroid hormone determination in water using an environmentally friendly membrane based extraction technique
- 2008
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1192:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- In this study, a method was developed for determination of steroid hormones (17beta-estradiol, estrone, 17alpha-ethynylestradiol) in tap and sewage water samples from Sweden. Sample preparation and analysis were performed by a hollow-fibre microporous membrane liquid-liquid extraction (HF-MMLLE) set-up combined with gas chromatography-mass spectrometry (GC-MS). In this approach, only the organic liquid in the lumen (10microL) of the hollow-fibre membrane was utilised for depleting extraction. Several parameters were studied, including: type of organic solvent, sample pH, salt and humic acid content. The optimised method allowed the determination of the analyte at the low ngL(-1) level in tap and sewage water. A linear plot gave correlation coefficients better than 0.995 and resulted in a method limit of detection of 1.6, 3 and 10ngL(-1) for 17beta-estradiol, estrone, and 17alpha-ethynylestradiol, respectively, in sewage water. Enrichment factors were over 1400 after derivatisation. The repeatabilities at 50 and 600ngL(-1) were better than 10% and 6%, respectively.
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| 4. |
- Nasir, Irem, et al.
(författare)
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Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions
- 2015
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Ingår i: ACS Chemical Neuroscience. - : The American Chemical Society (ACS). - 1948-7193 .- 1948-7193. ; 6:8, s. 1436-44
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (t1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration.
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| 5. |
- Sävblom, Charlotta, et al.
(författare)
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Genetic variation in KLK2 and KLK3 is associated with concentrations of hK2 and PSA in serum and seminal plasma in young men
- 2014
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Ingår i: Clinical Chemistry. - : American Association for Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 60:3, s. 490-499
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Genetic variants in KLK2 and KLK3 have been associated with increased serum concentrations of their encoded proteins, human kallikrein-related peptidase 2 (hK2) and prostate-specific antigen (PSA), and with prostate cancer in older men. Low PSA concentrations in seminal plasma (SP) have been associated with low sperm motility. To evaluate whether KLK2 and KLK3 genetic variants affect physiological prostatic secretion, we studied the association of SNPs with hK2 and PSA concentrations in SP and serum of young, healthy men.METHODS: Leukocyte DNA was extracted from 303 male military conscripts (median age 18.1 years). Nine SNPs across KLK2-KLK3 were genotyped. We measured PSA and hK2 in SP and serum using immunofluorometric assays. The association of genotype frequencies with hK2 and PSA concentrations was tested with the Kruskal-Wallis test.RESULTS: Four KLK2 SNPs (rs198972, rs198977, rs198978, and rs80050017) were strongly associated with hK2 concentrations in SP and serum, with individuals homozygous for the major alleles having 3- to 7-fold higher concentrations than the intermediate concentrations found in other homozygotes and heterozygotes (all P < 0.001). Three of these SNPs were significantly associated with percentage of free PSA (%fPSA) in serum (all P < 0.007). Three KLK3 SNPs showed associations with PSA in SP, and the rs1058205 SNP was associated with total PSA in serum (P = 0.001) and %fPSA (P = 0.015).CONCLUSIONS: Associations observed in young, healthy men between the SP and serum concentrations of hK2 and PSA and several genetic variants in KLK2 and KLK3 could be useful to refine models of PSA cutoff values in prostate cancer testing.
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| 6. |
- Ter Schure, Arnout F.H., et al.
(författare)
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Latitudinal Fractionation of Polybrominated Diphenyl Ethers and Polychlorinated Biphenyls in Frogs (Rana temporaria)
- 2002
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Ingår i: Environmental Science and Technology. - 0013-936X .- 1520-5851. ; 36:23, s. 5057-5061
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Tidskriftsartikel (refereegranskat)abstract
- The flame retardant polybrominated diphenyl ethers (PBDEs) have become ubiquitous environmental pollutants. The environmental distribution of PBDEs is much less studied than that of the polychlorinated biphenyls (PCBs).To compare the environmental fate of the PCBs withtheir partial substitute, the PBDEs, common frogs (Ranatemporaria) were collected along a 1500-km-long latitudinal gradient of the Scandinavian Peninsula and their livers analyzed for PCBs and PBDEs. Mean concentrations of total PCBs and BDE47 ranged from 9200 to 92 900 and 30 to120 ng kg-1 fresh weight, respectively, whereas BDE99 was detected in less than 50% of the frogs. PCB concentrations were higher than that of the PBDEs, and the differences decreased in the northern latitudes. Moreover, the pollutant concentrations in frog livers were negative functions of latitude. The observed scatter and regression slopes imply several influencing factors (such as habitat, exposure route, uptake, metabolism, excretion, etc.) and indicate release events instead of the grasshopper effect. Biological variables such as gender, age, body size, and lipid content did not influence pollutant concentrations in the frog livers. The total PCB and BDE47 concentrations in frogs were highly correlated. Hence, their environmental fate is analogous and our results highlight the need to examine the potential role of xenobiotics on amphibian populations.
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| 7. |
- Vrhovac, Lidija, 1994-
(författare)
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Structural characterization of Deinococcus radiodurans phytochrome in solution
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Phytochromes are red/far-red photoreceptors which control a wide spectrum of biological processes inplants, fungi, and bacteria. Although phytochromes have been extensively studied, their mechanism ofaction still remains elusive. The signaling mechanism suggested by crystal structures involves refoldingof the so-called PHY tongue. However, currently, the involvement of the other two prominent structuralelements, the so-called helical spine and a knot in the peptide chain, remains unclear.Here, the first backbone assignment of the photosensory module of D. radiodurans phytochrome ispresented. To achieve a higher degree of the backbone assignment and thus gain further knowledge onthe conformational changes in the chromophore binding domain, an efficient method for refolding ofthe CBDPHY phytochrome fragment was developed. While further NMR measurements on therefolded phytochrome are needed, preliminary results already show the value of the extended backboneassignment for the future structural studies. Previously unreported changes in the knot region of thephotosensory module of D. radiodurans phytochrome were captured by solution NMR. All NMRobservables suggest photoinduced structural changes in the aforementioned region, implying that thesignal is carried from the chromophore to the helical spine, and through it, to the PHY domain and theoutput module. Furthermore, a study of the region near the bottom of the helical spine has beenconducted. Using several sets of the residual dipolar coupling (RDC) measurements, together with themolecular dynamics (MD) simulations, we gained a better understanding of the network of interactionsspanning from the chromophore to the dimer contact surface.Additionally, a procedure for structural analysis of nanoscale particles at XFELs using angular crosscorrelationsis outlined here. This approach was applied to the scattering data of ideal icosahedralparticles, a phytochrome and the human pre-ribosomal (pre-40S) particle. Condor was used to predictX-ray scattering amplitudes of these particles for customized experimental designs. Angularcorrelations can be accurately extracted from multiple-particle fluctuation X-ray scatteringexperiments. We concluded that angular cross-correlation functions (CCFs) preserve a substantialamount of structural information which enables the observation of structural features of particles at thenanometer scale. Consequently, correlation maps could be useful to follow fast dynamical changesin the structure, for instance, as a response to external stimuli.
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