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1.
Magnus Abrahamson Sif Jonsdottir Isleifur Olafsson Olafur Jensson Anders Grubb 《Human genetics》1992,89(4):377-380
Summary Hereditary cystatin C amyloid angiopathy (HCCAA) is a dominantly inherited disease characterized by amyloidosis, dementia and fatal cerebral hemorrhage of young adults. A method for rapid and simple diagnosis of HCCAA is described. It is based upon oligonucleotide-directed enzymatic amplification of a 275-bp genomic DNA segment containing exon 2 of the cystatin C gene from a blood sample, followed by digestion of the amplification product with AluI. Loss of an AluI recognition site in the amplified DNA segment from HCCAA patients results in a deviating band-pattern at agarose gel electrophoresis, compared with that obtained from normal subjects or unaffected HCCAA family members. In a population of 9 patients with manifest HCCAA, 14 patients with other causes of brain hemorrhage and 16 healthy individuals, the diagnostic procedure displayed a sensitivity and specificity for HCCAA of 100%. Amplified DNA segments from 4 HCCAA patients of four different families were analyzed by nucleotide sequencing; the HCCAA-causing mutation in all families was found to be a single TA substitution in the codon for amino acid residue 68 of cystatin C. 相似文献
2.
The abundance and distribution of Candea larvae was studiedin Ísafjord-deep, north-west Iceland, at approximatelymonthly intervals from February 1987 to February 1988 Zooplanktonsampling was made at nine stations along the length of the fjord,while temperature and chlorophyll a measurements from one ofthe stations are also presented Larvae of six species occurredin the samples, Eualus pusiolus and Pandalus borealis were mostnumerous, constituting 62 8 and 25 9% of the larvae respectively.The other species were, in declining order of abundance, Pandalusmontagui, Spirontocaris spp. (S spinus and s lilljeborgii) andSabinea septemcarinata. Eualus pusiolus was of highest abundancein the outer and middle parts of the fjord, while P.borealiswas most common in the middle and inner parts The onset of hatchingof all species in AprilMay appeared closely linked tothe phytoplankton spring bloom, while the temperature in thefjord was by then near the annual low (23°C). Exceptfor E pusiolus, of which a small part of the population produceda second brood during the summer, most of the larvae had disappearedfrom the plankton by the middle of August The monthly carapacegrowth of P.borealis larvae during the summer months was estimatedto be 1.0 mm. 相似文献
3.
Evaluation of mass spectrometric techniques for charaterization of engineered proteins 总被引:7,自引:0,他引:7
Roepstorff Peter Schram Karl H. Andersen Jens S. Rafn Kate Baldursson Trausti Krøll Jenny Poulsen Kjeld Knudsen Jens Kristiansen Karsten 《Molecular biotechnology》1995,3(1):1-7
A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described.
The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used
as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation.
The various aspects of the procedure and its application is described in detail. 相似文献
4.
Wattanachaisaereekul S Lantz AE Nielsen ML Andrésson OS Nielsen J 《Biotechnology and bioengineering》2007,97(4):893-900
Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes. 相似文献
5.
Baldursson O Ostedgaard LS Rokhlina T Cotten JF Welsh MJ 《The Journal of biological chemistry》2001,276(3):1904-1910
Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel. 相似文献
6.
S-3',4'-anhydroadenosyl-l-methionine is an analogue of the S-adenosyl-l-methionine coenzyme. Here we report on a rapid solvent exchange of the methylene protons at the 5'-position of this analogue. The rate of H/D exchange was measured by nuclear magnetic resonance spectroscopy under buffered conditions in deuterium oxide. The reaction is specific base catalyzed and displays a second-order rate constant of 2 x 10(4) M(-1) s(-1), which corresponds to a rate enhancement of 10(12) compared to solvent exchange of alpha-methylene protons in acyclic, aliphatic sulfonium ions. No other carbon bonded hydrogens in the molecule exchange with solvent under the experimental conditions. Allylic stabilization of a carbanionic-like transition state for the solvent exchange process can account for these results. Solvent exchange under these mild conditions provides a simple way to prepare a 5'-2H-labeled form of the coenzyme analogue. 相似文献
7.
3',4'-Anhydroadenosylcobalamin (anAdoCbl) is an analogue of the adenosylcobalamin (AdoCbl) coenzyme (Magnusson, O.Th., and Frey, P. A. (2000) J. Am. Chem. Soc. 122, 8807-8813). This compound supports activity for diol dehydrase at 0.02% of that observed with AdoCbl. In a side reaction, however, anAdoCbl induces suicide inactivation by an electron-transfer mechanism. Homolytic cleavage of the Co-C bond of anAdoCbl at the active site of diol dehydrase was observed by spectrophotometric detection of cob(II)alamin. Anaerobic conversion of enzyme bound cob(II)alamin to cob(III)alamin, both in the absence and presence of substrate, indicates that the coenzyme derived 5'-deoxy-3',4'-anhydroadenosine-5'-yl serves as the oxidizing agent. This hypothesis is supported by the stoichiometric formation of 3',5'-dideoxyadenosine-4',5'-ene as the nucleoside cleavage product, as determined by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Experiments performed in deuterium oxide show that a single solvent exchangeable proton is incorporated into the product. These data are consistent with the intermediate formation of a transient allylic anion formed after one electron transfer from cob(II)alamin to the allylic 5'-deoxy-3',4'-anhydroadenosyl radical. Selective protonation at C3' was demonstrated by spectroscopic characterization of the purified product. This study provides an example of suicide inactivation of a radical enzyme brought about by a side reaction of an analogue of the radical intermediate. 相似文献
8.
Daniel F. Gudbjartsson Hilma Holm Olafur S. Indridason Gudmar Thorleifsson Vidar Edvardsson Patrick Sulem Femmie de Vegt Frank C. H. d'Ancona Martin den Heijer Leifur Franzson Thorunn Rafnar Kristleifur Kristjansson Unnur S. Bjornsdottir Gudmundur I. Eyjolfsson Lambertus A. Kiemeney Augustine Kong Runolfur Palsson Unnur Thorsteinsdottir Kari Stefansson 《PLoS genetics》2010,6(7)
Chronic kidney disease (CKD) is a worldwide public health problem that is associated with substantial morbidity and mortality. To search for sequence variants that associate with CKD, we conducted a genome-wide association study (GWAS) that included a total of 3,203 Icelandic cases and 38,782 controls. We observed an association between CKD and a variant with 80% population frequency, rs4293393-T, positioned next to the UMOD gene (GeneID: 7369) on chromosome 16p12 (OR = 1.25, P = 4.1×10−10). This gene encodes uromodulin (Tamm-Horsfall protein), the most abundant protein in mammalian urine. The variant also associates significantly with serum creatinine concentration (SCr) in Icelandic subjects (N = 24,635, P = 1.3×10−23) but not in a smaller set of healthy Dutch controls (N = 1,819, P = 0.39). Our findings validate the association between the UMOD variant and both CKD and SCr recently discovered in a large GWAS. In the Icelandic dataset, we demonstrate that the effect on SCr increases substantially with both age (P = 3.0×10−17) and number of comorbid diseases (P = 0.008). The association with CKD is also stronger in the older age groups. These results suggest that the UMOD variant may influence the adaptation of the kidney to age-related risk factors of kidney disease such as hypertension and diabetes. The variant also associates with serum urea (P = 1.0×10−6), uric acid (P = 0.0064), and suggestively with gout. In contrast to CKD, the UMOD variant confers protection against kidney stones when studied in 3,617 Icelandic and Dutch kidney stone cases and 43,201 controls (OR = 0.88, P = 5.7×10−5). 相似文献
9.
Duplicated sequence motif in the long terminal repeat of maedi-visna virus extends cell tropism and is associated with neurovirulence 总被引:2,自引:0,他引:2
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Oskarsson T Hreggvidsdóttir HS Agnarsdóttir G Matthíasdóttir S Ogmundsdóttir MH Jónsson SR Georgsson G Ingvarsson S Andrésson OS Andrésdóttir V 《Journal of virology》2007,81(8):4052-4057
Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence. 相似文献
10.