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- Andersson, Charlotte I J, et al.
(författare)
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Error-prone PCR of Vitreoscilla hemoglobin (VHb) to support the growth of microaerobic Escherichia coli
- 2000
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Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 70:4, s. 55-446
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Tidskriftsartikel (refereegranskat)abstract
- Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions. It is very likely that the properties of VHb are not optimized for foreign hosts; therefore, we used error-prone PCR to generate a number of randomly mutated vhb genes to be expressed and studied in Escherichia coli. In addition, the mutated VHb proteins also contained an extension of eight residues (MTMITPSF) at the amino terminus. VHb mutants were screened for improved growth properties under microaerobic conditions and 15 clones expressing mutated hemoglobin protein were selected for further characterization and cultivated in a microaerobic bioreactor to analyze the physiological effects of novel VHb proteins on cell growth. The expression of four VHb mutants, carried by pVM20, pVM50, pVM104, and pVM134, were able to enhance microaerobic growth of E. coli by approximately 22%, 155%, 50%, and 90%, respectively, with a concomitant decrease of acetate excretion into the culture medium. The vhb gene in pVM20 contains two mutations substituting residues Glu19(A17) and Glu137(H23) to Gly. pVM50 expresses a VHb protein carrying two mutations: His36(C1) to Arg36 and Gln66(E20) to Arg66. pVM104 and pVM134 express VHb proteins carrying the mutations Ala56(E10) to Gly and Ile24(B5) to Thr, respectively. Our experiments also indicate that the positive effects elicited by mutant VHb-expression from pVM20 and pVM50 are linked to the peptide tail. Removal of the N-terminal sequence reduced cell growth approximately 23% and 53%, respectively, relative to wild-type controls. These results clearly demonstrate that it is possible to obtain mutated VHb proteins with improved characteristics for improving microaerobic growth of E. coli by using combined mutation techniques, addition of a peptide tail, and random error-prone PCR.
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| 5. |
- Vasen, HFA, et al.
(författare)
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Decision analysis in the surgical treatment of patients with familial adenomatous polyposis: a Dutch-Scandinavian collaborative study including 659 patients
- 2001
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Ingår i: Gut. - : BMJ. - 0017-5749 .- 1468-3288. ; 49:2, s. 231-235
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Tidskriftsartikel (refereegranskat)abstract
- The choice of colorectal surgery in patients with familial adenomatous polyposis lies between the morbidity of proctocolectomy and ileum-pouch-anal anastomosis (IPAA) and the mortality from rectal cancer after total colectomy and ileorectal anastomosis (IRA). The aims of the present study were: (1) to assess the risk of dying from rectal cancer after IRA, (2) to compare the life expectancy between patients with an IRA and those with an IPAA, and (3) to investigate whether regular endoscopic examination of the rectum leads to detection of cancer at an earlier stage.METHODSClinical and pathological data on 659 patients who underwent colectomy and ileorectal anastomosis were collected from four national polyposis registries—that is, in Denmark, Finland, Sweden, and the Netherlands. Data were analysed using survival analysis methods. Decision analysis was used to compare the life expectancy between patients with an IRA and those with an IPAA.RESULTSA total of 47 patients developed rectal cancer after IRA. The risk of dying from rectal cancer was 12.5% (95% confidence interval 7.1–17.9%) by age 65. Compared with IRA, IPAA would lead to an increase in life expectancy of 1.8 years. Seventy five per cent of patients with rectal cancer had a negative rectoscopy within 12 months before the diagnosis.CONCLUSIONIRA is associated with substantial mortality due to rectal cancer. Follow up examinations of the rectum does not have sufficient preventive effect on morbidity and mortality of rectal cancer.
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| 6. |
- Bülow, Elinor, et al.
(författare)
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Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor.
- 2002
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Ingår i: Journal of Leukocyte Biology. - 1938-3673. ; 71:2, s. 279-288
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Tidskriftsartikel (refereegranskat)abstract
- During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein alpha(1)-microglobulin (alpha(1)-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to alpha(1)-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of alpha(1)-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.
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| 7. |
- Bulow, P., et al.
(författare)
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Long-term consequences of the reformation of psychiatric care : A 15-year follow-up study
- 2002
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Ingår i: Nordic Journal of Psychiatry. - : Informa UK Limited. - 0803-9488 .- 1502-4725. ; 56:1, s. 15-21
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Tidskriftsartikel (refereegranskat)abstract
- Although there are a great many epidemiological studies of psychiatric diseases and the outcomes of controlled rehabilitation programmes, there is a shortage of studies of routine psychiatric care. This study is a retrospective cohort study of the total number (n = 138) of patients receiving inpatient care at a psychiatric rehabilitation clinic in Jönköping during 1984. The follow-up period is 15 years, 1984-98. The group had a considerable amount of inpatient care, but this decreased dramatically during the time of investigation. Positive indications were that there were no suicides during the last 5 years of the period and that the number of registered crimes showed a marked decrease from 1992. Negative indications were low levels of work and occupation and few social contacts.
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| 9. |
- Fexby, Sara, et al.
(författare)
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N-Terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition
- 2004
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 807:1, s. 25-31
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Tidskriftsartikel (refereegranskat)abstract
- The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R-2 = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed. (C) 2004 Elsevier B.V. All rights reserved.
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