| 1. |
- Carlsson, Jörgen, et al.
(författare)
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Conjugate chemistry and cellular processing of EGF-dextran
- 1999
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Ingår i: Acta Oncologica. - 0284-186X .- 1651-226X. ; 38:3, s. 313-321
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Tidskriftsartikel (refereegranskat)abstract
- Conjugates with specific binding to the epidermal growth factor receptor, EGFR, of interest for radionuclide based imaging and therapy were prepared using mouse epidermal growth factor, mEGF, and dextran. In one type of conjugate, mEGF was coupled to dextran by reductive amination in which the free amino group on the mEGF N-terminal reacted with the aldehyde group on the reductive end of dextran. The end-end coupled conjugate could be further activated by the cyanopyridinium agent CDAP, thereby introducing tyrosines to the dextran part. In the other type of conjugate, the cyanylating procedure using CDAP was applied, first to activate dextran and then allowing for the amino terminus of mEGF to randomly attach to the dextran. In the latter case, radionuclide-labelled tyrosines or glycines could be added in the same conjugation step. All types of mEGF-dextran conjugates had EGFR-specific binding since the binding could be displaced by an excess of non-radioactive mEGF. The conjugates were to a large extent internalized in the test cells and the associated radioactivity was retained intracellularly for different times depending on both the type of cells and conjugate applied. Different intracellular 'traffic routes' for the radionuclides are discussed as well as applications for both imaging and therapy.
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| 2. |
- Arvidsson, Per-Ola, et al.
(författare)
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Purification and identification of the violaxanthin de-epoxidase as a 43 kDa protein
- 1996
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Ingår i: Photosynthesis Research. - 0166-8595. ; 49:2, s. 119-129
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Tidskriftsartikel (refereegranskat)abstract
- Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g–1 s–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b 6 and an internal sequence of polyphenol oxidase.
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| 3. |
- Arvidsson, Per-Ola, et al.
(författare)
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Violaxanthin accessibility and temperature dependency for de-epoxidation in spinach thylakoid membranes
- 1997
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Ingår i: Photosynthesis Research. - 0166-8595. ; 52:1, s. 39-48
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Tidskriftsartikel (refereegranskat)abstract
- Using DTT and iodoacetamide as a novel irreversible method to inhibit endogenous violaxanthin de-epoxidase, we found that violaxanthin could be converted into zeaxanthin from both sides of the thylakoid membrane provided that purified violaxanthin de-epoxidase was added. The maximum conversion was the same from both sides of the membrane. Temperature was found to have a strong influence both on the rate and degree of maximal violaxanthin to zeaxanthin conversion. Thus only 50% conversion of violaxanthin was detected at 4 degreesC, whereas at 25 degreesC and 37 degreesC the degree of conversion was 70% and 80%, respectively. These results were obtained with isolated thylakoids from non-cold acclimated leafs. Pigment analysis of sub-thylakoid membrane domains showed that violaxanthin was evenly distributed between stroma lamellae and grana partitions. This was in contrast to chlorophyll a and beta-carotene which were enriched in stroma lamellae fractions while chlorophyll b, lutein and neoxanthin were enriched in the grana membranes. In combination with added violaxanthin de-epoxidase we found almost the same degree of conversion of violaxanthin to zeaxanthin (73-78%) for different domains of the thylakoid membrane. We conclude that violaxanthin de-epoxidase converts violaxanthin in the lipid matrix and not at the proteins, that violaxanthin does not prefer one particular membrane region or one particular chlorophyll protein complex, and that the xanthophyll cycle pigments are oriented in a vertical manner in order to be accessible from both sides of the membrane when located in the lipid matrix.
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| 4. |
- Bratt, Charlotte Eva, et al.
(författare)
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Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentration
- 1995
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Ingår i: Photosynthesis Research. - 0166-8595. ; 45:2, s. 169-175
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Tidskriftsartikel (refereegranskat)abstract
- The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a Km for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single Km±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.
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| 6. |
- Hagelin, Joakim, et al.
(författare)
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Dutch university scientists increase research productivity using fewer animals per project
- 1999
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Ingår i: Netherlands Journal of Medicine. - 0300-2977 .- 1872-9061. ; 55:5, s. 212-214
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Tidskriftsartikel (refereegranskat)abstract
- The proposed general reduction in the number of animals used in researchwithin the EU is inappropriate. if a reduction is warranted it should be related to the scientific requirements. We show that the numbers of laboratory animals used per published paper in the Netherlands has decreased by 23% between 1990 and 1997. A further reduction might be detrimental to scientific progress. (See Editorial p. 206)
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