| 1. |
- Abrahamson, Magnus, et al.
(författare)
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Structure and expression of the human cystatin C gene
- 1990
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Ingår i: Biochemical Journal. - 1470-8728. ; 268:2, s. 287-294
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Tidskriftsartikel (refereegranskat)abstract
- The structural organization of the gene for the human cysteine-proteinase inhibitor cystatin C was studied. Restriction-endonuclease digests of human genomic DNA hybridized with human cystatin C cDNA and genomic probes produced patterns consistent with a single cystatin C gene and, also, the presence of six closely related sequences in the human genome. A 30 kb restriction map covering the genomic region of the cystatin C gene was constructed. The positions of three polymorphic restriction sites, found at examination of digests of genomic DNA from 79 subjects, were localized in the flanking regions of the gene. The gene was cloned and the nucleotide sequence of a 7.3 kb genomic segment was determined, containing the three exons of the cystatin C structural gene as well as 1.0 kb of 5'-flanking and 2.0 kb of 3'-flanking sequences. Northern-blot experiments revealed that the cystatin C gene is expressed in every human tissue examined, including kidney, liver, pancreas, intestine, stomach, antrum, lung and placenta. The highest cystatin C expression was seen in seminal vesicles. The apparently non-tissue-specific expression of this cysteine-proteinase inhibitor gene is discussed with respect to the structure of its 5'-flanking region, which shares several features with those of housekeeping genes.
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| 2. |
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| 3. |
- Barderi, P, et al.
(författare)
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The NADP+ linked glutamate dehydrogenase from Trypanosoma cruzi : sequence, genomic organization and expression
- 1998
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 330:2, s. 951-958
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Tidskriftsartikel (refereegranskat)abstract
- NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.
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| 4. |
- BARKER, CJ, et al.
(författare)
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Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells
- 1995
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 306306 ( Pt 2), s. 557-564
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Tidskriftsartikel (refereegranskat)abstract
- 1. An inositol trisphosphate (InsP3) distinct from Ins(1,4,5)P3 and Ins(1,3,4)P3, which we previously observed in myeloid and lymphoid cells [French, Bunce, Stephens, Lord, McConnell, Brown, Creba and Michell (1991) Proc R. Soc. London B 245, 193-201; Bunce, French, Allen, Mountford, Moore, Greaves, Michell and Brown (1993) Biochem. J. 289, 667-673], is present in WRK1 rat mammary tumour cells and pancreatic endocrine beta-cells. 2. It has been identified as Ins(1,2,3)P3 by a combination of oxidation to ribitol, a structurally diagnostic polyol, and ammoniacal hydrolysis to identified inositol monophosphates. 3. Ins(1,2,3)P3 concentration in HL60 cells changed little during stimulation by ATP or fMetLeuPhe or during neutrophilic or monocytic differentiation, and Ins(1,2,3)P3 was unresponsive to vasopressin in WRK1 cells. 4. Ins(1,2,3)P3 was usually more abundant than Ins(1,4,5)P3, often being present at concentrations between approximately 1 microM and approximately 10 microM. 5. HL60, WRK-1 and lymphoid cells also contain Ins(1,2)P2 or Ins(2,3)P2, or a mixture of these two enantiomers, as a major InsP2 species. 6. Ins(1,2,3)P3 and Ins(1,2)P2/Ins(2,3)P2 are readily detected in cells labelled for long periods, but not in acutely labelled cells. This behaviour resembles that of InsP6, the most abundant cellular inositol polyphosphate that includes the 1,2,3-trisphosphate motif, which also achieves isotopic equilibrium with inositol only slowly. 7. Ins(1,2,3)P3 is the major InsP3 that accumulates during metabolism of InsP6 by WRK-1 cell homogenates. 8. Possible metabolic relationships between Ins(1,2,3)P3, Ins(1,2)P2/Ins(2,3)P2 and other inositol polyphosphates in cells, and a possible role for Ins(1,2,3)P3 in cellular iron handling, are considered.
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| 5. |
- Ben Nasr, Abdelhakim, et al.
(författare)
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Absorption of kininogen from human plasma by Streptococcus pyogenes is followed by the release of bradykinin
- 1997
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 326:3, s. 657-660
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Tidskriftsartikel (refereegranskat)abstract
- H-kininogen (high-molecular-mass kininogen, HK) is the precursor of the vasoactive peptide hormone bradykinin (BK). Previous work has demonstrated that HK binds to Streptococcus pyogenes through M-proteins, fibrous surface proteins and important virulence factors of these bacteria. Here we find that M-protein-expressing bacteria absorb HK from human plasma. The HK bound to the bacteria was found to be cleaved, and analysis of the degradation pattern suggested that the cleavage of HK at the bacterial surface is associated with the release of BK. Moreover, addition of activated plasma prekallikrein to bacteria preincubated with human plasma, resulted in BK release. This mechanism, by which a potent vasoactive and proinflammatory peptide is generated at the site of infection, should influence the host-parasite relationship during S. pyogenes infections.
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| 6. |
- Benthin, G, et al.
(författare)
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Transformation of subcutaneous nitric oxide into nitrate in the rat
- 1997
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 323323 ( Pt 3), s. 853-858
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Tidskriftsartikel (refereegranskat)abstract
- Following its addition to arterialized blood in vitro, nitric oxide (NO) is transformed into nitrate in the erythrocytes. Inhaled NO is similarly transformed into nitrate in the blood in vivo. These observations suggest that nitrate is a universal end-metabolite of NO, i.e. of endogenously formed NO as well. However, endogenous NO may also be inactivated in tissues, i.e. outside the vascular lumen. To study the fate of NO metabolized with delayed access to the blood, rats were given subcutaneous injections of 15NO or K15NO3, and the plasma concentrations of 15NO3- were followed for 450 min after injection. The values for the distribution volume and plasma decay (t½) of 15NO3- did not differ between rats given 15N-labelled NO and NO3-. The area under the plasma decay curve for rats given 15NO amounted to 89% of the corresponding area for animals given K15NO3. This demonstrates that 15NO, when given extravascularly in millimolar concentrations, is mainly transformed into 15N-labelled nitrate. Other rats were kept in an atmosphere containing a mixture of 16O2 and 18O2. Nitrate residues containing either one or two 18O atoms were isolated from the blood, indicating that inhaled oxygen was incorporated during both the formation of NO and the subsequent transformation of NO into nitrate. The fraction of nitrate residues containing two 18O atoms was larger than that containing one 18O atom. We propose that nitrate is a major stable metabolite of endogenous NO that does not primarily diffuse into the vascular lumen following formation. Hence nitrate seems to be the quantitatively most important end-product of the metabolism of endogenous NO. The transformation of endogenous NO into nitrate involves the incorporation of inhaled oxygen.
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| 7. |
- Birve, Simon, 1964-, et al.
(författare)
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Secondary structure of NADPH : protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods
- 1996
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 317:2, s. 549-555
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Tidskriftsartikel (refereegranskat)abstract
- To study the secondary structure of the enzyme NADPH:protochlorophyllide oxidoreductase (PCOR), a novel method of enzyme isolation was developed. The detergent isotridecyl poly(ethylene glycol) ether (Genapol X-080) selectively solubilizes the enzyme from a prolamellar-body fraction isolated from wheat (Triticum aestivum L.). The solubilized fraction was further purified by ion-exchange chromatography. The isolated enzyme was studied by fluorescence spectroscopy at 77 K, and by CD spectroscopy. The fluorescence-emission spectra revealed that the binding properties of the substrate and co-substrate were preserved and that photo-reduction occurred. The CD spectra of PCOR were analysed for the relative amounts of the secondary structures, alpha-helix, beta-sheet, turn and random coil. The secondary structure composition was estimated to be 33% alpha-helix, 19% beta-sheet, 20% turn and 28% random coil. These values are in agreement with those predicted by the Predict Heidelberg Deutschland and self-optimized prediction method from alignments methods. The enzyme has some amino acid identity with other NADPH-binding enzymes containing the Rossmann fold. The Rossmann-fold fingerprint motif is localized in the N-terminal region and at the expected positions in the predicted secondary structure. It is suggested that PCOR is anchored to the interfacial region of the membrane by either a beta-sheet or an alpha-helical region containing tryptophan residues. A hydrophobic loop-region could also be involved in membrane anchoring.
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| 8. |
- Brown, GR, et al.
(författare)
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Parallel changes in nuclear and cytosolic calcium in mouse pancreatic beta-cells
- 1997
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 325325 ( Pt 3), s. 771-778
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Tidskriftsartikel (refereegranskat)abstract
- In the neuroendocrine pancreatic β-cell, elevations in intracellular Ca2+ lead to insulin secretion and the initiation of gene transcription. However, the relationship between cytosolic and nuclear Ca2+ in these cells is unknown. The Ca2+ permeability of the nuclear membrane would therefore determine if Ca2+ could play a direct role in Ca2+-dependent nuclear processes. Using confocal fluorescence microscopy with the ratiometric Ca2+ indicator indo-1 and carefully correcting for compartmentalized indicator, we now demonstrate that there is no difference between the nuclear Ca2+ concentration and the cytosolic Ca2+ concentration ([Ca2+]c) in the resting β-cell. Slow Ca2+ oscillations induced by glucose, fast oscillations induced by glucagon-like peptide-1 and responses to potassium and carbachol all indicate that changes in cytosolic Ca2+ are reflected within the nucleus. We conclude that there are no restrictions on Ca2+ entry into the nucleus of the pancreatic β-cell subsequent to increases in [Ca2+]c. This implies that any signal involved in increasing [Ca2+]c, and thereby insulin release, may also promote nuclear Ca2+-induced gene transcription.
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| 9. |
- Burkitt, MJ, et al.
(författare)
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1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions
- 1996
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 313313 ( Pt 1), s. 163-169
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Tidskriftsartikel (refereegranskat)abstract
- Isolated rat-liver nuclei were incubated with a series of membrane-permeable metal-ion-complexing agents and examined for DNA damage. Of the reagents tested, only 1,10-phenanthroline (OP) and neocuproine (NC) were found to induce DNA fragmentation. Agarose-gel electrophoresis of the DNA fragments generated in the presence of OP revealed internucleosomal cleavage, which is widely considered to be a hallmark for the enzymic DNA digestion that occurs during apoptosis. Ascorbate, particularly in the presence of hydrogen peroxide, increased the levels of fragmentation induced by OP. As well as undergoing fragmentation, the DNA from nuclei was also found to contain 8-hydroxydeoxyguanosine, which indicates attack (oxidation) by the hydroxyl radical. Complementary experiments in vitro involving ESR determinations of hydroxyl radical formation and measurements of DNA oxidation under biomimetic conditions demonstrated that Cu2+, but not Fe3+, forms a complex with either OP or NC (but not the other complexing agents tested) that stimulates hydroxyl radical formation and DNA damage in the presence of hydrogen peroxide and ascorbate. It is therefore proposed that OP in the nuclei incubations binds to Cu2+, which exists naturally in chromosomes, forming a complex that promotes hydroxyl-radical-dependent DNA fragmentation. These findings demonstrate the promotion of hydroxyl-radical-mediated DNA damage by endogenous Cu2+ and, perhaps more significantly, demonstrate that the internucleosomal DNA ‘laddering’ that is often used as an indicator of apoptosis may also result from DNA fragmentation by non-enzymic processes.
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| 10. |
- Chevreuil, O, et al.
(författare)
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Heparin-decasaccharides impair the catabolism of chylomicrons.
- 1996
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 320 ( Pt 2), s. 437-44
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Tidskriftsartikel (refereegranskat)abstract
- On intravenous injection to rats, decasaccharides gave rise to a short-lived peak of lipoprotein lipase (LPL) activity, whereas octa- and hexasaccharides caused only marginal increases. In isolated hearts perfused by a single pass, decasaccharides released LPL more efficiently than conventional heparin on a mass basis. Octa- and hexasaccharides were much less efficient. Similar results were obtained for hepatic lipase, which was studied both in vivo and by liver perfusion. In the intact rat, the heparin fragments themselves disappeared rapidly from the circulating blood. The decay of hepatic lipase activity after the early peak roughly paralleled the decay of decasaccharide concentration, but for LPL the decay was faster, presumably because the liver extracted this lipase from plasma. To assess the lipase activities remaining in contact with blood a large dose of conventional heparin was injected at a series of times after the decasaccharides. LPL was decreased by 40% after 1 h. At that time, the LPL activity that could be released from isolated hearts by single-pass perfusion with heparin for 2 min ("functional LPL') was decreased by 75%. Chylomicrons labelled in vivo with [14C]oleic acid (primarily in triacylglycerols, providing a tracer for lipolysis) and [3H]retinol (primarily in ester form, providing a tracer for the particles) were injected intravenously to explore the effects of the LPL depletion on lipoprotein metabolism. Triacylglycerol lipolysis and particle clearance was markedly delayed from 30 min to 2 h after injection of decasaccharides. After 1 h the fractional catabolic rate was only one-third of the control value and the catabolism of chylomicron triacylglycerols by perfused hearts was delayed to a similar extent. Thus injection of decasaccharides leads to accelerated turnover of LPL with loss of functional LPL from extrahepatic tissues. This in turn leads to a period of delayed lipolysis and removal of chylomicron particles.
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