| 1. |
- Kononenko, Olga, et al.
(author)
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Opioid precursor protein isoform is targeted to the cell nuclei in the human brain
- 2017
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165 .- 1872-8006. ; 1861:2, s. 246-255
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Journal article (peer-reviewed)abstract
- BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain.METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus.RESULTS: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging.CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.
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| 2. |
- Belyaev, I Y, et al.
(author)
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Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis
- 1999
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In: Biochimica et Biophysica Acta - General Subjects. - 0304-4165 .- 1872-8006. ; 1428:2-3, s. 348-356
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Journal article (peer-reviewed)abstract
- The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2. mg/ml, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with gamma-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation.
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| 3. |
- Dahlqvist, Ulla, et al.
(author)
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Endogenous substrates of protein kinase in rat liver cell sap under different dietary conditions
- 1978
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 540:1, s. 13-23
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Journal article (peer-reviewed)abstract
- Liver cell sap from normally fed rats, rats fed with a high-carbohydrate diet and fasted rats was chromatographed on DEAE-cellulose (pH 7.0). The chromatogram from each diet group was analyzed for pyruvate kinase activity and endogenous substrates of cyclic AMP-stimulated protein kinase. The materials were pooled into five phosphorylatable fractions, in each of which phosphate incorporation at 0.1 mM and 1.0 mM [32P]ATP in the presence of cyclic AMP and protein kinase was determined. For characterization of the phosphorylatable components, thin-layer gel chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis in detergent were used for determination of native and minimal molecular weights, respectively. Except for pyruvate kinase, eight components which incorporated at least 0.05 nmol of [32P]phosphate/g of liver were detected. The phosphorylation of four of them was stimulated by cyclic AMP. Their minimal molecular weights were 42000, 21000, 52000 and 49000. The component with a minimal molecular weight of 42000 seemed to have a native molecular weight of 160000. Both the 21000 and the 52000 component had a native molecular weight of about 110000-120000. The protein with a minimal molecular weight of 49000 could not be correlated with certainty to a native molecular weight. The proteins whose phosphorylation was not stimulated by cyclic AMP had minimal molecular weights of 54000, 39000, 34000 and 22000.
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| 4. |
- Ahmadpour, Doryaneh, 1973, et al.
(author)
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Yeast reveals unexpected roles and regulatory features of aquaporins and aquaglyceroporins
- 2014
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In: Biochimica et Biophysica Acta. General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006 .- 0006-3002. ; 1840:5, s. 1482-1491
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Research review (peer-reviewed)abstract
- Background: The yeast Saccharomyces cerevisiae provides unique opportunities to study roles and regulation of aqua/glyceroporins using frontline tools of genetics and genomics as well as molecular cell and systems biology. Scope of review: S. cerevisiae has two similar orthodox aquaporins. Based on phenotypes mediated by gene deletion or overexpression as well as on their expression pattern, the yeast aquaporins play important roles in key aspects of yeast biology: establishment of freeze tolerance, during spore formation as well as determination of cell surface properties for substrate adhesion and colony formation. Exactly how the aquaporins perform those roles and the mechanisms that regulate their function under such conditions remain to be elucidated. S. cerevisiae also has two different aquaglyceroporins. While the role of one of them, Yfl054c, remains to be determined, Fps1 plays critical roles in osmoregulation by controlling the accumulation of the osmolyte glycerol. Fpsl communicates with two osmo-sensing MAPK signalling pathways to perform its functions but the details of Fps1 regulation remain to be determined. Major conclusions: Several phenotypes associated with aqua/glyceroporin function in yeasts have been established. However, how water and glycerol transport contribute to the observed effects is not understood in detail. Also many of the basic principles of regulation of yeast aqua/glyceroporins remain to be elucidated. General significance: Studying the yeast aquaporins and aquaglyceroporins offers rich insight into the life style, evolution and adaptive responses of yeast and rewards us with discoveries of unexpected roles and regulatory mechanisms of members of this ancient protein family. This article is part of a Special Issue entitled Aquaporins. (c) 2013 Elsevier B.V. All rights reserved.
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| 5. |
- Blikstad, Cecilia, et al.
(author)
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Emergence of a novel highly specific and catalytically efficient enzyme from a naturally promiscuous glutathione transferase
- 2008
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1780:12, s. 1458-63
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Journal article (peer-reviewed)abstract
- Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in kcat/Km values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A4-4 have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A4-4 and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and androstenedione are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 94% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A4-4.
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| 6. |
- Carlsson, Michael, et al.
(author)
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Different fractions of human serum glycoproteins bind galectin-1 or galectin-8, and their ratio may provide a refined biomarker for pathophysiological conditions in cancer and inflammatory disease
- 2012
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In: Biochimica et Biophysica Acta - General Subjects. - : Elsevier. - 0304-4165 .- 1872-8006 .- 0006-3002. ; 1820:9, s. 1366-1372
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Journal article (peer-reviewed)abstract
- Background: Changes in glycosylation of serum proteins are common, and various glycoforms are being explored as biomarkers in cancer and inflammation. We recently showed that glycoforms detected by endogenous galectins not only provide potential biomarkers, but also have different functions when they encounter galectins in tissue cells. Now we have explored the use of a combination of two galectins with different specificities, to further increase biomarker sensitivity and specificity. less thanbrgreater than less thanbrgreater thanMethods: Sera from 14 women with metastatic breast cancer, 12 healthy controls, 14 patients with IgA-nephritis (IgAN), and 12 patients with other glomerulonephritis were fractionated by affinity chromatography on immobilized human galectin-1 or galectin-8N, and the protein amounts of the bound and unbound fractions for each galectin were determined. less thanbrgreater than less thanbrgreater thanResults: Each galectin bound largely different fractions of the serum glycoproteins, including different glycoforms of haptoglobin. In the cancer sera, the level of galectin-1 bound glycoproteins was higher and galectin-8N bound glycoproteins lower compared to the other patients groups, whereas in IgAN sera the level of galectin-8N bound glycoproteins were higher. less thanbrgreater than less thanbrgreater thanConclusion: The ratio of galectin-1 bound/galectin-8N bound glycoproteins showed high discriminatory power between cancer patients and healthy, with AUC of 0.98 in ROC analysis, and thus provides an interesting novel cancer biomarker candidate. less thanbrgreater than less thanbrgreater thanGeneral significance: The galectin-binding ability of a glycoprotein is not only a promising biomarker candidate but may also have a specific function when the glycoprotein encounters the galectin in tissue cells, and thus be related to the pathophysiological state of the patient. This article is part of a Special Issue entitled Glycoproteomics.
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| 7. |
- Guan, Na N., et al.
(author)
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Release and inhibitory effects of prostaglandin D2 in guinea pig urinary bladder and the role of urothelium
- 2014
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In: Biochimica et Biophysica Acta. - Amsterdam, Neterhlands : Elsevier. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1840:12, s. 3443-3451
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Journal article (peer-reviewed)abstract
- BACKGROUND: While studying a urothelium-derived inhibitory factor in guinea pig urinary bladders we observed considerable release of prostanoids, including PGD2-like activity. The present study was carried out to identify the prostanoids and to study their roles in modulating guinea pig urinary bladder motility.METHODS: Release of PGE2 and PGD2 in isolated guinea pig urinary bladder preparations was analyzed by high performance liquid chromatography (HPLC) combined with bioassay on bladder strips. Isolated urothelium-intact (UI) or -denuded (UD) bladder strips were subjected to electrical field stimulation (EFS) and applications of PGE2 and PGD2.RESULTS: A resting release of 95±9 (n=5) nggtissue(-1)h(-1) PGE2-like activity and 210±34 (n=4) nggtissue(-1)h(-1) PGD2-like activity was found, where PGD2-like was subject to marked spontaneous inactivation during isolation. Prostanoids release was decreased by 70-90% by the cyclo-oxygenase inhibitor diclofenac in UI preparations. Urothelium removal decreased prostanoids release by more than 90%. PGE2 increased basal tone and spontaneous contractions, whereas PGD2 had little or no effect on these. Exogenous PGE2 enhanced and PGD2 inhibited contractile responses to EFS, exogenous acetylcholine- and ATP, whereas PGD2 caused marked dose-dependent inhibition. PGE2 and PGD2 effects were more pronounced in diclofenac-treated UD tissues.CONCLUSIONS: PGD2 and PGE2 are released from guinea pig bladder urothelium and PGD2 has inhibitory effects on bladder motility, mainly through a postjunctional action on smooth muscle responsiveness.GENERAL SIGNIFICANCE: The release and inhibitory effects merit further studies in relation to normal biological function as well as overactive bladder syndrome.
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| 8. |
- Gustafsson, Tomas N., et al.
(author)
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Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase and bactericidal antibacterials targeting Bacillus species, Staphylococcus aureus and Mycobacterium tuberculosis
- 2016
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In: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006 .- 0006-3002. ; 1860:6, s. 1265-1271
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Journal article (peer-reviewed)abstract
- Background: Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. Methods: We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. Results: The most potent compounds in the series gave IC50 values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 mu M (0.12 mu g/ml) for B. subtilis,1.5 mu M (0.64 mu g/ml) for S. aureus, 2 mu M (0.86 mu g/ml) for B. cereus and 10 mu g/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. Conclusions: These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. General significance: We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens.
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| 9. |
- Jensen, Lasse
(author)
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The circadian clock and hypoxia in tumor cell de-differentiation and metastasis
- 2015
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In: Biochimica et Biophysica Acta - General Subjects. - : Elsevier. - 0304-4165 .- 1872-8006 .- 0006-3002. ; 1850:8, s. 1633-1641
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Research review (peer-reviewed)abstract
- Background: Cancer is considered to develop due to disruptions in the tissue microenvironment in addition to genetic disruptions in the tumor cells themselves. The two most important microenvironmental disruptions in cancer are arguably tissue hypoxia and disrupted circadian rhythmicity. Endothelial cells, which line the luminal side of all blood vessels transport oxygen or endocrine circadian regulators to the tissue and are therefore of key importance for circadian disruption and hypoxia in tumors. Scope of review: Here I review recent findings on the role of circadian rhythms and hypoxia in cancer and metastasis, with particular emphasis on how these pathways link tumor metastasis to pathological functions of blood vessels. The involvement of disrupted cell metabolism and redox homeostasis in this context and the use of novel zebrafish models for such studies will be discussed. Major conclusions: Circadian rhythms and hypoxia are involved in tumor metastasis on all levels from pathological deregulation of the cell to the tissue and the whole organism. Pathological tumor blood vessels cause hypoxia and disruption in circadian rhythmicity which in turn drives tumor metastasis. Zebrafish models may be used to increase our understanding of the mechanisms behind hypoxia and circadian regulation of metastasis. General significance: Disrupted blood flow in tumors is currently seen as a therapeutic goal in cancer treatment but may drive invasion and metastasis via pathological hypoxia and circadian clock signaling. Understanding the molecular details behind such regulation is important to optimize treatment for patients with solid tumors in the future. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. (C) 2014 Elsevier B.V. All rights reserved.
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| 10. |
- Lepp, Håkan, et al.
(author)
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Internal charge transfer in cytochrome c oxidase at a limited proton supply : proton pumping ceases at high pH.
- 2009
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1790:6, s. 552-7
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Journal article (peer-reviewed)abstract
- BACKGROUND: In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O(2) is linked to proton uptake from solution to form H(2)O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane. METHODS: In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O(2). These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles. RESULTS: The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O(2) reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH. CONCLUSIONS: The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the "wrong" side of the membrane) and that at this pH the net proton-uptake stoichiometry is approximately 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme. GENERAL SIGNIFICANCE: These results provide new insights into the function of cytochrome c oxidase.
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