| 1. |
- Dahlqvist, Ulla, et al.
(författare)
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Endogenous substrates of protein kinase in rat liver cell sap under different dietary conditions
- 1978
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 540:1, s. 13-23
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Blikstad, Cecilia, et al.
(författare)
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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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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1780:12, s. 1458-63
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Guan, Na N., et al.
(författare)
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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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Ingår i: Biochimica et Biophysica Acta. - Amsterdam, Neterhlands : Elsevier. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1840:12, s. 3443-3451
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Kononenko, Olga, et al.
(författare)
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Opioid precursor protein isoform is targeted to the cell nuclei in the human brain
- 2017
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165 .- 1872-8006. ; 1861:2, s. 246-255
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Lepp, Håkan, et al.
(författare)
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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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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1790:6, s. 552-7
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Pasupuleti, Mukesh, et al.
(författare)
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Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa
- 2009
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1790:8, s. 800-808
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Due to increasing antibiotics resistance, antimicrobial peptides (AMPs) are receiving increased attention. Pseudomonas aeruginosa is a major pathogen in this context, involved, e.g., in keratitis and wound infections. Novel bactericidal agents against this pathogen are therefore needed. METHODS: Bactericidal potency was monitored by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was probed by hemolysis. Mechanistic information was obtained from assays on peptide-induced vesicle disruption and lipopolysaccharide binding. RESULTS: End-tagging by hydrophobic amino acids yields increased potency of AMPs against P. aeruginosa, irrespective of bacterial proteinase production. Exemplifying this by two peptides from kininogen, GKHKNKGKKNGKHNGWK and KNKGKKNGKH, potency increased with tag length, correlating to more efficient bacterial wall and vesicle rupture, and to more pronounced P. aeruginosa lipopolysaccharide binding. End-tag effects remained at high electrolyte concentration and in the presence of plasma or anionic macromolecular scavengers. The tagged peptides displayed stability against P. aeruginosa elastase, and were potent ex vivo, both in a contact lens model and in a skin wound model. GENERAL SIGNIFICANCE: End-tagging, without need for post-peptide synthesis modification, may be employed to enhance AMP potency against P. aeruginosa at maintained limited toxicity.
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| 7. |
- Periyathambi, Prabu, et al.
(författare)
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Macrophages mediated diagnosis of rheumatoid arthritis using fibrin based magnetic nanoparticles as MRI contrast agents.
- 2017
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1861:1 Pt A, s. 2992-3001
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: A variety of bioimaging tools assists in the diagnosis and evaluation of rheumatoid arthritis (RA) and other osteoarthritis. However, detection of RA in the early stages by targeting its macrophages with suitable contrast agents will help in arresting the progression of the disease.METHODS: In the present study, we investigated the effectiveness of using magnetic fibrin nanoparticles (MFNPs) conjugated with folic acid (FA-MFNPs) as a specific contrast agent to target the activated macrophages, which overexpress the folate receptors (FR) in the knee joints of rats with antigen-induced arthritis (AIA).RESULTS: FA-MFNPs were spherical with an average size of 18.3±1.6nm. In vitro studies have shown effective internalization of FA-MFNPs into the Raw264.7 macrophage cells. In vivo studies were carried out by injecting FA-MFNPs intravenously into the arthritic rats. The results showed enhanced MR imaging in the synovium of arthritic joints. Prussian blue histological staining confirmed uptake of FA-MFNPs by macrophages in the synovial tissue.CONCLUSION: The animal experiment results indicate that FA-MFNPs can be used as a specific MRI contrast agent in identifying phagocytic active macrophages in the synovial joints.GENERAL SIGNIFICANCE: Blood is the precursor source for synthesising the fibrin-based iron oxide (magnetic) nanoparticles (MFNPs) with diameters between 12 and 15nm. It has excellent superparamagnetic behaviour, biocompatibility, osteogenic potency, hemocompatibility, and biodegradable properties. MFNPs-based nanocomposites might be a promising contrast agent for bioimaging.
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| 8. |
- Poliakov, Anton, et al.
(författare)
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Structure-activity relationships for the selectivity of hepatitis C virus NS3 protease inhibitors
- 2004
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1672:1, s. 51-59
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Tidskriftsartikel (refereegranskat)abstract
- The selectivity of hepatitis C virus (HCV) non-structural protein 3 (NS3) protease inhibitors was determined by evaluating their inhibitory effect on other serine proteases (human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine pancreatic chymotrypsin (BPC)) and a cysteine protease (cathepsin B). For these peptide inhibitors, the P1-side chain and the C-terminal group were the major determinants of selectivity. Inhibitors with electrophilic C-terminal residues were generally non-selective while compounds with non-electrophilic C-terminal residues were more selective. Furthermore, compounds with P1 aminobutyric acid residues were non-selective, while 1-aminocyclopropane-1-carboxylic acid (ACPC) and norvaline-based inhibitors were generally selective. The most potent and selective inhibitors of NS3 protease tested contained a non-electrophilic phenyl acyl sulfonamide C-terminal residue. HLE was most likely to be inhibited by the HCV protease inhibitors, in agreement with similar substrate specificities for these enzymes. The identified structure-activity relationships for selectivity are of significance for design of selective HCV NS3 protease inhibitors.
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| 9. |
- Tolmachev, Vladimir, et al.
(författare)
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Radiolabelled proteins for positron emission tomography : pros and cons of labelling methods
- 2010
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1800:5, s. 487-510
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Forskningsöversikt (refereegranskat)abstract
- BACKGROUND: Dynamic biomedical research is currently yielding a wealth of information about disease-associated molecular alterations on cell surfaces and in the extracellular space. The ability to visualize and quantify these alterations in vivo could provide important diagnostic information and be used to guide individually-optimized therapy. Biotechnology can provide proteinaceous molecular probes with highly specific target recognitions. Suitably labelled, these may be used as tracers for radionuclide-based imaging of molecular disease signatures. If the labels are positron-emitting radionuclides, the superior resolution, sensitivity and quantification capability of positron emission tomography (PET) can be exploited. SCOPE OF REVIEW: This article discusses different approaches to labelling proteins with positron-emitting nuclides with suggestions made depending on the biological features of the tracers. MAJOR CONCLUSIONS: Factors such as matching biological and physical half-lives, availability of the nuclide, labelling yields, and influences of labelling on targeting properties (affinity, charge and lipophilicity, cellular processing and retention of catabolites) should be considered when selecting a labelling strategy for each proteinaceous tracer. GENERAL SIGNIFICANCE: The labelling strategy used can make all the difference between success and failure in a tracer application. This review emphasises chemical, biological and pharmacological considerations in labelling proteins with positron-emitting radionuclides.
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| 10. |
- Vedakumari, Weslen S, et al.
(författare)
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Fibrin nanoparticles as Possible vehicles for drug delivery.
- 2013
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 1830:8, s. 4244-4253
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Several issues have been raised emphasizing the harmful toxic effects of metal nanoparticles towards biological systems. Search of biological nanoparticles with excellent biocompatibility and bioavailability could address this problem.METHODS: Fibrin nanoparticles (FNP) were prepared using a novel technique and characterized for their physico-chemical properties. In vitro studies were performed to examine cytotoxicity and cellular uptake of FNP. Innate immune response to FNP was studied by (i) estimating in vitro generation of complement split products, C3a and C4d and (ii) in vivo expression of pro-inflammatory cytokines, TNF-α, IL-1 and IL-6. In vivo biodistribution study was carried out by intravenous administration of FITC-labelled FNP in mice.RESULTS: FNP were spherical with size ranging from 25 to 28nm. In vitro studies proved the biocompatibility of the nanoparticles, with their distribution across the cytoplasm and nucleus of treated cells. Complement activation studies showed insignificant increase in the level of C3a when compared with positive control. RT-PCR results revealed significant upregulation of TNF-α and downregulation of IL-6 cytokines after 6h of FNP administration. In vivo biodistribution studies showed moderate blood circulation time, with predominant distribution of nanoparticles in the liver followed by the lungs, kidney and spleen. Haematology, serum biochemistry, and histopathology analyses demonstrated that FNP were non-toxic.CONCLUSION: Owing to their small size, low cost, ease of preparation and excellent biocompatibility, FNP might be a promising novel material for drug delivery applications.GENERAL SIGNIFICANCE: Our results demonstrate the safe and promising use of FNP for biomedical applications.
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