| 1. |
- Berti, Paul J, et al.
(författare)
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Affinity purification and elimination of methionine oxidation in recombinant human cystatin C
- 1997
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 11:1, s. 111-118
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.
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| 2. |
- Lindahl, Peter, et al.
(författare)
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Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B
- 1994
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 33:14, s. 4384-4392
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Tidskriftsartikel (refereegranskat)abstract
- The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (VallOArg cystatin C), the inhibition constant, Ki, increased 3 1-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin B. When the wild-type form of the inhibitor was used, the corresponding effect on Ki was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-17 1, no such difference in how Ki is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on Ki indicate that the additional positive charge introduced in VallOArg cystatin C is interacting with the negative charge on Glu-245 in cathepsin B when these two proteins form a complex; the cystatin variant is thus binding in a substratelike manner with this region of the enzyme. Indirectly, these results suggest that when native cystatin C and cathepsin B form a complex, Val-10 in the inhibitor interacts with the S2 subsite of the enzyme. A Ki value of 0.13 nM was obtained for the interaction of VallOArg cystatin C with papain. Compared to the dissociation equilibrium constant for wild-type cystatin C and papain, this value represents a 12 000-fold decrease in affinity. The corresponding effect with cathepsin B was only 15-fold, which presumably reflects the importance of the P2 position in binding of the N-terminal region of cystatin C to different cysteine proteinases.
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| 3. |
- Morley, John E., et al.
(författare)
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Sarcopenia With Limited Mobility : An International Consensus
- 2011
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Ingår i: Journal of the American Medical Directors Association. - : Elsevier BV. - 1525-8610 .- 1538-9375. ; 12:6, s. 403-409
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Tidskriftsartikel (refereegranskat)abstract
- A consensus conference convened by the Society of Sarcopenia, Cachexia and Wasting Disorders has concluded that "Sarcopenia, le, reduced muscle mass, with limited mobility" should be considered an important clinical entity and that most older persons should be screened for this condition. "Sarcopenia with limited mobility" is defined as a person with muscle loss whose walking speed is equal to or less than 1 m/s or who walks less than 400 m during a 6-minute walk, and who has a lean appendicular mass corrected for height squared of 2 standard deviations or more below the mean of healthy persons between 20 and 30 years of age of the same ethnic group. The limitation in mobility should not clearly be a result of otherwise defined specific diseases of muscle, peripheral vascular disease with intermittent claudication, central and peripheral nervous system disorders, or cachexia. Clinically significant interventions are defined as an increase in the 6-minute walk of at least 50 meters or an increase of walking speed of at least 0.1 m/s.
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| 4. |
- Wannigama, Dhammika Leshan, et al.
(författare)
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Tracing the transmission of mpox through wastewater surveillance in Southeast Asia
- 2023
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Ingår i: JOURNAL OF TRAVEL MEDICINE. - 1195-1982 .- 1708-8305. ; 30:5
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Tidskriftsartikel (refereegranskat)abstract
- High population density and tourism in Southeast Asia increase the risk of mpox due to frequent interpersonal contacts. Our wastewater surveillance in six Southeast Asian countries revealed positive signals for Monkeypox virus (MPXV) DNA, indicating local transmission. This alerts clinicians and helps allocate resources like testing, vaccines and therapeutics in resource-limited countries.
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