This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl ...methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 μg mL⁻¹ and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL⁻¹. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.
A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to ...enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (∼5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50°C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB–RNase H scheme can be applied to a broad range of nucleic acid amplification methods.
The key events in regulating cardiac muscle contraction involve Ca2+ binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca2+ ...movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca2+ binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca2+-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca2+. L29Q had no effect on the closing rate of the N-domain triggered by release of Ca2+, but reduced the Ca2+-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca2+ dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.
Our recent molecular studies revealed two divergent PEP-carboxylase (PEPC Ppc) encoding genes in the green microalga
Chlamydomonas reinhardtii,
CrPpc1 and
CrPpc2, which are coordinately responsive to ...changes in inorganic-N and -C supply at the transcript level Mamedov, T.G., Moellering, E.R. and Chollet, R. (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga
C. reinhardtii, Plant J. 42, 832–843. Here, we report the distribution of these two encoded catalytic subunits in the minor Class-1 and predominant Class-2 PEPC enzyme-forms, the latter of which is a novel high-molecular-mass, hetero-oligomeric complex containing
both CrPpc1 (p109) and CrPpc2 (p131) polypeptides. The Class-1 enzyme, however, is a typical PEPC homotetramer comprised solely of p109. We also document that the amount of both CrPpc1/2 catalytic subunits is up-/down-regulated by varying levels of
NH
4
+
supplied to the culture medium.
A strain of the cyanobacterium Arthrospira was isolated from Lake Chahannaoer in northern China and was characterized according to microscopic morphology, photosynthetic oxygen-evolving activity, ...growth rate, and nutritional profile. Compared with thermophilic Arthrospira species occurring naturally in tropical and subtropical lakes, this isolate is mesophilic and grows optimally at ~20℃. The total protein, fatty acid, phycocyanin, carotenoid, and chlorophyll a contents were 67.6, 6.1, 4.32, 0.29, and 0.76 grams per 100 grams of dry weight, respectively. The strain is rich in polyunsaturated fatty acids (PUFAs). An essential omega-3 fatty acid, docosahexaenoic acid (DHA), was detected, and γ-linolenic acid (GLA) and DHA accounted for 28.3% of the total fatty acid content. These features of this newly isolated strain make it potentially useful in commercial mass culture in local areas or as a biofuel feedstock. It is also an alternative resource for studying the metabolic PUFA pathways and mechanisms of cold stress tolerance in cyanobacteria.
The Chloroplast Rieske Iron-Sulfur Protein de Vitry, Catherine; Ouyang, Yexin; Finazzi, Giovanni ...
Journal of biological chemistry/The Journal of biological chemistry,
10/2004, Letnik:
279, Številka:
43
Journal Article
Recenzirano
Odprti dostop
We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur protein; that
is, the flexible hinge that connects the transmembrane helix to the soluble ...cluster-bearing domain, the N-terminal stromal
protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b f complex, perform plastoquinol oxidation, and signal 6 redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo . Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex
accumulation or electron transfer efficiency. Deletion of three residues (Î3G) dramatically decreased reaction rates by a
factor of â¼10. These data indicate that the chloroplast iron-sulfur protein-linking domain is much more flexible than that
of its counterpart in mitochondria. Despite greatly slowed catalysis in the Î3G mutant, there was no apparent delay in light-harvesting
complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske protein
did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with
mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske protein transmembrane helix
resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske protein transmembrane helix plays an active role in assembly-mediated control
of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b 6 f structure.
The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain ...(MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca2+ binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca2+-bound to the Ca2+-free dynamic states were significantly faster (53.2–116.8 s−1) than that of the IR (14.1 s−1). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca2+ dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca2+ signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation.
The kinetics and dynamics of the C-domain of cTnI were studied.
Fluorescence anisotropy data show support for the fly casting model and a fourth state of thin filament activation.
The fly casting model holds true in the thin filament, but the presence of S1 modulates the process.
Our study provides information on the role of the cTnI C-domain in thin filament regulation.
•cTnT phosphorylation by PKC was mimicked in in vitro thin filaments.•I–C switching was monitored by FRET for these phospho-mimics.•Phospho-mimics altered I–C switching Ca2+-sensitivity, structure, ...and kinetics.•cTnT(T204E) significantly reduced Ca2+-sensitivity of I–C switching.•cTnT(3M) and (4M) increased Ca2+-sensitivity, reduced I–C separation and kinetics.
FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca2+ and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca2+ and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca2+ sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca2+-sensitivity. The rate of Ca2+-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca2+-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca2+-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca2+-sensitivity and likely play an important role in cardiac regulation.
Display omitted
•FRET was used to monitor Ca2+-dependent N-cTnC opening in skinned myocardial fibers.•Inhibition of strong cross-bridges reduced the maximal extent of N-cTnC opening.•Cycling ...cross-bridges enhanced the overall exposure of the N-cTnC hydrophobic patch.•Non-cycling strong cross-bridges stimulated N-cTnC opening under nanomolar Ca2+.•The overall stability of N-cTnC opening correlated positively with Ca2+ sensitivity.
The in situ structural coupling between the cardiac troponin (cTn) Ca2+-sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using Förster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca2+ titrations of cTnC(T13C/N51C)AEDENS/DDPM-reconstituted fibers showed that the Ca2+-dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force−Ca2+ relationship. N-cTnC opening still occurred steeply during Ca2+ titrations in the presence of 1mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca2+-sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca2+ levels, treatment with ADP·Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca2+ titrations in the presence of ADP·Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca2+ with increased Ca2+-sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca2+-sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI.
This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) ...(PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both "peak mode" and "plateau mode" and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL(-1) in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms.
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