Physical exercise stimulates salivary secretion of cystatins.

Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins.

Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca-binding affinity in a C-domain mutant of cardiac troponin C.

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca-binding affinity for D145E but only small changes for the wild-type protein.

Balance between -nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation.

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form -nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of -nitrosoglutathione reductase (GSNOR) during myogenesis is understood.

Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca Ion.

Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype.

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+)Sensitivity in Intact Cardiomyocytes.

Aims: The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S-nitrosoglutathione (GSNO), a protein S-nitrosylating agent. Although S-nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S-nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments.

Brazilian Science and Research Integrity: Where are We? What Next?

Building a world-class scientific community requires first-class ingredients at many different levels: funding, training, management, international collaborations, creativity, ethics, and an understanding of research integrity practices. All over the world, addressing these practices has been high on the science policy agenda of major research systems. Universities have a central role in fostering a culture of research integrity, which has posed additional challenges for faculty, students and administrators - but also opportunities.

Exogenous β-amyloid peptide interferes with GLUT4 localization in neurons.

Aging represents a major risk factor for numerous illnesses that are of increasing importance to society, including two of the most prevalent: diabetes and Alzheimer's disease. Studies have shown that diabetes is a risk factor for spontaneous Alzheimer's disease. While these studies suggest that diabetes can contribute to Alzheimer's disease, the implications of AD on diabetes are practically unexplored.

Insights into the intramolecular coupling between the N- and C-domains of troponin C derived from high-pressure, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering studies.

Troponin C (TnC), the Ca(2+)-binding component of the troponin complex of vertebrate skeletal muscle, consists of two structurally homologous domains, the N- and C-domains; these domains are connected by an exposed α-helix. Mutants of full-length TnC and of its isolated domains have been constructed using site-directed mutagenesis to replace different Phe residues with Trp. Previous studies utilizing these mutants and high hydrostatic pressure have shown that the apo form of the C-domain is less stable than the N-domain and that the N-domain has no effect on the stability of the C-domain [Rocha, C.

The impact of stem cells on electron fluxes, proton translocation, and ATP synthesis in kidney mitochondria after ischemia/reperfusion.

Tissue damage by ischemia/reperfusion (I/R) results from a temporary cessation of blood flow followed by the restoration of circulation. The injury depresses mitochondrial respiration, increases the production of reactive oxygen species (ROS), decreases the mitochondrial transmembrane potential, and stimulates invasion by inflammatory cells. The primary objective of this work was to address the potential use of bone marrow stem cells (BMSCs) to preserve and restore mitochondrial function in the kidney after I/R.

Redox balance and mitochondrial glycerol phosphate dehydrogenase activity in trained rats.

Free radical production is increased in many disease states and during exercise, but in the latter the concurrent stimulation of the antioxidant defense system seems to protect the organism from excessive production of reactive oxygen species. Chronic exercise can exert negative effects on the activity of mitochondrial glycerol phosphate dehydrogenase (mGPdH), which may offer some explanation for the antioxidant effects of training, since this enzyme is a relevant producer of free radicals. To test this correlation, we compared mGPdH activity, two antioxidant defense markers and two markers of oxidative stress in sedentary and trained (Tr) rats.

β-Amyloid peptide is internalized into chick retinal neurons and alters the distribution of myosin Vb.

The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease (AD). A major hallmark of AD is dementia from a loss of neuronal function, attributed to the presence and accumulation of β-amyloid (Aβ) peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking.

Redox state of troponin C cysteine in the D/E helix alters the C-domain affinity for the thin filament of vertebrate striated muscle.

Background: Despite a broad spectrum of structural studies, it is not yet clear whether the D/E helix of troponin C (TnC) contributes to the interaction of TnC with troponin I (TnI). Redox modifications at Cys 98 in the D/E helix were explored for clues to TnC binding to the thin filament off-state, using recombinant wild-type TnC and an engineered mutant without Cys (Cys98Leu).

Methods: Recombinant proteins and rabbit psoas skinned fibres were reduced with dithiothreitol (DTT) and variously recombined.

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach.

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity.

2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1.

2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg(2+)ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS).

Myosin is reversibly inhibited by S-nitrosylation.

Nitric oxide (NO*) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO* or NO* derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes.

Free-energy linkage between folding and calcium binding in EF-hand proteins.

Troponin is the singular Ca(2+)-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca(2+)-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90).

Betaine protects urea-induced denaturation of myosin subfragment-1.

We have demonstrated previously that urea inhibits the activity and alters the tertiary structure of skeletal muscle myosin in a biphasic manner. This was attributed to differential effects on its globular and filamentous portion. The inhibition of catalytic activity was counteracted by methylamines.

Volume and free energy of folding for troponin C C-domain: linkage to ion binding and N-domain interaction.

Troponin C (TnC) is an 18-kDa acidic protein of the EF-hand family that serves as the trigger for muscle contraction. In this study, we investigated the thermodynamic stability of the C-domain of TnC in all its occupancy states (apo, Mg (2+)-, and Ca (2+)-bound states) using a fluorescent mutant with Phe 105 replaced by Trp (F105W/C-domain, residues 88-162) and (1)H NMR spectroscopy. High hydrostatic pressure was employed as a perturbing agent, in combination with urea or without it.

Modulation of troponin C affinity for the thin filament by different cross-bridge states in skinned skeletal muscle fibers.

In vertebrate skeletal muscle, the C-domain of troponin C (TnC) serves as an anchor; the N-domain regulates the position of troponin-tropomyosin on the thin filament after changes in intracellular Ca2+. Another type of thin-filament regulation is provided by cross-bridges. In this study, we use skinned fibers reconstituted with chicken recombinant TnC (rTnC) to examine TnC-thin filament affinity when cross-bridges containing different ligands are formed.

Ionic interventions that alter the association of troponin C C-domain with the thin filaments of vertebrate striated muscle.

The regulatory complex of vertebrate skeletal muscle integrates information about cross-bridge binding, divalent cations and other intracellular ionic conditions to control activation of muscle contraction. Relatively little is known about the role of the troponin C (TnC) C-domain in the absence of Ca2+. Here, we use a standardized condition for measuring isometric tension in rabbit psoas skinned fibers to track TnC attachment and detachment in the absence of Ca2+ under different conditions of ionic strength, pH and MgATP.

Troponin C/calmodulin chimeras as erythrocyte plasma membrane Ca2+-ATPase activators.

Calmodulin (CaM) and troponin C (TnC) are EF-hand proteins that play fundamentally different roles in animal physiology. TnC has a very low affinity for the plasma membrane Ca2+-ATPase and is a poor substitute for CaM in increasing the enzyme's affinity for Ca2+ and the rate of ATP hydrolysis. We use a series of recombinant TnC (rTnC)/CaM chimeras to clarify the importance of the CaM carboxyl-terminal domain in the activation of the plasma membrane Ca2+-ATPase.

Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament.

The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites.