23 research outputs found
Altered protein expression and protein nitration pattern during d-galactosamine-induced cell death in human hepatocytes: a proteomic analysis
BACKGROUND/AIMS:
Hepatic injury by d-galactosamine (d-GalN) is a suitable experimental model of hepatocellular injury. The induction of oxidative and nitrosative stress participates during d-GalN-induced cell death in cultured rat hepatocytes. This study aimed to identify protein expression changes during the induction of apoptosis and necrosis by d-GalN in cultured human hepatocytes.
METHODS:
A proteomic approach was used to identify the proteins involved and those altered by tyrosine nitration. A high dose of d-GalN (40 mM) was used to induce apoptosis and necrosis in primary culture of human hepatocytes. Cellular lysates prepared at different times after addition of d-GalN were separated by two-dimensional electrophoresis. Gel spots with an altered expression and those matching nitrotyrosine-immunopositive proteins were excised and analyzed by mass spectrometry.
RESULTS:
d-GalN treatment upregulated microsomal cytochrome b5, fatty acid binding protein and manganese superoxide dismutase, and enhanced annexin degradation. d-GalN increased tyrosine nitration of four cytosolic (Hsc70, Hsp70, annexin A4 and carbonyl reductase) and three mitochondrial (glycine amidinotransferase, ATP synthase beta chain, and thiosulfate sulfurtransferase) proteins in human hepatocytes.
CONCLUSIONS:
The results provide evidences that oxidative stress and nitric oxide-derived reactive oxygen intermediates induce specific alterations in protein expression that may be critical for the induction of apoptosis and necrosis by d-GalN in cultured human hepatocytes
Hepatocellular carcinoma risk-stratification based on ASGR1 in circulating epithelial cells for cancer interception
Purpose: Lack of diagnostic and prognostic biomarkers in hepatocellular
carcinoma impedes stratifying patients based on their risk of developing
cancer. The aim of this study was to evaluate phenotypic and genetic
heterogeneity of circulating epithelial cells (CECs) based on
asialoglycoprotein receptor 1 (ASGR1) and miR-122-5p expression as
potential diagnostic and prognostic tools in patients with hepatocellular
carcinoma (HCC) and liver cirrhosis (LC).
Methods: Peripheral blood samples were extracted from LC and HCC patients
at different disease stages. CECs were isolated using positive immunomagnetic
selection. Genetic and phenotypic characterization was validated by double
immunocytochemistry for cytokeratin (CK) and ASGR1 or by in situ hybridization
with miR-122-5p and CECs were visualized by confocal microscopy.
Results: The presence of CECs increased HCC risk by 2.58-fold, however, this
was only significant for patients with previous LC (p = 0.028) and not for those
without prior LC (p = 0.23). Furthermore, the number of CECs lacking
ASGR1 expression correlated significantly with HCC incidence and absence
of miR-122-5p expression (p = 0.014; r = 0.23). Finally, overall survival was
significantly greater for patients at earlier cancer stages (p = 0.018), but this difference was only maintained in the group with the presence of CECs (p =
0.021) whereas progression-free survival was influenced by the absence of
ASGR1 expression.
Conclusion: Identification and characterization of CECs by ASGR1 and/or miR-
122-5p expression may be used as a risk-stratification tool in LC patients, as it
was shown to be an independent prognostic and risk-stratification marker in LC
and early disease stage HCC patients
Mitochondrial physiology
As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery
Mitochondrial physiology
As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery
Therapeutic Applications of Nitric Oxide and Derivatives
-Provides updated reviews on the chemistry and signaling of newly derived therapeutic nitric oxide (NO) donors/inhibitors and their complexes in liposomes or nanospheres in both pre-clinical and clinical activities
-Discusses the application of NO in monotherapy or in combination with conventional therapies in a variety of cancers and inflammatory diseases
-Encompasses real-world examples of recent research related to NO and cance
Detection and proteomic identification of S-nitrosated proteins in human hepatocytes
The S-nitrosation of protein thiols is a redox-based posttranslational modification that modulates protein function and cell phenotype. Although the detection of S-nitrosated proteins is problematical because of the lability of S-nitrosothiols, an increasing range of proteins has been shown to undergo S-nitrosation with the improvement of molecular tools. This chapter describes the methodology used to identify potential targets of S-nitrosation in cultured primary human hepatocytes using proteomic approaches. This methodology is based on the biotin switch method, which labels S-nitrosated proteins with an affinity tag, allowing their selective detection and proteomic identification
Detection and proteomic identification of S-nitrosated proteins in human hepatocytes
The S-nitrosation of protein thiols is a redox-based posttranslational modification that modulates protein function and cell phenotype. Although the detection of S-nitrosated proteins is problematical because of the lability of S-nitrosothiols, an increasing range of proteins has been shown to undergo S-nitrosation with the improvement of molecular tools. This chapter describes the methodology used to identify potential targets of S-nitrosation in cultured primary human hepatocytes using proteomic approaches. This methodology is based on the biotin switch method, which labels S-nitrosated proteins with an affinity tag, allowing their selective detection and proteomic identification
Proteomic analysis for developing new biomarkers of hepatocellular carcinoma
AIM: To identify new markers of hepatocellular carcinoma (HCC) using a proteomic analysis
S-Nitrosation of proteins during D-galactosamine-induced cell death in human hepatocytes
Nitric oxide (NO) participates in the cell death induced by d-Galactosamine (d-GalN) in hepatocytes, and NO-derived reactive oxygen intermediates are critical contributors to protein modification and hepatocellular injury. It is anticipated that S-nitrosation of proteins will participate in the mechanisms leading to cell death in d-GalN-treated human hepatocytes. In the present study, d-GalN-induced cell death was related to augmented levels of NO production and S-nitrosothiol (SNO) content. The biotin switch assay confirmed that d-GalN increased the levels of S-nitrosated proteins in human hepatocytes. S-nitrosocysteine (CSNO) enhanced protein S-nitrosation and altered cell death parameters that were related to S-nitrosation of the executioner caspase-3. Fifteen S-nitrosated proteins participating in metabolism, antioxidative defense and cellular homeostasis were identified in human hepatocytes treated with CSNO. Among them, seven were also identified in d-GalN-treated hepatocytes. The results here reported underline the importance of the alteration of SNO homeostasis during d-GalN-induced cell death in human hepatocyte