Abstract
Mass spectrometric studies of the interactions of cisplatin and transplatin with myoglobin (Mb) provide information concerning interaction kinetics, Mb adduct identity, and cisplatin and transplatin binding sites on Mb.
Although the Mb–cisplatin interaction is faster than the Mb–transplatin interaction, monoadducts and diadducts were formed in both the interactions over 30 h. In order to locate the binding sites of cisplatin and transplatin on Mb, digests of free Mb, Mb–cisplatin and Mb–transplatin adducts were subjected to analysis by Fourier transform mass spectrometry (FT-MS). This analysis revealed that two fragment ions, 1313.275+ and 1316.685+, were obtained only from the Mb–cisplatin and Mb–transplatin adduct digests.
Tandem mass spectrometry (MS/MS and MS3) of the 1313.275+ and 1316.685+ ions indicate that these ions arise from [Pt(NH3)]2+ and [Pt(NH3)2]2+, respectively, bound to peptide His97-Gly153. The product-ion spectra of the MS/MS and MS3 analyses of the 1313.275+ ion indicate a common binding site of cisplatin and transplatin on His116-His119 residues. The interactions of cisplatin and transplatin with a dipeptide His-Ser and the three dimensional (3D) structure of native Mb suggest that cisplatin and transplatin coordinate to His116 and His119.
Ting Zhaoa and Fred L. King, a,
a C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506-6045, USA
Thursday
Tuesday
Expression and Functional Regulation of Myoglobin in Epithelial Cancers
(American Journal of Pathology. 2009;175:201-206.)
Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging has been extensively characterized. We hypothesized that solid tumors could take advantage of proteins such as myoglobin to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species.
We therefore sought to establish whether myoglobin might be expressed and functionally regulated in epithelial tumors that are known to face hypoxia and oxidative stress during disease progression. We analyzed the expression of myoglobin in human epithelial cancers at both transcriptional and protein levels; moreover, we investigated the expression levels of myoglobin in cancer cell lines subjected to different conditions, including hypoxia, oxidative stress, and mitogenic stimuli. We provide evidence that human epithelial tumors, including breast, lung, ovary, and colon carcinomas, express high levels of myoglobin from the earliest stages of disease development.
In human cancer cells, myoglobin is induced by a variety of signals associated with tumor progression, including mitogenic stimuli, oxidative stress, and hypoxia. This study provides evidence that myoglobin, previously thought to be restricted to myocytes, is expressed at high levels by human carcinoma cells. We suggest that myoglobin expression is part of a cellular program aimed at coping with changed metabolic and environmental conditions associated with neoplastic growth.
Expression and Functional Regulation of Myoglobin in Epithelial Cancers
Simona Emilia Flonta*, Sabrina Arena*, Alberto Pisacane, Paolo Michieli and Alberto Bardelli* From the Laboratory of Molecular Genetics,* the Unit of Pathology, and the Division of Molecular Oncology, Institute for Cancer Research and Treatment, University of Turin Medical School, Turin; and the FIRC Institute of Molecular Oncology, Milan, Italy
Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging has been extensively characterized. We hypothesized that solid tumors could take advantage of proteins such as myoglobin to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species.
We therefore sought to establish whether myoglobin might be expressed and functionally regulated in epithelial tumors that are known to face hypoxia and oxidative stress during disease progression. We analyzed the expression of myoglobin in human epithelial cancers at both transcriptional and protein levels; moreover, we investigated the expression levels of myoglobin in cancer cell lines subjected to different conditions, including hypoxia, oxidative stress, and mitogenic stimuli. We provide evidence that human epithelial tumors, including breast, lung, ovary, and colon carcinomas, express high levels of myoglobin from the earliest stages of disease development.
In human cancer cells, myoglobin is induced by a variety of signals associated with tumor progression, including mitogenic stimuli, oxidative stress, and hypoxia. This study provides evidence that myoglobin, previously thought to be restricted to myocytes, is expressed at high levels by human carcinoma cells. We suggest that myoglobin expression is part of a cellular program aimed at coping with changed metabolic and environmental conditions associated with neoplastic growth.
Expression and Functional Regulation of Myoglobin in Epithelial Cancers
Simona Emilia Flonta*, Sabrina Arena*, Alberto Pisacane, Paolo Michieli and Alberto Bardelli* From the Laboratory of Molecular Genetics,* the Unit of Pathology, and the Division of Molecular Oncology, Institute for Cancer Research and Treatment, University of Turin Medical School, Turin; and the FIRC Institute of Molecular Oncology, Milan, Italy
Thursday
Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney's mitochondria
Plotnikov EY, Chupyrkina AA, Pevzner IB, Isaev NK, Zorov DB.
A.N.Belozersky Institute of Physico-Chemical Biology, and Institute of Mitoingeneering, Moscow State University, Moscow 119991, Russia.
Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology
A.N.Belozersky Institute of Physico-Chemical Biology, and Institute of Mitoingeneering, Moscow State University, Moscow 119991, Russia.
Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology
Monday
Serum myoglobin and renal morbidity and mortality
OBJECTIVES: The intractability of renal dysfunction following thoracic and thoraco-abdominal aortic repair leads us to believe that the accepted mechanisms of renal injury - ischaemia and embolism - are incompletely explanatory. We studied postoperative myoglobinaemia and renal dysfunction following aortic surgery.
METHODS: Between September 2006 and February 2008, we studied serum myoglobin in 109 patients requiring thoracic/thoraco-abdominal repair for three postoperative days. Forty-two of the 109 (38%) patients were female. The median age was 67 years (range 23-84 years). As we have focussed more attention on renal function, our independent renal consultants have dialysed more aggressively. We divided dialysis into: (1) creatinine indication, (2) non-creatinine indication and (3) no dialysis.
RESULTS: Thirteen of the 109 (12%) patients met creatinine indication for dialysis (>4 mg dl(-1)) and an additional 28 (26%) were dialysed for other reasons. Overall mortality was 12 out of 109 (11%) cases: 11 out of 41 (27%) in dialysed patients and one out of 68 (1.5%) in non-dialysed patients. Mortality did not differ between the indications for dialysis. Predictors of mortality were baseline glomerular filtration rate (GFR), postoperative myoglobin and dialysis. The only predictor of dialysis was postoperative myoglobin.
CONCLUSION: A strong relationship between postoperative serum myoglobin and renal failure suggests a rhabdomyolysis-like contributing aetiology following thoraco-abdominal aortic repair. We postulate a novel mechanism of renal injury for which mitigation strategies should be developed.
Miller CC 3rd, Villa MA, Sutton J, Lau D, Keyhani K, Estrera AL, Azizzadeh A, Coogan SM, Safi HJ.
Department of Cardiothoracic and Vascular Surgery, University of Texas Medical School at Houston, Memorial Hermann Heart and Vascular Institute, Houston, TX 77030,
METHODS: Between September 2006 and February 2008, we studied serum myoglobin in 109 patients requiring thoracic/thoraco-abdominal repair for three postoperative days. Forty-two of the 109 (38%) patients were female. The median age was 67 years (range 23-84 years). As we have focussed more attention on renal function, our independent renal consultants have dialysed more aggressively. We divided dialysis into: (1) creatinine indication, (2) non-creatinine indication and (3) no dialysis.
RESULTS: Thirteen of the 109 (12%) patients met creatinine indication for dialysis (>4 mg dl(-1)) and an additional 28 (26%) were dialysed for other reasons. Overall mortality was 12 out of 109 (11%) cases: 11 out of 41 (27%) in dialysed patients and one out of 68 (1.5%) in non-dialysed patients. Mortality did not differ between the indications for dialysis. Predictors of mortality were baseline glomerular filtration rate (GFR), postoperative myoglobin and dialysis. The only predictor of dialysis was postoperative myoglobin.
CONCLUSION: A strong relationship between postoperative serum myoglobin and renal failure suggests a rhabdomyolysis-like contributing aetiology following thoraco-abdominal aortic repair. We postulate a novel mechanism of renal injury for which mitigation strategies should be developed.
Miller CC 3rd, Villa MA, Sutton J, Lau D, Keyhani K, Estrera AL, Azizzadeh A, Coogan SM, Safi HJ.
Department of Cardiothoracic and Vascular Surgery, University of Texas Medical School at Houston, Memorial Hermann Heart and Vascular Institute, Houston, TX 77030,
Tuesday
Roles of the creatine kinase system and myoglobin in maintaining energetic state in the working heart
.Wu F, Beard DA.
Biotechnology and Bioengineering Center, Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
BACKGROUND: The heart is capable of maintaining contractile function despite a transient decrease in blood flow and increase in cardiac ATP demand during systole. This study analyzes a previously developed model of cardiac energetics and oxygen transport to understand the roles of the creatine kinase system and myoglobin in maintaining the ATP hydrolysis potential during beat-to-beat transient changes in blood flow and ATP hydrolysis rate.
RESULTS: The theoretical investigation demonstrates that elimination of myoglobin only slightly increases the predicted range of oscillation of cardiac oxygenation level during beat-to-beat transients in blood flow and ATP utilization. In silico elimination of myoglobin has almost no impact on the cytoplasmic ATP hydrolysis potential (DeltaGATPase). In contrast, disabling the creatine kinase system results in considerable oscillations of cytoplasmic ADP and ATP levels and seriously deteriorates the stability of DeltaGATPase in the beating heart.
CONCLUSION: The CK system stabilizes DeltaGATPase by both buffering ATP and ADP concentrations and enhancing the feedback signal of inorganic phosphate in regulating mitochondrial oxidative phosphorylation.
Biotechnology and Bioengineering Center, Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
BACKGROUND: The heart is capable of maintaining contractile function despite a transient decrease in blood flow and increase in cardiac ATP demand during systole. This study analyzes a previously developed model of cardiac energetics and oxygen transport to understand the roles of the creatine kinase system and myoglobin in maintaining the ATP hydrolysis potential during beat-to-beat transient changes in blood flow and ATP hydrolysis rate.
RESULTS: The theoretical investigation demonstrates that elimination of myoglobin only slightly increases the predicted range of oscillation of cardiac oxygenation level during beat-to-beat transients in blood flow and ATP utilization. In silico elimination of myoglobin has almost no impact on the cytoplasmic ATP hydrolysis potential (DeltaGATPase). In contrast, disabling the creatine kinase system results in considerable oscillations of cytoplasmic ADP and ATP levels and seriously deteriorates the stability of DeltaGATPase in the beating heart.
CONCLUSION: The CK system stabilizes DeltaGATPase by both buffering ATP and ADP concentrations and enhancing the feedback signal of inorganic phosphate in regulating mitochondrial oxidative phosphorylation.
Thursday
A potentiometric protein sensor built with surface molecular imprinting method
Surface molecular imprinting, as compared to molecular imprinted bulk polymers, has the advantages of higher re-occupation percentage of the reception sites, fast response, integration of sensing element and transducer, etc. In this study, a potentiometric protein sensor was developed based on the surface molecular imprinting technique. Using the self-assembled monolayers of alkanethiol with hydroxyl terminal groups as the matrix material, and target protein molecules as the template, the sensing layer was created on the surface of the gold-coated silicon chip-an electrochemical transducer. Potentiometric measurement demonstrated that the sensor could selectively detect myoglobin or hemoglobin molecules, either with or without the presence of other protein molecules in the same solution.
ARTICLE
ARTICLE
Monday
Biochemical impact of a soccer match — analysis of oxidative stress and muscle damage markers throughout recovery
Background
Exercise is a prone condition to enhanced oxidative stress and damage and the specific activity pattern of a soccer match may favour additional pro-oxidant redox alterations. To date, no studies have reported the impact of a soccer match on oxidative stress and muscle damage markers.
Aim
To analyse the effect of a competitive soccer match on plasma levels of oxidative stress and muscle damage markers, and to relate these findings with lower limb functional data.
Methods
Blood samples, leg muscle strength, sprint ability and delayed-onset muscle soreness (DOMS) were obtained in 16 soccer players before, at 30 min, 24, 48 and 72 h after a soccer match. Plasma creatine kinase (CK), myoglobin (Mb), malondialdehyde (MDA), sulfhydryl (–SH) groups, total antioxidant status (TAS), uric acid (UA) and blood leukocyte counts were determined.
Results
A soccer match elevated plasma Mb following 30 min and creatine kinase (CK) levels throughout the 72 h-recovery period. MDA increased throughout the recovery period and –SH decreased until 48 h post-match. TAS increased at 30 min and UA increased throughout the 72 h recovery. Blood neutrophils increased at 30 min whereas lymphocytes decreased and returned to baseline from 24 to 72 h. DOMS was higher than baseline until 72 h. Lower limb strength and sprint ability were lower than baseline until 72 h recovery.
Conclusion
The present data suggest that a soccer match increases the levels of oxidative stress and muscle damage throughout the 72 h-recovery period. The extent to which the redox alterations are associated with the recovery of muscle function should be further analysed.
ARTICLE
Exercise is a prone condition to enhanced oxidative stress and damage and the specific activity pattern of a soccer match may favour additional pro-oxidant redox alterations. To date, no studies have reported the impact of a soccer match on oxidative stress and muscle damage markers.
Aim
To analyse the effect of a competitive soccer match on plasma levels of oxidative stress and muscle damage markers, and to relate these findings with lower limb functional data.
Methods
Blood samples, leg muscle strength, sprint ability and delayed-onset muscle soreness (DOMS) were obtained in 16 soccer players before, at 30 min, 24, 48 and 72 h after a soccer match. Plasma creatine kinase (CK), myoglobin (Mb), malondialdehyde (MDA), sulfhydryl (–SH) groups, total antioxidant status (TAS), uric acid (UA) and blood leukocyte counts were determined.
Results
A soccer match elevated plasma Mb following 30 min and creatine kinase (CK) levels throughout the 72 h-recovery period. MDA increased throughout the recovery period and –SH decreased until 48 h post-match. TAS increased at 30 min and UA increased throughout the 72 h recovery. Blood neutrophils increased at 30 min whereas lymphocytes decreased and returned to baseline from 24 to 72 h. DOMS was higher than baseline until 72 h. Lower limb strength and sprint ability were lower than baseline until 72 h recovery.
Conclusion
The present data suggest that a soccer match increases the levels of oxidative stress and muscle damage throughout the 72 h-recovery period. The extent to which the redox alterations are associated with the recovery of muscle function should be further analysed.
ARTICLE
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