Enzyme activity and AGE formation in a model of AST glycoxidation by D-fructose in vitro

Neenzimska glikacija je proces kojim se reducirajući šećeri kovalentnim vezama povezuju s amino skupinama proteina, bez posredovanja enzima. Taj proces dijelom je i oksidativan pa se često naziva glikooksidacija. Konačni produkti glikacije (AGE) nastaju polimerizacijom mnogih intermedijarnih produkata. U ovom radu, praćen je u in vitro uvjetima, mjerenjem katalitičke aktivnosti enzima i fluorescencije nastalih ukupnih AGE produkata i pentozidina, utjecaj D-fruktoze kao reaktivnog šećera na aspartat aminotransferazu (EC 2.6.1.1.) kao modelni protein. Promjena katalitičke aktivnosti enzima pokazatelj je promjenjene funkcije proteina glikacijom dok je intenzitet fluorescencije ukupnih AGE produkata i pentozidina samo djelomičan pokazatelj glikoksidacijskih promjena. Katalitička aktivnost AST izmjerena IFCC preporučenom metodom u inkubacijskoj smjesi koja je sadržavala 50 mmol L-1 fruktoze, smanjena je na 42% (p<0.05) petog dana, te na 11% (p<0.05) dvadeset i prvog dana. Statistički su značajne razlike u katalitičkoj aktivnosti enzima inkubiranog sa i bez fruktoze već nakon petog dana.Stvaranje ukupnih AGE produkata i pentozidina statistički je značajno nakon pet, odnosno 15 dana inkubacije enzima s fruktozom. Katalitička aktivnost enzima jasno ukazuje na funkcionalne promjene uzrokovane glikiranjem, dok je evaluacija stvaranja ukupnih AGE produkata, a pogotovo pentozidina, mjerenjem fluorescencije manje pouzdan pokazatelj promjena.Non-enzymatic glycation as the reaction chain between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L-1), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentrations were significant higher since 5th day of incubation (110 %, p < 0.05) and the fluorescent pentosidine concentrations from 15th day of incubation (117 %, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable

Enzyme activity and AGE formation in a model of AST glycoxidation by D-fructose in vitro

Neenzimska glikacija je proces kojim se reducirajući šećeri kovalentnim vezama povezuju s amino skupinama proteina, bez posredovanja enzima. Taj proces dijelom je i oksidativan pa se često naziva glikooksidacija. Konačni produkti glikacije (AGE) nastaju polimerizacijom mnogih intermedijarnih produkata. U ovom radu, praćen je u in vitro uvjetima, mjerenjem katalitičke aktivnosti enzima i fluorescencije nastalih ukupnih AGE produkata i pentozidina, utjecaj D-fruktoze kao reaktivnog šećera na aspartat aminotransferazu (EC 2.6.1.1.) kao modelni protein. Promjena katalitičke aktivnosti enzima pokazatelj je promjenjene funkcije proteina glikacijom dok je intenzitet fluorescencije ukupnih AGE produkata i pentozidina samo djelomičan pokazatelj glikoksidacijskih promjena. Katalitička aktivnost AST izmjerena IFCC preporučenom metodom u inkubacijskoj smjesi koja je sadržavala 50 mmol L-1 fruktoze, smanjena je na 42% (p<0.05) petog dana, te na 11% (p<0.05) dvadeset i prvog dana. Statistički su značajne razlike u katalitičkoj aktivnosti enzima inkubiranog sa i bez fruktoze već nakon petog dana.Stvaranje ukupnih AGE produkata i pentozidina statistički je značajno nakon pet, odnosno 15 dana inkubacije enzima s fruktozom. Katalitička aktivnost enzima jasno ukazuje na funkcionalne promjene uzrokovane glikiranjem, dok je evaluacija stvaranja ukupnih AGE produkata, a pogotovo pentozidina, mjerenjem fluorescencije manje pouzdan pokazatelj promjena.Non-enzymatic glycation as the reaction chain between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L-1), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentrations were significant higher since 5th day of incubation (110 %, p < 0.05) and the fluorescent pentosidine concentrations from 15th day of incubation (117 %, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney

Background: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. Results: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. Conclusions: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.Peer reviewe

Katalitička aktivnost i stvaranje AGE-produkata u modelu glikoksidacije AST D-fruktozom in vitro

Non-enzymatic glycation as the reaction chain between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L-1), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentrations were significant higher since 5th day of incubation (110 %, p < 0.05) and the fluorescent pentosidine concentrations from 15th day of incubation (117 %, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable.Neenzimska glikacija je proces kojim se reducirajući šećeri kovalentnim vezama povezuju s amino skupinama proteina, bez posredovanja enzima. Taj proces dijelom je i oksidativan pa se često naziva glikooksidacija. Konačni produkti glikacije (AGE) nastaju polimerizacijom mnogih intermedijarnih produkata. U ovom radu, praćen je u in vitro uvjetima, mjerenjem katalitičke aktivnosti enzima i fluorescencije nastalih ukupnih AGE produkata i pentozidina, utjecaj D-fruktoze kao reaktivnog šećera na aspartat aminotransferazu (EC 2.6.1.1.) kao modelni protein. Promjena katalitičke aktivnosti enzima pokazatelj je promjenjene funkcije proteina glikacijom dok je intenzitet fluorescencije ukupnih AGE produkata i pentozidina samo djelomičan pokazatelj glikoksidacijskih promjena. Katalitička aktivnost AST izmjerena IFCC preporučenom metodom u inkubacijskoj smjesi koja je sadržavala 50 mmol L-1 fruktoze, smanjena je na 42% (p<0.05) petog dana, te na 11% (p<0.05) dvadeset i prvog dana. Statistički su značajne razlike u katalitičkoj aktivnosti enzima inkubiranog sa i bez fruktoze već nakon petog dana.Stvaranje ukupnih AGE produkata i pentozidina statistički je značajno nakon pet, odnosno 15 dana inkubacije enzima s fruktozom. Katalitička aktivnost enzima jasno ukazuje na funkcionalne promjene uzrokovane glikiranjem, dok je evaluacija stvaranja ukupnih AGE produkata, a pogotovo pentozidina, mjerenjem fluorescencije manje pouzdan pokazatelj promjena

Oxidative stress parameters in different systemic rheumatic diseases

The involvement of oxidative stress in the pathogenesis of rheumatic disorders, such as systemic sclerosis (SSc) and chronic polyarthritides, has been suggested yet not thoroughly verified experimentally. We analysed 4 plasmatic parameters of oxidative stress in patients with SSc (n = 17), psoriatic arthritis (PsA) (n = 10) and rheumatoid arthritis (RA) (n = 9) compared with healthy subjects (n = 22). The biomarkers were: total antioxidant capacity (TAC) measured by ferric reducing antioxidant power (FRAP) method, hydroperoxides determined by ferrous ion oxidation in presence of xylenol orange (FOX) method and sulfhydryl and carbonyl groups assessed by spectrophotometric assays. The results showed significantly increased hydroperoxides in SSc, PsA and RA (3.97 +/- 2.25, 4.87 +/- 2.18 and 5.13 +/- 2.36 mu mol L-1, respectively) compared with the control group (2.31 +/- 1.40 mu mol L-1; P < 0.05). Sulfhydryls were significantly lower in SSc (0.466 +/- 0.081 mmol L-1), PsA (0.477 +/- 0.059 mmol L-1) and RA (0.439 +/- 0.065 mmol L-1) compared with the control group (0.547 +/- 0.066 mmol L-1; P < 0.05). TAC in all three diseases showed no difference in comparison with controls. Carbonyls were significantly higher in RA than in the control group (32.1 +/- 42 vs 2.21 +/- 1.0 nmol (mg protein)(-1); P < 0.05). The obtained data indicate augmented free radical-mediated injury in these rheumatic diseases and suggest a role for the use of antioxidants in prevention and treatment of these pathologies

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney

Background: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. Results: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. Conclusions: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.N