Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of ... more Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of degradation products from artificial and natural chitin substrates such as 4-NP-(GlcNAc) 2 , (GlcNAc) 4 and colloidal chitin. The FACE method has been improved by our group for analysis of chitooligosaccharides in the presence of several buffer systems commonly used in the biochemical evaluation of chitinolytic activities of enzymes at pH 2.0-8.0. FACE is a very sensitive technique detecting picomolar amounts of molecules. We optimized the detection conditions as follows: exposure type, precision; sensitivity, high resolution; exposure time, 5 s. We evaluated the (GlcNAc) 2 levels using a standard curve that allows chitooligosaccharides quantification at up to 10 nmol amounts. Using the method presented here, the chitinolytic properties of different chitinases can be compared directly. Serratia chitinase A (ChiA) and chitinase B (ChiB), two well-studied bacterial chitinases, have been shown by HPLC to have a synergistic effect on the chitin degradation rate. Using the FACE method, we determined the combinatory effects of mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) in natural chitin substrates processing. • FACE is a simple and quantitative method. • Our improved procedure enables the quantification of chitooligosaccharides produced by chitinases at pH 2.0-8.0. • FACE is able to quantify chitooligosaccharides at up to 10 nmol amounts.
International Journal of Biological Macromolecules, Aug 1, 2019
Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food proc... more Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food processing and various pathophysiological conditions such as chronic inflammatory diseases. By combination of the colorimetric analysis and fluorophore-assisted carbohydrate electrophoresis (FACE) method, we directly compared the chitinolytic properties of mouse Chit1 and AMCase and determined their combinatory effects in artificial and natural chitin substrates processing. Chit1 and AMCase display different dynamics of chitinolytic properties through acidic to neutral conditions. At pH 2.0, the activity of AMCase was higher than that of Chit1 and stronger or comparable with that of Serratia marcescens chitinase B, a well-characterized bacterium chitinase. Changes of degradation products using different substrates indicate that AMCase and Chit1 have diverse properties under various pH conditions. Exposure of the chitin substrates to both Chit1 and AMCase did not indicate any mutual interference of these enzymes and showed no synergistic effect, in contrast to observations regarding some bacterial chitinases. Our results suggest that Chit1 and AMCase have no synergistic effect under physiological conditions.
Objectives: Depression has been reported to be an independent risk factor for coronary heart dise... more Objectives: Depression has been reported to be an independent risk factor for coronary heart disease (CHD). We investigated the association of depressive symptoms with lipids and coagulation factors in young individuals free of CHD. Methods: We recruited 1191 young healthy individuals candidates for military academies (mean age=18.44-0.8 years, males 762) coming from all social classes and geographical compartments of Greece. Depression was assessed by using the depression scale of Minnesota Multiphasic Personality Invento~ test. The T-score for depression scale ranged from 22 to 90. Total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, lipoprotein (a), fibrinogen, factor VII, factor VIII and factor X were measured. Body mass index (BMI), physical activity and smoking were also reported. Results: The participants were divided in qnartiles according to the depression score. Three hundred twenty-two subjects were classified in the upper quartile of the depression score (score>48) and 269 were classified in the lower quartile (score<37). Factor VII (102.95 vs 98.5%) and X levels (92 vs 89.7) were significantly higher in individuals in the upper quartile of the depression score compared to the lower quartile of depression score while the other studied parameters did not show significant difference. In a logistic regression model with factor VII as dependent variable (upper vs lower quartile) and depression score, age, gender, BMI, smoking as predictor variables, depression was an independent predictor of factor VII levels with an adjusted odds ratio for high levels of factor VII of 1.05 (95% confidence interval 1.008-1.09, p=0.01). Factor VII was associated with triglycerides (r=-0.21, p=0.001), while factor X with triglycerides (r=-0.22, p<0.001) and cholesterol levels (r=-0.12, p<0.001). Conclusions: Depressed mood is associated with a hypercoagulant profile as it is expressed by the higher levels of coagulation factors VII and X. This might partially explain the higher propensity for CHD of people with depressive symptoms.
May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare ... more May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.
Free papers 04-Dementia Neuroprotective potential of Solanesol and Epigallocatechin gallate on IC... more Free papers 04-Dementia Neuroprotective potential of Solanesol and Epigallocatechin gallate on ICV-STZ induced cognitive dysfunction in rats
and protein expression of molecules involved in degenerative pathways related to AD such as autop... more and protein expression of molecules involved in degenerative pathways related to AD such as autophagy, proteosomal pathway, oxidative stress, survival, apoptosis, AB levels and phosphorylation of tau protein among the different groups of fibroblasts. Results: We characterize the 12 fibroblasts lines, by karyotyping, DNA sequencing and perform proteomic analyses. We found activation in the GSK3B pathway, tau hyperphosphorylation, with an increase in total tau expression and CatD overexpression in fibroblasts from AD patients compared to healthy controls. Conclusions: The fibroblasts of individuals with Alzheimer’s disease represent a suitable in vitro model for further molecular studies on the pathogenic process of AD, that could be used for the search of biomarkers of the disease and to validate potential new therapies. Additionally, these cells could be reprogrammed into iPCS, that had a broad differentiation potential, from which is possible to generate neurons, that would recapitulate the pathogenesis of AD brain cells.
It has been accepted that the hypermutation status of immunoglobulin heavy chain genes (IgVH) is ... more It has been accepted that the hypermutation status of immunoglobulin heavy chain genes (IgVH) is one of the most important independent prognostic factors in chronic lymphocytic leukemia (CLL). According to the degree ofIgVH hypermutaion, CLL patients can be stratified into prognostic groups. Given the impact ofIgVH mutation status on clinical setting, it has become highly desirable to standardize the laboratory methodologies used for IgVH mutation status determination. To check the reliability of our laboratory results, we performed a random interlaboratory testing. From 10 CLL samples tested, in 9 cases identical results were obtained in both laboratories. In one case, the result was discordant. The discrepancy was caused by theIgVH database used. This finding prompted us to double-check our cohort of 624 CLL patients, using IgBLAST and IMGT databases. The results showed 7.5% (47/624) discrepancies between both databases. In 21 out of 47 cases, the degree of hypermutation has changed in regard to the database used, resulting in major changes in the prognostic subgroup. Other irregularities between both databases were identified, with yet to be determined significance. In the light of presented data we would like to stress the necessity to identify/compile the most comprehensiveIgVH database to be used for the determination ofIgVH mutation status in CLL.
BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with an increased risk of Alzheimer... more BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with an increased risk of Alzheimer’s disease (AD), episodic memory deficits and less accurate spatial navigation in non‐demented older adults. The brain‐derived neurotrophic factor (BDNF) Val66Met polymorphism is also related to the increased risk of AD and more pronounced memory impairment but its role in APOE ɛ4‐related spatial navigation deficits has not been established. Our aim was to examine the influence of combination of APOE and BDNF Val66Met polymorphisms on spatial navigation and other cognitive functions in individuals with amnestic mild cognitive impairment (aMCI).MethodIn total, 116 older adults with aMCI from the Czech Brain Aging Study were stratified based on APOE and BDNF Val66Met polymorphisms into four groups: ɛ4–/BDNF Val/Val (n = 29), ɛ4–/BDNF Met (n = 11), ɛ4+/BDNF Val/Val (n = 52), and ɛ4+/BDNF Met (n = 24). All participants underwent comprehensive neuropsychological examination, brain MRI and spa...
Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, a... more Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N-acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and rando...
BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with episodic memory deficits, less... more BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with episodic memory deficits, less accurate spatial navigation and hippocampal atrophy in nondemented older adults. The brain‐derived neurotrophic factor (BDNF) Met allele may further worsen memory impairment and reduce hippocampal activation in APOE ɛ4 carriers but its role in APOE ɛ4‐related spatial navigation deficits has not been established. We aimed to evaluate the influence of combination of the APOE and BDNF Val66Met polymorphisms on spatial navigation, cognitive functions and volumes of selected brain regions associated with spatial navigation including the hippocampus, entorhinal cortex, precuneus, inferior parietal and posterior cingulate cortex in individuals with subjective cognitive decline (SCD).MethodIn total, 71 older adults with SCD from the Czech Brain Aging Study were stratified based on the APOE and BDNF Val66Met polymorphisms into four groups: ɛ4–/BDNF Val/Val (n =27), ɛ4–/BDNF Met (n = 15), ɛ4+/BDN...
Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase... more Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys resid...
Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenes... more Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenesis reduction in mammals. The mechanism of the chitooligosaccharides’ formation in vivo has been, however, poorly understood. Here we report that mouse acidic chitinase (Chia), which is widely expressed in mouse tissues, can produce chitooligosaccharides from deacetylated chitin (chitosan) at pH levels corresponding to stomach and lung tissues. Chia degraded chitin to produce N-acetyl-d-glucosamine (GlcNAc) dimers. The block-type chitosan (heterogenous deacetylation) is soluble at pH 2.0 (optimal condition for mouse Chia) and was degraded into chitooligosaccharides with various sizes ranging from di- to nonamers. The random-type chitosan (homogenous deacetylation) is soluble in water that enables us to examine its degradation at pH 2.0, 5.0, and 7.0. Incubation of these substrates with Chia resulted in the more efficient production of chitooligosaccharides with more variable sizes was fro...
Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, includi... more Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0–7.0) and temperature (30–70 ℃) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications.
Genetic discoveries of Alzheimer’s disease are the drivers of our understanding, and together wit... more Genetic discoveries of Alzheimer’s disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer’s disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer’s disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer’s disease.
Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of ... more Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of degradation products from artificial and natural chitin substrates such as 4-NP-(GlcNAc) 2 , (GlcNAc) 4 and colloidal chitin. The FACE method has been improved by our group for analysis of chitooligosaccharides in the presence of several buffer systems commonly used in the biochemical evaluation of chitinolytic activities of enzymes at pH 2.0-8.0. FACE is a very sensitive technique detecting picomolar amounts of molecules. We optimized the detection conditions as follows: exposure type, precision; sensitivity, high resolution; exposure time, 5 s. We evaluated the (GlcNAc) 2 levels using a standard curve that allows chitooligosaccharides quantification at up to 10 nmol amounts. Using the method presented here, the chitinolytic properties of different chitinases can be compared directly. Serratia chitinase A (ChiA) and chitinase B (ChiB), two well-studied bacterial chitinases, have been shown by HPLC to have a synergistic effect on the chitin degradation rate. Using the FACE method, we determined the combinatory effects of mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) in natural chitin substrates processing. • FACE is a simple and quantitative method. • Our improved procedure enables the quantification of chitooligosaccharides produced by chitinases at pH 2.0-8.0. • FACE is able to quantify chitooligosaccharides at up to 10 nmol amounts.
International Journal of Biological Macromolecules, Aug 1, 2019
Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food proc... more Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food processing and various pathophysiological conditions such as chronic inflammatory diseases. By combination of the colorimetric analysis and fluorophore-assisted carbohydrate electrophoresis (FACE) method, we directly compared the chitinolytic properties of mouse Chit1 and AMCase and determined their combinatory effects in artificial and natural chitin substrates processing. Chit1 and AMCase display different dynamics of chitinolytic properties through acidic to neutral conditions. At pH 2.0, the activity of AMCase was higher than that of Chit1 and stronger or comparable with that of Serratia marcescens chitinase B, a well-characterized bacterium chitinase. Changes of degradation products using different substrates indicate that AMCase and Chit1 have diverse properties under various pH conditions. Exposure of the chitin substrates to both Chit1 and AMCase did not indicate any mutual interference of these enzymes and showed no synergistic effect, in contrast to observations regarding some bacterial chitinases. Our results suggest that Chit1 and AMCase have no synergistic effect under physiological conditions.
Objectives: Depression has been reported to be an independent risk factor for coronary heart dise... more Objectives: Depression has been reported to be an independent risk factor for coronary heart disease (CHD). We investigated the association of depressive symptoms with lipids and coagulation factors in young individuals free of CHD. Methods: We recruited 1191 young healthy individuals candidates for military academies (mean age=18.44-0.8 years, males 762) coming from all social classes and geographical compartments of Greece. Depression was assessed by using the depression scale of Minnesota Multiphasic Personality Invento~ test. The T-score for depression scale ranged from 22 to 90. Total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, lipoprotein (a), fibrinogen, factor VII, factor VIII and factor X were measured. Body mass index (BMI), physical activity and smoking were also reported. Results: The participants were divided in qnartiles according to the depression score. Three hundred twenty-two subjects were classified in the upper quartile of the depression score (score>48) and 269 were classified in the lower quartile (score<37). Factor VII (102.95 vs 98.5%) and X levels (92 vs 89.7) were significantly higher in individuals in the upper quartile of the depression score compared to the lower quartile of depression score while the other studied parameters did not show significant difference. In a logistic regression model with factor VII as dependent variable (upper vs lower quartile) and depression score, age, gender, BMI, smoking as predictor variables, depression was an independent predictor of factor VII levels with an adjusted odds ratio for high levels of factor VII of 1.05 (95% confidence interval 1.008-1.09, p=0.01). Factor VII was associated with triglycerides (r=-0.21, p=0.001), while factor X with triglycerides (r=-0.22, p<0.001) and cholesterol levels (r=-0.12, p<0.001). Conclusions: Depressed mood is associated with a hypercoagulant profile as it is expressed by the higher levels of coagulation factors VII and X. This might partially explain the higher propensity for CHD of people with depressive symptoms.
May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare ... more May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.
Free papers 04-Dementia Neuroprotective potential of Solanesol and Epigallocatechin gallate on IC... more Free papers 04-Dementia Neuroprotective potential of Solanesol and Epigallocatechin gallate on ICV-STZ induced cognitive dysfunction in rats
and protein expression of molecules involved in degenerative pathways related to AD such as autop... more and protein expression of molecules involved in degenerative pathways related to AD such as autophagy, proteosomal pathway, oxidative stress, survival, apoptosis, AB levels and phosphorylation of tau protein among the different groups of fibroblasts. Results: We characterize the 12 fibroblasts lines, by karyotyping, DNA sequencing and perform proteomic analyses. We found activation in the GSK3B pathway, tau hyperphosphorylation, with an increase in total tau expression and CatD overexpression in fibroblasts from AD patients compared to healthy controls. Conclusions: The fibroblasts of individuals with Alzheimer’s disease represent a suitable in vitro model for further molecular studies on the pathogenic process of AD, that could be used for the search of biomarkers of the disease and to validate potential new therapies. Additionally, these cells could be reprogrammed into iPCS, that had a broad differentiation potential, from which is possible to generate neurons, that would recapitulate the pathogenesis of AD brain cells.
It has been accepted that the hypermutation status of immunoglobulin heavy chain genes (IgVH) is ... more It has been accepted that the hypermutation status of immunoglobulin heavy chain genes (IgVH) is one of the most important independent prognostic factors in chronic lymphocytic leukemia (CLL). According to the degree ofIgVH hypermutaion, CLL patients can be stratified into prognostic groups. Given the impact ofIgVH mutation status on clinical setting, it has become highly desirable to standardize the laboratory methodologies used for IgVH mutation status determination. To check the reliability of our laboratory results, we performed a random interlaboratory testing. From 10 CLL samples tested, in 9 cases identical results were obtained in both laboratories. In one case, the result was discordant. The discrepancy was caused by theIgVH database used. This finding prompted us to double-check our cohort of 624 CLL patients, using IgBLAST and IMGT databases. The results showed 7.5% (47/624) discrepancies between both databases. In 21 out of 47 cases, the degree of hypermutation has changed in regard to the database used, resulting in major changes in the prognostic subgroup. Other irregularities between both databases were identified, with yet to be determined significance. In the light of presented data we would like to stress the necessity to identify/compile the most comprehensiveIgVH database to be used for the determination ofIgVH mutation status in CLL.
BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with an increased risk of Alzheimer... more BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with an increased risk of Alzheimer’s disease (AD), episodic memory deficits and less accurate spatial navigation in non‐demented older adults. The brain‐derived neurotrophic factor (BDNF) Val66Met polymorphism is also related to the increased risk of AD and more pronounced memory impairment but its role in APOE ɛ4‐related spatial navigation deficits has not been established. Our aim was to examine the influence of combination of APOE and BDNF Val66Met polymorphisms on spatial navigation and other cognitive functions in individuals with amnestic mild cognitive impairment (aMCI).MethodIn total, 116 older adults with aMCI from the Czech Brain Aging Study were stratified based on APOE and BDNF Val66Met polymorphisms into four groups: ɛ4–/BDNF Val/Val (n = 29), ɛ4–/BDNF Met (n = 11), ɛ4+/BDNF Val/Val (n = 52), and ɛ4+/BDNF Met (n = 24). All participants underwent comprehensive neuropsychological examination, brain MRI and spa...
Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, a... more Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N-acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and rando...
BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with episodic memory deficits, less... more BackgroundThe apolipoprotein E (APOE) ɛ4 allele is associated with episodic memory deficits, less accurate spatial navigation and hippocampal atrophy in nondemented older adults. The brain‐derived neurotrophic factor (BDNF) Met allele may further worsen memory impairment and reduce hippocampal activation in APOE ɛ4 carriers but its role in APOE ɛ4‐related spatial navigation deficits has not been established. We aimed to evaluate the influence of combination of the APOE and BDNF Val66Met polymorphisms on spatial navigation, cognitive functions and volumes of selected brain regions associated with spatial navigation including the hippocampus, entorhinal cortex, precuneus, inferior parietal and posterior cingulate cortex in individuals with subjective cognitive decline (SCD).MethodIn total, 71 older adults with SCD from the Czech Brain Aging Study were stratified based on the APOE and BDNF Val66Met polymorphisms into four groups: ɛ4–/BDNF Val/Val (n =27), ɛ4–/BDNF Met (n = 15), ɛ4+/BDN...
Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase... more Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys resid...
Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenes... more Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenesis reduction in mammals. The mechanism of the chitooligosaccharides’ formation in vivo has been, however, poorly understood. Here we report that mouse acidic chitinase (Chia), which is widely expressed in mouse tissues, can produce chitooligosaccharides from deacetylated chitin (chitosan) at pH levels corresponding to stomach and lung tissues. Chia degraded chitin to produce N-acetyl-d-glucosamine (GlcNAc) dimers. The block-type chitosan (heterogenous deacetylation) is soluble at pH 2.0 (optimal condition for mouse Chia) and was degraded into chitooligosaccharides with various sizes ranging from di- to nonamers. The random-type chitosan (homogenous deacetylation) is soluble in water that enables us to examine its degradation at pH 2.0, 5.0, and 7.0. Incubation of these substrates with Chia resulted in the more efficient production of chitooligosaccharides with more variable sizes was fro...
Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, includi... more Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0–7.0) and temperature (30–70 ℃) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications.
Genetic discoveries of Alzheimer’s disease are the drivers of our understanding, and together wit... more Genetic discoveries of Alzheimer’s disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer’s disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer’s disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer’s disease.
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Papers by Václav Maťoška