Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encep...Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.展开更多
We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, ...We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.展开更多
Previous studies showed that rabbit muscle phosphofructokinase-1 (PFK-1) activity losses due to dilution, due to inhibition by ascorbate, and due to some lithium salts were prevented by rabbit muscle aldolase. Chicken...Previous studies showed that rabbit muscle phosphofructokinase-1 (PFK-1) activity losses due to dilution, due to inhibition by ascorbate, and due to some lithium salts were prevented by rabbit muscle aldolase. Chicken PFK-1 and fish PFK-1 interacted with ascorbate and were inhibited, consistent with a previously proposed function that ascorbate facilitates glycogen in resting muscle by inhibiting glycolysis. This report shows that a plant enzyme, spinach aldolase, has the same ability to prevent rabbit muscle PFK-1 activity loses as rabbit muscle aldolase and in some instances it was a better protector from activity losses than rabbit aldolase. Spinach aldolase also protected chicken and fish PFK-1s from inhibitions by ascorbate and from activity losses due to dilution. Prevention of losses PFK-1 activities from animal species by a plant protein, spinach aldolase, suggests an evolutionary conservative relationship between PFK-1s and aldolases.展开更多
After gun-shot wound with high velocity steel-ball pellet was inflicted of one hind limb ofthe pigs, the changes of hemodynamics, blood gases, chest x-ray films, serum T3 and T4,fibronectin, protein content of lung la...After gun-shot wound with high velocity steel-ball pellet was inflicted of one hind limb ofthe pigs, the changes of hemodynamics, blood gases, chest x-ray films, serum T3 and T4,fibronectin, protein content of lung lavage, lung weight, etc were observed within the 72-hour-peri-od postinjury or when the animal was killed or died 72 h postinjury. The pathological changes wereexamined as soon as possible with both optic and electron microscope. Changes of the above-men-tioned parameters in different intensity were found especially in those animals which diedspontaneously. In addition, the relationship between the occurrence of respiratory distresssyndrome(RDS) after trauma and the trauma index-injury severity score was discussed and it waspointed out that attention should be paid to the subclinical type of RDS.展开更多
Hippophae rhamnoides L.is a plant of immense ethnopharmacological importance and is a known source for various valuable biochemicals and nutraceuticals.The production of folate,a vitamin involved in several vital func...Hippophae rhamnoides L.is a plant of immense ethnopharmacological importance and is a known source for various valuable biochemicals and nutraceuticals.The production of folate,a vitamin involved in several vital functions,in this plant is rather poorly understood.Herein,we investigate the hypothesis that rhizobial bacteria serve the plant in this essential vitamin’s biosynthesis.Bacterial strains of Bacillus,Azorhizobium,Frankia,Paenibacillus,Brevibacillus and Pseudomonas,were isolated from the rhizosphere of the plant.HPLC and LCMS were used to trace the production of intra and extra-cellular folate by representative rhizospheric bacterial strains in vitro.From the seventeen functionally characterized bacterial strains of the plant’s rhizosphere,thirteen produced significant amounts of folate.Azorhizobium BR5401 produced the maximum amount of folic acid(424μg/mL),and Bacillus GY779 was the only strain capable of producing both intracellular and extra-cellular folic acid.The Open Reading Frame coding for dihydroneopterin aldolase,an enzyme involved in folate biosynthesis,was found in one of the representative isolates.Our experimental findings help us to suggest that the folate synthesized by rhizobial bacteria is transported to the plant,highlighting a significant benefit of coexistence.展开更多
Hereditary fructose intolerance(HFI)is a rare autosomal recessive inherited disorder that occurs due to the mutation of enzyme aldolase B located on chromosome 9q22.3.A fructose load leads to the rapid accumulation of...Hereditary fructose intolerance(HFI)is a rare autosomal recessive inherited disorder that occurs due to the mutation of enzyme aldolase B located on chromosome 9q22.3.A fructose load leads to the rapid accumulation of fructose 1-phosphate and manifests with its downstream effects.Most commonly children are affected with gastrointestinal symptoms,feeding issues,aversion to sweets and hypoglycemia.Liver manifestations include an asymptomatic increase of transaminases,steatohepatitis and rarely liver failure.Renal involvement usually occurs in the form of proximal renal tubular acidosis and may lead to chronic renal insufficiency.For confirmation,a genetic test is favored over the measurement of aldolase B activity in the liver biopsy specimen.The crux of HFI management lies in the absolute avoidance of foods containing fructose,sucrose,and sorbitol(FSS).There are many dilemmas regarding tolerance,dietary restriction and occurrence of steatohepatitis.Patients with HFI who adhere strictly to FSS free diet have an excellent prognosis with a normal lifespan.This review attempts to increase awareness and provide a comprehensive review of this rare but treatable disorder.展开更多
l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT24...l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT2440(l-PpTA)in Escherichia coli BL21(DE3)and improved the activity and thermostability by protein engineering.Five amino acid residues(Ser10,His89,Asp93,Arg177,and Arg321)located in the substrate-binding pocket were selected and for mutation.Eight mutants(D93A,D93G,D93M,D93F,D93S,D93Q,D93Y and D93H)with increased enzyme activity were identified and their k_(cat)/K_(M)values showed about 1-7-fold higher than wild-type.Among all the variants,D93H showed the highest catalytic efficiency with 2925 and 4515 s^(−1)mM^(−1)of k_(cat)/K_(M)values toward l-threonine and l-allo-threonine,respectively.In addition,circular dichroism spectrum exhibited that the melting temperature of D93H(54.2℃)was 5℃higher than wild-type(49.2℃).Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate,which facilitated the proton extraction and promote the enzymatic reaction.This work affords a candidate for the synthesis ofβ-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.展开更多
Background Cell metabolism plays a pivotal role in tumor progression,and targeting cancer metabolism might effectively kill cancer cells.We aimed to investigate the role of hexokinases in prostate cancer(PCa)and ident...Background Cell metabolism plays a pivotal role in tumor progression,and targeting cancer metabolism might effectively kill cancer cells.We aimed to investigate the role of hexokinases in prostate cancer(PCa)and identify a crucial target for PCa treatment.Methods The Cancer Genome Atlas(TCGA)database,online tools and clinical samples were used to assess the expression and prognostic role of ADP-dependent glucokinase(ADPGK)in PCa.The effect of ADPGK expression on PCa cell malignant phenotypes was validated in vitro and in vivo.Quantitative proteomics,metabolomics,and extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)tests were performed to evaluate the impact of ADPGK on PCa metabolism.The underlying mechanisms were explored through ADPGK overexpression and knockdown,co-immunoprecipitation(Co-IP),ECAR analysis and cell counting kit-8(CCK-8)assays.Results ADPGK was the only glucokinase that was both upregulated and predicted worse overall survival(OS)in prostate adenocarcinoma(PRAD).Clinical sample analysis demonstrated that ADPGK was markedly upregulated in PCa tissues vs.non-PCa tissues.High ADPGK expression indicates worse survival outcomes,and ADPGK serves as an independent factor of biochemical recurrence.In vitro and in vivo experiments showed that ADPGK overexpression promoted PCa cell proliferation and migration,and ADPGK inhibition suppressed malignant phenotypes.Metabolomics,proteomics,and ECAR and OCR tests revealed that ADPGK significantly accelerated glycolysis in PCa.Mechanistically,ADPGK binds aldolase C(ALDOC)to promote glycolysis via AMP-activated protein kinase(AMPK)phosphorylation.ALDOC was positively correlated with ADPGK,and high ALDOC expression was associated with worse survival outcomes in PCa.Conclusions In summary,ADPGK is a driving factor in PCa progression,and its high expression contributes to a poor prognosis in PCa patients.ADPGK accelerates PCa glycolysis and progression by activating ALDOC-AMPK signaling,suggesting that ADPGK might be an effective target and marker for PCa treatment and prognosis evaluation.展开更多
Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA en-coding a NaCl-induced fructose-1, 6- diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homo...Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA en-coding a NaCl-induced fructose-1, 6- diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homology (66%—73%) to chloroplast fructose-1, 6-diphos- phate aldolase (AldP) in many plants according to their amino acid sequences. The phylogenetic analysis further confirmed that AldP in alga is the nearest to DsALDP. As to its expression pattern, DsALDP was de novo synthesized by NaCl induction. Its expression level was significantly changed with inducing time. After the selected DsALDP cDNA subcloned into a binary vector pBI121, the new construct was introduced into tobacco by Agrobacterium tumefaciens. The results of Southern blot and RT-PCR analysis of four transgenic T1 plants indicated that DsALDP was integrated into genome of these transgenic plants and effectively expressed. Aldolase activities have been detected in T1-1, T1-2 and T1-3 plants by bioassay under 100—200 mmol/L NaCl. It was also observed that proline contents in them were differentially increased.展开更多
Recent investigations indicated that the absorbed water in the hydrated pro- teins was in different states. A portion of molecules of the absorbed water were bound up with hydrophilic sites on the protein surface by h...Recent investigations indicated that the absorbed water in the hydrated pro- teins was in different states. A portion of molecules of the absorbed water were bound up with hydrophilic sites on the protein surface by hydrogen bonds and did not join in the ice lattice. The water in this state was called nonfreezable water. The other water was called freezable water, but still it might be in different展开更多
Over the past few years,it is observed an increased interest for oleaginous microorganisms in the perspective to produce microbial oils of great commercial interest through the consumption of low/zero cost substrates....Over the past few years,it is observed an increased interest for oleaginous microorganisms in the perspective to produce microbial oils of great commercial interest through the consumption of low/zero cost substrates.In this paper,the physiology of the fungus Umbelopsis isabellina growing on blends of glycerol and glucose was investigated.In all experiments the fungus completely consumed glucose and produced satisfactory quantities of biomass containing reserve lipids in high percentages.However,glycerol concentration in the growth medium was negatively correlated to glucose assimilation rate,mainly during the balanced-growth phase.Nevertheless,at high initial concentrations,glycerol was partially consumed and seemed to contribute positively to the suppression of lipid degradation.Following the discovery of this complex regulatory mechanism regarding glucose and glycerol co-assimilation,the activity of three key-enzymes namely aldolase,glycerol kinase and glycerol dehydrogenase,which are implicated in glycerol and glucose assimilation,was investigated.The experiments revealed a clear preference of the fungus for glucose over glycerol.On the other hand,storage polysaccharides are degraded instead of storage lipid at the late oleaginous phase for maintenance purpose.These new biochemical features will enable the design of appropriate growth media for the co-fermentation of these two substrates by U.isabellina with the aim to maximize lipid accumulation.展开更多
To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have b...To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. In previous studies, sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid （ABA）, and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic in the GA-induced growth of roots. In among GA, aldolase, and root growth. pathway and may play an important role this review, we discuss the relationship among GA, aldolase, and root growth.展开更多
In plants, the shoot apical meristem (SAM) is essential for the growth of aboveground organs. However, little is known about its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thali...In plants, the shoot apical meristem (SAM) is essential for the growth of aboveground organs. However, little is known about its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thaliana displays an autonomous heat-stress (HS) memory of a previous non-lethal HS, allowing the SAM to regain growth after exposure to an otherwise lethal HS several days later. Using RNA sequencing, we identified genes participating in establishing the SAM's HS transcriptional memory, including the stem cell (SC) regulators CLAVATA1 (CLV1) and CLV3, HEAT SHOCK PROTEIN 17.6A (HSP17.6A), and the primary carbohydrate metabolism gene FRUCTOSE-BISPHOSPHATE ALDOLASE 6 (FBA6). We demonstrate that sugar availability is essential for survival of plants at high temperature. HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2A) directly regulates the expression of HSP17.6A and FBA6 by binding to the heat-shock elements in their promoters, indicating that HSFA2 is required for transcriptional activation of SAM memory genes. Collectively, these findings indicate that plants have evolved a sophisticated protection mechanism to maintain SCs and, hence, their capacity to re-initiate shoot growth after stress release.展开更多
文摘Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.
文摘We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.
文摘Previous studies showed that rabbit muscle phosphofructokinase-1 (PFK-1) activity losses due to dilution, due to inhibition by ascorbate, and due to some lithium salts were prevented by rabbit muscle aldolase. Chicken PFK-1 and fish PFK-1 interacted with ascorbate and were inhibited, consistent with a previously proposed function that ascorbate facilitates glycogen in resting muscle by inhibiting glycolysis. This report shows that a plant enzyme, spinach aldolase, has the same ability to prevent rabbit muscle PFK-1 activity loses as rabbit muscle aldolase and in some instances it was a better protector from activity losses than rabbit aldolase. Spinach aldolase also protected chicken and fish PFK-1s from inhibitions by ascorbate and from activity losses due to dilution. Prevention of losses PFK-1 activities from animal species by a plant protein, spinach aldolase, suggests an evolutionary conservative relationship between PFK-1s and aldolases.
文摘After gun-shot wound with high velocity steel-ball pellet was inflicted of one hind limb ofthe pigs, the changes of hemodynamics, blood gases, chest x-ray films, serum T3 and T4,fibronectin, protein content of lung lavage, lung weight, etc were observed within the 72-hour-peri-od postinjury or when the animal was killed or died 72 h postinjury. The pathological changes wereexamined as soon as possible with both optic and electron microscope. Changes of the above-men-tioned parameters in different intensity were found especially in those animals which diedspontaneously. In addition, the relationship between the occurrence of respiratory distresssyndrome(RDS) after trauma and the trauma index-injury severity score was discussed and it waspointed out that attention should be paid to the subclinical type of RDS.
基金The authors are grateful to the Defense Research Development Organization(Project No.TC/2519/INM/-04/2012/CARS of INM 311/1.2)for the financial support and opportunity to carry out these studies.
文摘Hippophae rhamnoides L.is a plant of immense ethnopharmacological importance and is a known source for various valuable biochemicals and nutraceuticals.The production of folate,a vitamin involved in several vital functions,in this plant is rather poorly understood.Herein,we investigate the hypothesis that rhizobial bacteria serve the plant in this essential vitamin’s biosynthesis.Bacterial strains of Bacillus,Azorhizobium,Frankia,Paenibacillus,Brevibacillus and Pseudomonas,were isolated from the rhizosphere of the plant.HPLC and LCMS were used to trace the production of intra and extra-cellular folate by representative rhizospheric bacterial strains in vitro.From the seventeen functionally characterized bacterial strains of the plant’s rhizosphere,thirteen produced significant amounts of folate.Azorhizobium BR5401 produced the maximum amount of folic acid(424μg/mL),and Bacillus GY779 was the only strain capable of producing both intracellular and extra-cellular folic acid.The Open Reading Frame coding for dihydroneopterin aldolase,an enzyme involved in folate biosynthesis,was found in one of the representative isolates.Our experimental findings help us to suggest that the folate synthesized by rhizobial bacteria is transported to the plant,highlighting a significant benefit of coexistence.
文摘Hereditary fructose intolerance(HFI)is a rare autosomal recessive inherited disorder that occurs due to the mutation of enzyme aldolase B located on chromosome 9q22.3.A fructose load leads to the rapid accumulation of fructose 1-phosphate and manifests with its downstream effects.Most commonly children are affected with gastrointestinal symptoms,feeding issues,aversion to sweets and hypoglycemia.Liver manifestations include an asymptomatic increase of transaminases,steatohepatitis and rarely liver failure.Renal involvement usually occurs in the form of proximal renal tubular acidosis and may lead to chronic renal insufficiency.For confirmation,a genetic test is favored over the measurement of aldolase B activity in the liver biopsy specimen.The crux of HFI management lies in the absolute avoidance of foods containing fructose,sucrose,and sorbitol(FSS).There are many dilemmas regarding tolerance,dietary restriction and occurrence of steatohepatitis.Patients with HFI who adhere strictly to FSS free diet have an excellent prognosis with a normal lifespan.This review attempts to increase awareness and provide a comprehensive review of this rare but treatable disorder.
基金the National Key research and Development Program of China(2018YFA0900302)the National Science Foundation of China(32271487,31970045)+2 种基金the National Firstclass Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06)Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT2440(l-PpTA)in Escherichia coli BL21(DE3)and improved the activity and thermostability by protein engineering.Five amino acid residues(Ser10,His89,Asp93,Arg177,and Arg321)located in the substrate-binding pocket were selected and for mutation.Eight mutants(D93A,D93G,D93M,D93F,D93S,D93Q,D93Y and D93H)with increased enzyme activity were identified and their k_(cat)/K_(M)values showed about 1-7-fold higher than wild-type.Among all the variants,D93H showed the highest catalytic efficiency with 2925 and 4515 s^(−1)mM^(−1)of k_(cat)/K_(M)values toward l-threonine and l-allo-threonine,respectively.In addition,circular dichroism spectrum exhibited that the melting temperature of D93H(54.2℃)was 5℃higher than wild-type(49.2℃).Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate,which facilitated the proton extraction and promote the enzymatic reaction.This work affords a candidate for the synthesis ofβ-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.
基金National Key R&D Plan(2023YFC3403200)National Natural Science Foundation of China(82070784,81702536,81974099 and 82170785)+4 种基金Science&Technology Department of Sichuan Province,China(2022JDRC0040,21GJHZ0246)Young Investigator Award of Sichuan University 2017(2017SCU04A17)Sichuan University-Panzhihua Science and Technology Cooperation Special Fund(2020CDPZH-4)China Postdoctoral Science Foundation(2021M692306)Post-Doctor Research Project of West China Hospital of Sichuan University(2021HXBH025).
文摘Background Cell metabolism plays a pivotal role in tumor progression,and targeting cancer metabolism might effectively kill cancer cells.We aimed to investigate the role of hexokinases in prostate cancer(PCa)and identify a crucial target for PCa treatment.Methods The Cancer Genome Atlas(TCGA)database,online tools and clinical samples were used to assess the expression and prognostic role of ADP-dependent glucokinase(ADPGK)in PCa.The effect of ADPGK expression on PCa cell malignant phenotypes was validated in vitro and in vivo.Quantitative proteomics,metabolomics,and extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)tests were performed to evaluate the impact of ADPGK on PCa metabolism.The underlying mechanisms were explored through ADPGK overexpression and knockdown,co-immunoprecipitation(Co-IP),ECAR analysis and cell counting kit-8(CCK-8)assays.Results ADPGK was the only glucokinase that was both upregulated and predicted worse overall survival(OS)in prostate adenocarcinoma(PRAD).Clinical sample analysis demonstrated that ADPGK was markedly upregulated in PCa tissues vs.non-PCa tissues.High ADPGK expression indicates worse survival outcomes,and ADPGK serves as an independent factor of biochemical recurrence.In vitro and in vivo experiments showed that ADPGK overexpression promoted PCa cell proliferation and migration,and ADPGK inhibition suppressed malignant phenotypes.Metabolomics,proteomics,and ECAR and OCR tests revealed that ADPGK significantly accelerated glycolysis in PCa.Mechanistically,ADPGK binds aldolase C(ALDOC)to promote glycolysis via AMP-activated protein kinase(AMPK)phosphorylation.ALDOC was positively correlated with ADPGK,and high ALDOC expression was associated with worse survival outcomes in PCa.Conclusions In summary,ADPGK is a driving factor in PCa progression,and its high expression contributes to a poor prognosis in PCa patients.ADPGK accelerates PCa glycolysis and progression by activating ALDOC-AMPK signaling,suggesting that ADPGK might be an effective target and marker for PCa treatment and prognosis evaluation.
文摘Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA en-coding a NaCl-induced fructose-1, 6- diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homology (66%—73%) to chloroplast fructose-1, 6-diphos- phate aldolase (AldP) in many plants according to their amino acid sequences. The phylogenetic analysis further confirmed that AldP in alga is the nearest to DsALDP. As to its expression pattern, DsALDP was de novo synthesized by NaCl induction. Its expression level was significantly changed with inducing time. After the selected DsALDP cDNA subcloned into a binary vector pBI121, the new construct was introduced into tobacco by Agrobacterium tumefaciens. The results of Southern blot and RT-PCR analysis of four transgenic T1 plants indicated that DsALDP was integrated into genome of these transgenic plants and effectively expressed. Aldolase activities have been detected in T1-1, T1-2 and T1-3 plants by bioassay under 100—200 mmol/L NaCl. It was also observed that proline contents in them were differentially increased.
文摘Recent investigations indicated that the absorbed water in the hydrated pro- teins was in different states. A portion of molecules of the absorbed water were bound up with hydrophilic sites on the protein surface by hydrogen bonds and did not join in the ice lattice. The water in this state was called nonfreezable water. The other water was called freezable water, but still it might be in different
基金financed by the Green Fund of the Hellenic Ministry of Environment and Energy,under the funding program“National Environment and Innovation Activities 2022”,Priority Axis“Research&Application”,Project“Sustainable technology for converting pomegranate residues into bioproducts and bioactive compounds”with the acronym“POMEGRANATE”.
文摘Over the past few years,it is observed an increased interest for oleaginous microorganisms in the perspective to produce microbial oils of great commercial interest through the consumption of low/zero cost substrates.In this paper,the physiology of the fungus Umbelopsis isabellina growing on blends of glycerol and glucose was investigated.In all experiments the fungus completely consumed glucose and produced satisfactory quantities of biomass containing reserve lipids in high percentages.However,glycerol concentration in the growth medium was negatively correlated to glucose assimilation rate,mainly during the balanced-growth phase.Nevertheless,at high initial concentrations,glycerol was partially consumed and seemed to contribute positively to the suppression of lipid degradation.Following the discovery of this complex regulatory mechanism regarding glucose and glycerol co-assimilation,the activity of three key-enzymes namely aldolase,glycerol kinase and glycerol dehydrogenase,which are implicated in glycerol and glucose assimilation,was investigated.The experiments revealed a clear preference of the fungus for glucose over glycerol.On the other hand,storage polysaccharides are degraded instead of storage lipid at the late oleaginous phase for maintenance purpose.These new biochemical features will enable the design of appropriate growth media for the co-fermentation of these two substrates by U.isabellina with the aim to maximize lipid accumulation.
文摘To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. In previous studies, sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid （ABA）, and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic in the GA-induced growth of roots. In among GA, aldolase, and root growth. pathway and may play an important role this review, we discuss the relationship among GA, aldolase, and root growth.
基金Sequencing datasets are available at the NCBI Sequencing Read Archive, BioProject ID PRJNA505602.
文摘In plants, the shoot apical meristem (SAM) is essential for the growth of aboveground organs. However, little is known about its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thaliana displays an autonomous heat-stress (HS) memory of a previous non-lethal HS, allowing the SAM to regain growth after exposure to an otherwise lethal HS several days later. Using RNA sequencing, we identified genes participating in establishing the SAM's HS transcriptional memory, including the stem cell (SC) regulators CLAVATA1 (CLV1) and CLV3, HEAT SHOCK PROTEIN 17.6A (HSP17.6A), and the primary carbohydrate metabolism gene FRUCTOSE-BISPHOSPHATE ALDOLASE 6 (FBA6). We demonstrate that sugar availability is essential for survival of plants at high temperature. HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2A) directly regulates the expression of HSP17.6A and FBA6 by binding to the heat-shock elements in their promoters, indicating that HSFA2 is required for transcriptional activation of SAM memory genes. Collectively, these findings indicate that plants have evolved a sophisticated protection mechanism to maintain SCs and, hence, their capacity to re-initiate shoot growth after stress release.
