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Objective:To explore the expression of fructose bisphosphate aldolase A (ALDOA) in the bone marrow of patients with acute myeloid leukemia (AML) and the correlation with clinical features and prognosis.Methods:The bone marrow samples of 90 newly diagnosed AML (non-acute promyelocytic leukemia) patients and 18 allogeneic hematopoietic stem cell transplantation donors who were treated from January 2013 to December 2015 in the First Affiliated Hospital of Zhengzhou University and the Children's Hospital Affiliated to Zhengzhou University were collected. The relative expression level of ALDOA mRNA in bone marrow samples was detected by using real-time quantitative polymerase chain reaction (qRT-PCR). Clinical data of these patients were retrospectively analyzed, and the patients were divided into continuous complete remission (CR) group and refractory recurrent (RR) group according to the clinical response and follow-up results. The differences of the relative expression level of ALDOA mRNA between AML group and the normal control group, CR group and RR group were analyzed. Univariate and multivariate Cox regression risk model were used for analysis of factors influencing prognosis of AML patients.Results:The relative expression level of ALDOA mRNA in AML group was higher than that in normal control group [(5.71±0.44) vs. (1.10±0.08), t = 4.74, P<0.001]. The relative expression level of ALDOA mRNA in the RR group was higher than that in the CR group [(6.69±0.67) vs. (4.30±0.36) , t = 2.79, P < 0.001]. In addition, there were statistically significant differences in the proportion of patients with ALDOA mRNA high expression and those with ALDOA mRNA low expression stratified by the number of white blood cell, the proportion of bone marrow blasts and whether complete remission could be achieved or not after 1 course of induction therapy (all P < 0.05). Overall survival in patients with ALDOA high expression was worse than that in patients with ALDOA low expression ( χ2 = 5.59, P = 0.018). Multivariate analysis showed that white blood cell count, prognosis stratification, whether complete remission could be achieved or not after 1 course of induction therapy and ALDOA expression were the independent prognostic factors for the death of AML patients (all P < 0.05). Conclusions:ALDOA may play an important role in the development and progression of AML, and the expression level of ALDOA in the bone marrow can be used as an index for the prognosis assessment of AML patients and may be a potential therapeutic target for AML.
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Threonine aldolases catalyze the aldol condensation of aldehydes with glycine to furnish β-hydroxy-α-amino acid with two stereogenic centers in a single reaction. This is one of the most promising green methods for the synthesis of optically pure β-hydroxy-α-amino acid with high atomic economy and less negative environmental impact. Several threonine aldolases from different origins have been identified and characterized. The insufficient -carbon stereoselectivity and the challenges of balancing kinetic versus thermodynamic control to achieve the optimal optical purity and yield hampered the application of threonine aldolases. This review summarizes the recent advances in discovery, catalytic mechanism, high-throughput screening, molecular engineering and applications of threonine aldolases, with the aim to provide some insights for further research in this field.
Subject(s)
Amino Acids , Catalysis , Glycine , Glycine Hydroxymethyltransferase/metabolism , Kinetics , Substrate Specificity , ThreonineABSTRACT
ABSTRACT Fructose-1,6-bisphosphate aldolase (FBAld) is an enzyme that catalyzes the cleavage of D-fructose-1,6-phosphate (FBP) to D-glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), and plays vital role in glycolysis and gluconeogenesis. However, molecular characterization and functional roles of FBAld remain unknown in sago palm. Here we report a modified CTAB-RNA extraction method was developed for the isolation of good quality RNA (RIN>8) from sago leaves and the isolation of FBAld cDNA from sago palm. The isolated sago FBAld (msFBAld) cDNA has total length of 1288 bp with an open reading frame of 1020 bp and a predicted to encode for a protein of 340 amino acid resides. The predicted protein shared a high degree of homology with Class-I FBAld from other plants. Meanwhile, the msFBAld gene spanned 2322 bp and consisted of five exons. Conserved domain search identified fifteen catalytically important amino acids at the active site and phylogenetic tree revealed localization of msFBAld in the chloroplast. A molecular 3D-structure of msFBAld was generated by homology modeling and a Ramachandran plot with 86.7% of the residues in the core region, 13.4% in the allowed region with no residues in the disallowed region. The modeled structure is a homotetramer containing an (/(-TIM-barrel at the center. Superimposition of the model with Class-I aldolases identified a catalytic dyad, Lys209-Glu167, which could be involved in the Schiff's base formation and aldol condensation. Apart from that, overproduction of the recombinant msFBAld in Escherichia coli resulted in increased tolerance towards salinity.
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Objective Invasive candidiasis is associated with a significant mortality clinically.The purpose of this study was to observe the T cells immune response to fructose bisphosphate aldolase (Fba), an immunodominant antigen of Candida albicans, and determine whether the antigen has the possibility of priming cellular immune protection in invasive candiasis.Methods Using ELISPOT assay, we determined the frequencies of positive spot-forming cells (SFCs) of Fba antigen-specific T cells secreting IFN-γ, IL-4 and IL-17A in the peripheral blood mononuclear cells (PBMCs) of 26 healthy individuals.Results After Fba stimulation, the frequencies of positive SFCs of IFN-γ, IL-4 and IL-17A in the 26 healthy subjects were 23 (9.75, 42.50), 0 (0, 0.25) and 1.5 (0.75, 8.25), respectively, with statistically significant differences among the three (P<0.01).The response rates of IFN-γ (100% [26/26]) and IL-17A (76.92% [20/26]) were significantly higher than that of IL-4 (15.38% [4/26]) (P<0.01).Fba-induced strong response (SCFs ≥20) for IFN-γ was observed in 57.69% (15/26) of the healthy individuals, that for IL-17A in only 1, while that for IL-4 in none.Responses of both Th1 and Th17 cells to Fba were found in 65.38% (17/26) of the subjects, that of Th1 cells in 19.23% (5/26), but that of Th2 cells in none.Conclusion Fba of Candida albicans can induce immunodominant responses of Th1 and Th17 cells and is a potential vaccine against invasive candiasis.
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Objective To preliminarily explore the effects of gene knock-out and overexpression of aldolase A on tumor cell proliferation, and then confirm the relationship between aldolase A and tumor growth during the process of anaerobic metabolism. Methods Through vector plasmid building and lentiviral infection, the cell lines stably overexpressing aldolase A and its gene knock- out cell lines were constructed respectively. The restructured cell lines were validated on gene and protein levels. Then, the changes of cell proliferation ratio were observed. Results Real time PCR results showed that aldolase A gene expression of the overexpressing cell line was about 3 times that of the normal cells. Among the five interfering targets of aldolase A designed according to aldolase A RNA sequences, aldolase A4 had more obvious interference effect on the target gene expression, the aldolase A mRNA level of which was about 1/50 of the normal cells. Compared with normal control group, the proliferation rate of aldolase A gene overexpression significantly increased by 40% in 48 h(P<0.05), while the proliferation rate of aldolase A gene knock out cells significantly de? creased about 86% in 24 h(P<0.05). Conclusion Aldolase A expressions have obvious effects on hepatocellular carcinoma cells.
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Objective To preliminarily explore the effects of gene knock-out and overexpression of aldolase A on tumor cell proliferation,and then confirm the relationship between aldolase A and tumor growth during the process of anaerobic metabolism. Methods Through vector plasmid building and lentiviral infection,the cell lines stably overexpressing aldolase A and its gene knock-out cell lines were constructed respectively. The restructured cell lines were validated on gene and protein levels. Then ,the changes of cell proliferation ratio were observed. Results Real time PCR results showed that aldolase A gene expression of the overex?pressing cell line was about 3 times that of the normal cells. Among the five interfering targets of aldolase A designed according to aldol?ase A RNA sequences,aldolase A4 had more obvious interference effect on the target gene expression,the aldolase A mRNA level of which was about 1/50 of the normal cells. Compared with normal control group,the proliferation rate of aldolase A gene overexpression significantly increased by 40% in 48 h(P<0.05),while the proliferation rate of aldolase A gene knock out cells significantly de?creased about 86%in 24 h(P<0.05). Conclusion Aldolase A expressions have obvious effects on hepatocellular carcinoma cells.
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Objective To clone express and purify Schistosoma japonicum fructose?1 6?bisphosphate aldolase SjFBPA in E. coli and observe its expression in different developmental stages of S. japonicum. Methods FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid and inducibly expressed with IPTG in E. coli BL21. SDS?PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA rSjFBPA . Then rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS?PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore SjFBPA mRNA was ana?lyzed in different developmental stages of S. japonicum by RT?PCR. Results SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro?tein could specifically reactive to the anti?His?tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT?PCR showed that SjFBPA mRNA was expressed in cercaria schistosomulum adult worm and egg of S. japonicum. Conclusion SjFBPA is successfully recombined and expressed in a prokaryotic system and SjFBPA mRNA is expressed in cercaria schistosomulum adult worm and egg of S. japonicum.
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In this study ,we aim to identify the protein interaction site of microneme protein 2 (MIC2) and aldolase in Toxoplasma gondii .The tryptophan (Trp ,W) at site 767 of carboxyl terminus of MIC2 (MIC2C) was mutated into alanine (Ala ,A) by site-directed mutagenesis to construct plasmid MIC2C W/A/pGEX-4T-1 .The mutant protein GST-MIC2C W/A was expressed in E .coli upon IPTG induction .Glutathione sepharose beads were incubated with GST-MIC2C W/A and GST-MIC2C respectively ,then incubated with tachyzoite lysates ,and bound proteins were eluted using sample buffer .Eluants were resolved by SDS-PAGE and Western blot .A protein band specifically recognized by anti-aldolase antibody was detected in prod-ucts coming from GST pull-down of GST-MIC2C ,but not in pull-down products coming from GST-MIC2C W/A .With muta-tion of MIC2C W767 to A ,MIC2 protein lost the binding ability to aldolase .Tryptophan (W767 ) was the protein interaction site of MIC2 and aldolase in T .gondii .
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Proximal muscle weakness can be induced by many diseases, such as muscular dystrophies, inflammatory muscle diseases, and polymyalgia rheumatica. Differential diagnosis of these diseases is important. The patient had proximal muscle weakness with a normal creatine kinase (CK) level. Our initial diagnosis was polymyalgia rheumatica because the CK level was normal. The patient was treated with low-dose corticosteroid. However, the muscle weakness did not improve. The diagnosis of polymyositis was confirmed by a muscle biopsy. We suggest that if the patient has typical symptoms with normal CK, then evaluations for inflammatory muscle diseases are essential.
Subject(s)
Humans , Biopsy , Creatine Kinase , Creatine , Diagnosis , Diagnosis, Differential , Fructose-Bisphosphate Aldolase , Muscle Weakness , Muscles , Muscular Dystrophies , Myositis , Polymyalgia Rheumatica , PolymyositisABSTRACT
This study aimed to evaluate the efficacy of fructose-1,6-bis phosphate aldolase (SMALDO) DNA vaccination against Schistosoma mansoni infection using different routes of injection. The SMALDO has been cloned into the eukaryotic expression vector pcDNA3.1/V5-His TOPO-TA and was used in injecting Swiss albino mice intramuscularly (IM), subcutaneously (SC), or intraperitoneally (IP) (50 microg/mouse). Mice vaccinated with non-recombinant pcDNA3.1 served as controls. Each group was immunized 4 times at weeks 0, 2, 4, and 6. Two weeks after the last booster dose, all mice groups were infected with 80 S. mansoni cercariae via tail immersion. At week 8 post-infection, animals were sacrificed for assessment of parasitological and histopathological parameters. High anti-SMALDO IgG antibody titers were detected in sera of all vaccinated groups (P<0.01) compared to the control group. Both the IP and SC vaccination routes resulted in a significant reduction in worm burden (46.2% and 28.9%, respectively, P<0.01). This was accompanied by a significant reduction in hepatic and intestinal egg counts (41.7% and 40.2%, respectively, P<0.01) in the IP group only. The number of dead eggs was significantly increased in both IP and IM groups (P<0.01). IP vaccination recorded the highest significant reduction in granuloma number and diameter (54.7% and 29.2%, respectively, P<0.01) and significant increase in dead miracidia (P<0.01). In conclusion, changing the injection route of SMALDO DNA vaccination significantly influenced the efficacy of vaccination. SMALDO DNA vaccination via IP route could be a promising protective and anti-pathology vaccine candidate against S. mansoni infection.
Subject(s)
Animals , Female , Mice , Antibodies, Helminth/blood , Disease Models, Animal , Fructose-Bisphosphate Aldolase/genetics , Histocytochemistry , Immunoglobulin G/blood , Injections, Intramuscular , Injections, Intraperitoneal , Injections, Subcutaneous , Parasite Load , Schistosoma mansoni/enzymology , Schistosomiasis mansoni/immunology , Vaccination/methods , Vaccines, DNA/administration & dosage , Vaccines, Synthetic/administration & dosageABSTRACT
El objetivo de este trabajo fue determinar la variabilidad biológica (VB) de Aldolasa (ALD) en individuos sanos, el índice de individualidad (II) del analito y evaluar la utilidad del intervalo de referencia poblacional (IRP) considerando al IRP de los analitos útil cuando el II es > 1,4 y de poca utilidad si es <0,6. También se evaluó el valor de referencia para el cambio (VRC) en la interpretación de dos determinaciones seriadas del mismo individuo, especialmente en el seguimiento de enfermedades músculo-esqueléticas en las que los niveles de creatinquinasa (CK) permanecen dentro del intervalo de referencia, mientras que los de ALD están elevados. Se obtuvieron 8 muestras de sangre de 10 individuos aparentemente sanos en días diferentes según protocolo preestablecido. Se determinó la actividad de ALD por método enzimático UV y se calcularon los parámetros de VB, II y VRC. Se obtuvo una VB intraindividual: 31%; VB interindividual: 21%, II 1.47 y VRC 90%. De acuerdo a los coeficientes de VB y el II obtenidos, los IRP son útiles para la interpretación de los resultados de ALD. Un resultado seriado será estadísticamente diferente del dato previo cuando el mismo tenga una diferencia mayor al 90% en relación al valor previo. El VRC entre dos determinaciones seriadas debe ser calculado en cada laboratorio con su propia variabilidad analítica para una correcta interpretación de los resultados.
The aim of this study was to determine the Biological Variation (BV) of Aldolase (ALD) in healthy patients, the individuality index (II) of the analyte and to assess how useful the population reference interval (PRI) is for the interpretation of the results, considering PRI useful when the II is >1.40 and useless when it is <0.6. Reference change values (RCV) were also assessed in the interpretation of two serial measurements of the same individual, specially in the monitoring of patients in which the serum level of Creatine Kinase (CK) remain within the reference range while ALD values are increased. Eight bloodsam-ples were obtained from ten apparently healthy subjects in different days according to a predetermined protocol. ALD activity was measured by an enzymatic UV method and BV, II and RCV were calculated. Intraindividual BV was 31 %, interindividual BV 21 %, II 1.47 and RCV 90%. Based on the results obtained for BV and II, PRIs are useful for the interpretation of ADL results. A serial result will be statistically different from the previous one when the variability between both results is higher than 90%. RCV must be determined in each laboratory using their own analytical variability in order to obtain a correct interpretation of the results.
O objetivo deste trabalho foi determinar a variabilidade biológica (VB) de Aldolase (ALD) em individuos saudáveis, o índice de individualidade (II) do analito e avaliar a utilidade do intervalo de referencia populacional (IRP) considerando o IRP dos analitos útil quando o II é > 1,4 e de pouca utilidade se é <0,6. Também foi avaliado o valor de referencia da variagáo (VRV) na interpretagáo de duas determinagóes seriadas do mesmo individuo, especialmente no seguimento de doengas músculo-esqueléticas nas quais os níveis de creatinoquinase (CK) permanecem dentro do intervalo de referencia, enquanto que os de ALD estáo elevados. Foram obtidas 8 amostras de sangue de 10 individuos aparentemente saudáveis em dias diferentes conforme protocolo preestabelecido. Determinou-se a atividade de ALD por método enzimático UV e se calcularam os parámetros de VB, II e VRC. Foi obtida uma VB intra-individual: 31 %; VB interindividual: 21 %, II 1.47 e VRV 90%. De acordo com os coeficientes de VB e o II obtidos, os IRP sáo úteis para a interpretagáo dos resultados de ALD. Um resultado seriado será estatisticamente diferente do dado prévio quando o mesmo tenha uma diferenga maior a 90% em relagáo ao valor prévio. O VRV entre duas determinagóes seriadas deve ser calculado em cada laboratório com sua própria variabilidade analítica para uma correta interpretagáo dos resultados.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Fructose-Bisphosphate Aldolase/blood , Biological Variation, Population , Chemistry Techniques, Analytical/standards , Reference ValuesABSTRACT
Hereditary fructose intolerance is an autosomal recessive disorder that is caused by a deficiency in fructose-1-phosphate aldolase (Aldolase B). Children can present with hypoglycemia, jaundice, elevated liver enzymes and hepatomegaly after intake of dietary fructose. Long-term intake of fructose in undiagnosed patients can result in hepatic failure or renal failure. We experienced a case of hereditary fructose intolerance presenting as recurrent hepatitis-like episodes. Detailed evaluation of her dietary habits revealed her avoidance of sweetened foods and fruits. Genetic analysis of ALDOB revealed that she is a homozygote for a novel frameshifting mutation c[758_759insT]+[758_759insT] (p.[val25 3fsX24]+[val253fsX24]). This report is the first of a Korean patient diagnosed with hereditary fructose intolerance using only molecular testing without undergoing intravenous fructose tolerance test or enzyme assay.
Subject(s)
Child , Humans , Enzyme Assays , Feeding Behavior , Frameshift Mutation , Fructose , Fructose Intolerance , Fructose-Bisphosphate Aldolase , Fructosephosphates , Fruit , Hepatitis , Hepatomegaly , Homozygote , Hypoglycemia , Jaundice , Liver , Liver Failure , Renal InsufficiencyABSTRACT
As células-tronco mesenquimais (MSCs) são células com grande potencial de diferenciação e estão sendo recentemente introduzidas na clínica para tratamento de várias doenças. Possuem várias vantagens incluindo sua estabilidade fenotípica in vitro. OBJETIVO: isolamento das MSCs de líquido amniótico, sua expansão e a demonstração da sua capacidade de se diferenciar em células miogênicas e adipogênicas, sem alterar a estabilidade cromossomal em meio de cultura. MÉTODOS: a fim de avaliar a mudança funcional destas células, foram avaliados parâmetros bioquímicos nas células adipogênicas já diferenciadas e antes da diferenciação através da dosagem de triglicérides. A diferenciação em células musculares foi avaliada comparando os níveis de creatinofosfoquinase - CK, desidrogenase lática - LDH e aldolase produzidas por estas células antes e após diferenciação. RESULTADOS: os níveis de triglicérides foram significativamente maiores nas células diferenciadas, mostrando ainda a formação de grânulos intracitoplasmáticos. Todos os outros valores obtidos foram significativamente maiores nas células miogênicas diferenciadas quando comparadas às não diferenciadas. CONCLUSÃO: os resultados sugerem que estes protocolos podem ser usados para avaliar diferenciação de células-tronco em células adipogênicas e miogênicas, e que o líquido amniótico pode ser uma fonte para obtenção destas células.
The mesenchymals stem cells (MSCs) are cells with the great potential of differentiation are being introduced in the clinic for treatment of several diseases. Mesenchymal stem cells have several advantages including the stability of their phenotype in vitro. BACKGROUND: isolation of MSCs in amniotic fluid, its expansion and the demonstration of the capacity of these cells to differentiate in adipogenic and miogenic cells, without to change the chromosomal stability of the MSCs in culture. METHODS: in order to evaluate the functional change of these cells, were gotten values of the differentiated adipogenic cells and not differentiated through the dosage of triglycerides. The miogenic nature of the differentiated cells was analyzed comparing the creatine kinase - CK, lactic dehydrogenase - LDH and aldolase produced by the cells. RESULTS: the values of triglycerides were significantly higher in differentiated cells, showing intracitoplasmatic granule form after differentiation. All the biochemical characters were significantly higher in differentiated miogenic cells. CONCLUSIONS: this study suggests that the standardized protocol of differentiation can be used in the attainment of cells with characteristics of adipogenic and muscular cells, from amniotic fluid.
Subject(s)
Female , Humans , Pregnancy , Amniotic Fluid/cytology , Cell Differentiation/physiology , Creatine Kinase/analysis , Mesenchymal Stem Cells , Amniotic Fluid/enzymology , Cell Culture Techniques , Cells, Cultured , Fructose-Bisphosphate Aldolase/analysis , Karyotyping , L-Lactate Dehydrogenase/analysis , Triglycerides/bloodABSTRACT
The complete genomes of more cyanobacterial strains have been completed for sequence, and genetic engineering of cyanobacteria has evolved in the post-genome era. Since Kaneko and colleagues had completed the sequence for the complete genome of Anabaena sp. PCC 7120 in 2001, functions of some genes in this genome, including fructose-1,6-bisphosphate aldolase (FBA) gene, have been predicated using the method of bioinformatics. However, little information is available regarding whether this gene can encode FBA and its product characteristics of related enzyme. Here, to explore this information, the predicted II-FBA gene-encoding region in Cyanobase database was cloned by PCR method and then ligated into pET-32a to generate the expression vector, pET-FBA-II. The results of SDS-PAGE indicated that the expression level of the expected target polypeptide was approximate 23.4 percent as compared to total protein and the molecular weight is about 40 kDa as compared to the protein molecular marker. The results of enzyme activity analysis showed that the activity of II-FBA was ~11.8 U per mg protein and owned a standard activity of II-FBA. To sum up, the results not only prove the functional prediction of this II-FBA gene from the Cyanobase database, but also provide the important conditions for further studying its physiological and biochemical characteristics and functions of the gene expression product.
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Objective To investigate the activity change pattern and corresponding significance of gly- colytic enzymes including alsolase A(ALDA)and lactate dehydrogenase M(LDH-M)regulated by hy- poxia-inducible factor 1?(HIF-1?)in spinal cord injury(SCI)of rats.Methods SD rats were ran- domly divided into control group and groups at 12 hours,1,2 and 3 days,1 and 2 weeks after compres- sive SCI,in which the activity changes of ALDA and LDH-M in the injured spinal cord were observed at different time points by means of enzyme histochemistry.Results Opitical density(A)value of AL- DA continued significant increase from two days to one week after SCI(P＜0.05)and decreased gradual- ly at 2 weeks after SCI.A value of LDH-M began significant increase at day 1 after SCI and recovered to normal level at 2 weeks after SCI(P＜0.05).Conclusion Activities of ALDA and LDH-M regulated by HIF-1?in spinal cord injury is significantly increased.
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Hereditary fructose intolerance(HFI) is an autosomal recessive disease caused by catalytic deficiency of aldolase B in which affected homozygotes develop hypoglycemia and abdominal symptoms after taking foods containing fructose. Chronic exposure to fructose may lead to progressive hepatic injury, renal injury, growth retardation, and ultimately to liver and kidney failure. Herein, we report a case of HFI with presentation of episodic vomiting, diarrhea, cold sweating, abnormal liver function and failure to thrive after 12 months of her age. She developed an aversion to fruits and sweet-tasting foods. When she was admitted to hospital at the age of 30 months, hepatomegaly, and dysfunction of proximal renal tubule with renal tubular acidosis were noted. We confirmed the diagnosis via enzyme assay on biopsied liver and intestine. A fructose restrictied diet was recommended. The patient has been symptom free with normal liver functions since then.
Subject(s)
Humans , Acidosis, Renal Tubular , Diagnosis , Diarrhea , Diet , Enzyme Assays , Failure to Thrive , Fructose , Fructose Intolerance , Fructose-Bisphosphate Aldolase , Fruit , Hepatomegaly , Homozygote , Hypoglycemia , Intestines , Kidney Tubules, Proximal , Liver , Renal Insufficiency , Sweat , Sweating , VomitingABSTRACT
Fructose 1,6-bisphosphate aldolase (FPA) was recently known as new member of heparin binding proteins and a new method for FPA purification has been proposed (Thanh Van Ta et all, J. Biochem. 125, 554-559,199) by measuring FPA - heparin binding inhibition caused by various glycosaminoglycans (GAGs), affinity of the two isoforms, aldolase A4 and C4, to the GAGs underphysiological ionic conditions was estimated. Among glycosaminoglycans employd, heparin was confirmed to be the unique one that could bind specifically these enzymes. In the lower ionic strength, the affinity order of both FPA isoforms (A4 and C4) to these GAGs appeared as heparin> chondroitin polysulfate> heparin sulfate > dermatan > chondrointin sulfate A > chondroin sulfate C. Employing the same techniques, the affinity of regioselectively desulfated heparins to FPA was estimated. Our results indicated that, among the sulfate groups is heparin, loss of N-sulfate group reduced most significantly the affinity to FPA A4 and C4. This sugests that FPA recognizes a specific heparin structure including the sulfo-amino group at C2 of the glucosamine residue as the vital factor in this interaction.
Subject(s)
Fructose-Bisphosphate Aldolase , GlycosaminoglycansABSTRACT
Synthesis of a radioactive photoactivable heterobifunctional reagent, Noxysuccinimide ester of 2-[14C]glycyl carboxy-9-diazofluorene is described. This reagent on photolysis gives rise to a reactive carbene which rapidly inserts into solvents like methanol. The probe can be easily linked to aldolase which on photolysis gives rise to aldolase dimer, trimer and tetramer depending on the density of linked probe. This probe has also been linked to concanavalin A. The radioactive concanavalin A so obtained was incubated with erythrocyte ghosts and photolysed. The membrane protein analysis by gel electrophoresis indicated that concanavalin A has been covalently crosslinked to band 3.
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Objective To identify the recombinant aldolase (ALD) of Plasmodium falciparum, and to develop monoclonal antibodies (McAbs) against the recombinant ALD. Methods ALD gene was amplified by PCR from genomic DNA of FCC1/HN strain, and expressed in E.coli DH5?. BALB/c mice were immunized with the recombinant ALD of P. falciparum via celiac injection for 3 times with 2 weeks interval. Three days after a booster injection, spleen cells of the immunized mice were used for producing McAbs. The immune serum was tested by IFAT and Western blotting. Results BALB/c mice immunized with purified aldolase protein developed strong immune response to the antigen, and the titer of specific antibody reached 1∶105 in all immune sera after the third immunization. Moreover, immune sera specifically recognized the cultured P. falciparum. Western blotting showed that the immune sera recognized specifically a Mr 41 000 band of crude malaria antigen. No cross-reaction with human red cells was detected. Seven positive hybridoma cell lines were obtained after 3 rows of selection. All the McAbs′subclasses belong to IgG1. IFAT showed that only 4 McAbs could recognize the cultured P.falciparum. Conclusion Plasmodial aldolase has been successfully expressed and purified, and the established hybridoma cell lines can secrete McAbs specific to the aldolase of P. falciparum.
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FDP/FIP (88.8%), respectively; (3) The diagnostic positivity of combination assay was 80.0% in low or negative AFP producing HCC, while 95.7% in those with elevated AFP. Our data suggested that the combination assay of serum tumor enzyme markers was valuable in the diagnosis of HCC, especially in low or negative AFP HCC patients