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Analytical and Bioanalytical Chemistry Jul 2021Nucleic acid aptamers are small fragments of DNA or RNA molecules binding specifically to targets, which can be obtained through in vitro screening via systematic...
Nucleic acid aptamers are small fragments of DNA or RNA molecules binding specifically to targets, which can be obtained through in vitro screening via systematic evolution of ligands by exponential enrichment (SELEX). Lactate dehydrogenase (LDH) is an important tumor marker, whose level in patients is of great significance for diagnosis of many diseases. Here, we report the identification of LDH aptamers by 9 rounds of screening from a length-mixed single-stranded DNA library using the SELEX technology. After the 3rd and 7th rounds of aptamer screening, affinity was significantly improved, and fluorescence quantitative analysis showed stronger affinity for the aptamers selected from the 7th to 9th rounds of screening. After high-throughput sequencing, motif analysis, and secondary structure prediction, we finally chose and further investigated 15 candidate LDH aptamer sequences with obvious differences in secondary structure in the 7th to 9th rounds of screening. Among them, LDH7-1, LDH7-9, LDH8-2, and LDH9-1 were shown to bind to LDH protein with high affinity and specificity with K < 25 nM. This study provides new ideas for rapid detection of LDH protein content and enzyme activity, thus contributing to the development of rapid medical detection.
Topics: Aptamers, Nucleotide; Base Sequence; Binding Sites; DNA, Single-Stranded; Humans; L-Lactate Dehydrogenase; SELEX Aptamer Technique
PubMed: 34028561
DOI: 10.1007/s00216-021-03397-2 -
Applied Microbiology and Biotechnology Jun 2023Acetoin, an important and high-value added bio-based platform chemical, has been widely applied in fields of foods, cosmetics, chemical synthesis, and agriculture....
Acetoin, an important and high-value added bio-based platform chemical, has been widely applied in fields of foods, cosmetics, chemical synthesis, and agriculture. Lactate is a significant intermediate short-chain carboxylate in the anaerobic breakdown of carbohydrates that comprise ~ 18% and ~ 70% in municipal wastewaters and some food processing wastewaters, respectively. In this work, a series of engineered Escherichia coli strains were constructed for efficient production of acetoin from cheaper and abundant lactate through heterogenous co-expression of fusion protein (α-acetolactate synthetase and α-acetolactate decarboxylase), lactate dehydrogenase and NADH oxidase, and blocking acetate synthesis pathways. After optimization of whole-cell bioconversion conditions, the engineered strain BL-11 produced 251.97 mM (22.20 g/L) acetoin with a yield of 0.434 mol/mol in shake flasks. Moreover, a titer of 648.97mM (57.18 g/L) acetoin was obtained in 30 h with a yield of 0.484 mol/mol lactic acid in a 1-L bioreactor. To the best of our knowledge, this is the first report on the production of acetoin from renewable lactate through whole-cell bioconversion with both high titer and yield, which demonstrates the economy and efficiency of acetoin production from lactate. Key Points • The lactate dehydrogenases from different organisms were expressed, purified, and assayed. • It is the first time that acetoin was produced from lactate by whole-cell biocatalysis. • The highest titer of 57.18 g/L acetoin was obtained with high theoretical yield in a 1-L bioreactor.
Topics: Acetoin; Lactic Acid; Escherichia coli; Wastewater; Bioreactors; L-Lactate Dehydrogenase
PubMed: 37178309
DOI: 10.1007/s00253-023-12560-x -
Investigative Ophthalmology & Visual... Jan 2016Like cancer cells, photoreceptor cells produce lactate aerobically, requiring lactate dehydrogenase A (LDH-A). Cancer cells also use glycolytic intermediates for...
PURPOSE
Like cancer cells, photoreceptor cells produce lactate aerobically, requiring lactate dehydrogenase A (LDH-A). Cancer cells also use glycolytic intermediates for biosynthesis. The molecular switch controlling glycolytic flow is thought to be an isoenzyme of pyruvate kinase (PKM2). Here, we determined the expression and localization of PKM2 and LDH-A in mammalian retina and make comparisons with the brain.
METHODS
Single- and double-labeling immunohistochemistry for PKM2, pyruvate kinase M1 (PKM1), and LDH-A were performed using retinal sections from C57BL/6 mice, Sprague-Dawley rats, rabbits, marmosets, and humans. Pyruvate kinase M1 and PKM2 mRNA and protein expression levels were quantified in rodent retina and brain by using qPCR and immunoblotting. The quaternary forms of PKM2 in rat retina were also determined.
RESULTS
Pyruvate kinase M2 was present in some glial cells and rod and cone photoreceptors in the retina of all species but was exclusively localized to glia in the brain. Pyruvate kinase M1 was confined to neurons in the retina and brain. Lactate dehydrogenase A was principally found in photoreceptors and inner portion of the avascular rabbit retina. Western blotting and qPCR confirmed high levels of PKM2 and LDH-A in the retina. There was a 6- to 9-fold greater expression of PKM2 mRNA in the rodent retina than in the brain. Both the dimeric (inactive, biosynthesis-driving form) and the active tetrameric (glycolytic-driving) forms of PKM2 were present in retina but not in brain.
CONCLUSIONS
Mammalian photoreceptors contain dimeric and tetrameric PKM2 and LDH-A. This is consistent with the ability to switch between energy production and biosynthesis like a proliferating tissue, possibly due to demands of opsin synthesis.
Topics: Aged; Animals; Blotting, Western; Callithrix; Cell Line; Energy Metabolism; Humans; Immunohistochemistry; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Mice; Mice, Inbred C57BL; Middle Aged; Pyruvate Kinase; RNA; Rabbits; Rats; Rats, Sprague-Dawley; Retina; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 26780311
DOI: 10.1167/iovs.15-17962 -
Journal of Neuro-oncology Sep 2022Lactate dehydrogenase (LDH) is a biomarker for cancer. However, the relationship between serum LDH levels and the survival of patients with brain metastasis has been...
BACKGROUND
Lactate dehydrogenase (LDH) is a biomarker for cancer. However, the relationship between serum LDH levels and the survival of patients with brain metastasis has been fully revealed. We aimed to evaluate the serum LDH levels and assess its prognostic value in patients with BM.
METHODS
The serum LDH levels were collected from 2507 patients with BM. Patients were categorized into four groups according to the quartile of serum LDH levels. The association between serum LDH levels and overall survival (OS) was evaluated using Cox regression models and Kaplan-Meier curves. Three predictive models were used to evaluate patients.
RESULTS
The Kaplan-Meier curve for survival by the serum LDH group demonstrates clear separation between four groups (P < 0.001). The participants in the lower group had longer OS than those in the higher group. After adjusting in multivariate Cox regression models remained significant for patients in the Q4 compared with patients in the Q1 (Q4:Q1 OR 1.58, 95% CI 1.38-1.80). Furthermore, the GPA-LDH model generates a pooled area under the curve of 0.630 (95% CI 0.600, 0.660).
CONCLUSIONS
Serum LDH levels and OS in patients with brain metastasis is an inverse association. Moreover, Serum LDH levels can improve the prognosis of the GPA model.
Topics: Biomarkers, Tumor; Brain Neoplasms; Humans; L-Lactate Dehydrogenase; Prognosis; Proportional Hazards Models; Retrospective Studies
PubMed: 35794505
DOI: 10.1007/s11060-022-04070-z -
The Journal of Biological Chemistry 2021Despite being initially regarded as a metabolic waste product, lactate is now considered to serve as a primary fuel for the tricarboxylic acid cycle in cancer cells. At...
Despite being initially regarded as a metabolic waste product, lactate is now considered to serve as a primary fuel for the tricarboxylic acid cycle in cancer cells. At the core of lactate metabolism, lactate dehydrogenases (LDHs) catalyze the interconversion of lactate to pyruvate and as such represent promising targets in cancer therapy. However, direct inhibition of the LDH active site is challenging from physicochemical and selectivity standpoints. However, LDHs are obligate tetramers. Thus, targeting the LDH tetrameric interface has emerged as an appealing strategy. In this work, we examine a dimeric construct of truncated human LDH to search for new druggable sites. We report the identification and characterization of a new cluster of interactions in the LDH tetrameric interface. Using nanoscale differential scanning fluorimetry, chemical denaturation, and mass photometry, we identified several residues (E62, D65, L71, and F72) essential for LDH tetrameric stability. Moreover, we report a family of peptide ligands based on this cluster of interactions. We next demonstrated these ligands to destabilize tetrameric LDHs through binding to this new tetrameric interface using nanoscale differential scanning fluorimetry, NMR water-ligand observed via gradient spectroscopy, and microscale thermophoresis. Altogether, this work provides new insights on the LDH tetrameric interface as well as valuable pharmacological tools for the development of LDH tetramer disruptors.
Topics: Epitope Mapping; Humans; L-Lactate Dehydrogenase; Lactate Dehydrogenases; Lactic Acid; Ligands; Magnetic Resonance Imaging; Peptides
PubMed: 33607109
DOI: 10.1016/j.jbc.2021.100422 -
Postgraduate Medicine May 2017Recent research demonstrates that lactate dehydrogenase (LDH) activity within the normal range may serve as a mediator in the (positive) relationship between physical...
OBJECTIVES
Recent research demonstrates that lactate dehydrogenase (LDH) activity within the normal range may serve as a mediator in the (positive) relationship between physical activity and cardiovascular disease risk. Emerging work supports deleterious associations between sedentary behavior and health, independent of physical activity. Thus, this study evaluated if sedentary behavior was associated with normal-range LDH activity, independent of physical activity.
METHODS
Data from the 2003-2006 NHANES were used (N = 2,087 adults; 40-79 yrs). LDH activity levels were estimated from a blood sample using LX20 and LDH reagent; participants were included if they had LDH activity levels within the normal range (105-333 IU/L). Physical activity and sedentary behavior were assessed via accelerometry.
RESULTS
Sedentary behavior was inversely associated with normal-range LDH activity when physical activity was excluded from the model (OR = 0.89; 95% CI: 0.83-0.97, P = 0.009 for LDH activity quartile 4 vs. 1). However, sedentary behavior was no longer associated with normal-range LDH activity after controlling for physical activity and other covariates (OR = 1.00, P = 0.49 for LDH activity quartile 2 vs. 1; OR = 1.00, P = 0.72 for LDH quartile 3 vs. 1; and OR = 0.99, P = 0.36 for LDH quartile 4 vs. 1).
CONCLUSION
Unlike physical activity, sedentary behavior is not independently associated with normal-range LDH activity.
Topics: Accelerometry; Female; Humans; L-Lactate Dehydrogenase; Male; Middle Aged; Nutrition Surveys; Reference Values; Sedentary Behavior; United States
PubMed: 28276789
DOI: 10.1080/00325481.2017.1298390 -
Drug Design, Development and Therapy 2016Praziquantel (PZQ) is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ), which is composed of (R)-PZQ and (S)-PZQ. In this work, the cytotoxicity of rac-PZQ and its...
Praziquantel (PZQ) is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ), which is composed of (R)-PZQ and (S)-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers (R)-PZQ and (S)-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that (R)-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2). However, in contrast to (R)-PZQ, the (S)-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, (R)-PZQ showed lower cytotoxicity on SH-SY5Y cells than (S)-PZQ. Meanwhile, (R)-PZQ at <80 μM concentration could promote proliferation of macrophage cells (Raw264.7). Our research revealed that (R)-PZQ has lower cytotoxicity than (S)-PZQ and has similar cytotoxicity with rac-PZQ. (S)-PZQ is the principal enantiomer to cause side effects on human definitive hosts. These findings gave the reasonable reasons for World Health Organization to produce (R)-PZQ as a replacement for rac-PZQ for the treatment of schistosomiasis.
Topics: Cell Line, Tumor; Hep G2 Cells; Humans; L-Lactate Dehydrogenase; Praziquantel; Schistosomiasis; Stereoisomerism; Tetrazolium Salts; Thiazoles
PubMed: 27445457
DOI: 10.2147/DDDT.S98096 -
Journal of Materials Chemistry. B Feb 2019Over the past ten years, considerable progress has been achieved in the field of nanomaterials-based enzymes (nanozymes). In comparison with natural enzymes, nanozymes... (Review)
Review
Over the past ten years, considerable progress has been achieved in the field of nanomaterials-based enzymes (nanozymes). In comparison with natural enzymes, nanozymes demonstrate significant advantages such as facile synthesis procedure, low price, long storage period, and high environmental stability. A variety of nanomaterials including nanocarbons, metals, metal oxides, metal chalcogenides, halogen compounds, metal-organic frameworks (MOFs), and layered double hydroxides (LDHs) have been extensively investigated for enzyme mimicking. In this review, the recent progresses made in the development of the enzymatic properties of these nanozymes have been discussed. We comprehensively discuss strategies to improve catalytic activity and substrate specificity, enzyme-like catalytic mechanism, and novel application of nanozymes in sensing techniques. In addition, the remaining challenges and some future directions have been addressed. With the fast development of nanozyme applications in bioscience and technology, research in this field has become more and more attractive, which is expected to be a long-term exciting subject in the near future.
Topics: Biomimetic Materials; Biosensing Techniques; Catalysis; Catalytic Domain; Graphite; L-Lactate Dehydrogenase; Metal-Organic Frameworks; Metals; Nanostructures; Oxides
PubMed: 32255092
DOI: 10.1039/c8tb02878h -
Protein Expression and Purification Dec 2019Lactate dehydrogenase (LDH) is a key enzyme to produce energy during hypoxia by anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, two protein subunits...
Lactate dehydrogenase (LDH) is a key enzyme to produce energy during hypoxia by anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, two protein subunits (LDH-1 and LDH-2) were previously identified, deduced from two different transcripts that come from the same LDH gene by processing via mutually exclusive alternative splicing. LDH-1 contains exon five and LDH-2 contains exon six and the two proteins differ only in 15 amino acid residues. Both subunits were independently cloned and overexpressed in E. coli as a fusion protein containing a chitin binding domain. Previously, recombinant LDH-2 was successfully purified and characterized, but LDH-1 was insoluble and aggregated forming inclusion bodies. We report the production of soluble LDH-1 by testing different pHs in the buffers used to lyse the bacterial cells before the purification step and the characterization of the purified protein to show that the cDNA indeed codes for a functional and active protein. The recombinant native protein is a homotetramer of approximately 140 kDa composed by 36 kDa subunits and has higher affinity for pyruvate than for lactate. LDH-1 has an optimum pH of 7.5 and is stable between pH 8.0 and 9.0; pH data analysis showed two pKa values of 6.1 ± 0.15 and 8.8 ± 0.15 suggesting a histidine and asparagine, respectively, involved in the active site. The enzyme optimal temperature was 44 °C and it was stable between 20 and 60 °C. LDH-1 was slightly activated by NaCl, KCl and MgCl and fully inhibited by ZnCl.
Topics: Animals; Cloning, Molecular; Isoenzymes; L-Lactate Dehydrogenase; Lactic Acid; Penaeidae; Protein Multimerization; Pyruvic Acid; Recombinant Proteins; Substrate Specificity
PubMed: 31351993
DOI: 10.1016/j.pep.2019.105461 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Feb 2022To analyze the polymorphism of lactate dehydrogenase () gene and predict B-cell epitopes in pLDH peptides in four species of human malaria parasites.
OBJECTIVE
To analyze the polymorphism of lactate dehydrogenase () gene and predict B-cell epitopes in pLDH peptides in four species of human malaria parasites.
METHODS
The blood samples and epidemiological characteristics were collected from malaria cases in Yunnan Province registered in the National Notifiable Disease Report System. The genes of four human species were amplified using nested PCR assay and sequenced. The polymorphisms of genes was analyzed using the software MEGA version 7.0.26 and DnaSP version 5.10, and the B-cell epitopes were predicted in pLDH peptides using the Immune Epitope Database (IEDB).
RESULTS
The sequences of LDH (), LDH (), LDH () and LDH () genes were obtained from 153, 29, 17 and 11 blood samples from patients with , , and malaria, respectively, which included 15, 2, 4 and 2 haplotypes and had a nucleotide diversity (π) of 0.104. A high level of intra-species differentiation was seen in the gene (π = 0.012), and the π values were all < 0.001 for , and genes. Active regions of B-cell antigen were predicted in the pLDH peptide chain of four human malaria parasites, of 4 to 5 in each chain, and the activity score was approximately 0.430. Among these peptide chains, the "86-PGKSDKEWNRD-96" short-peptide was a B-cell epitope shared by all four species of human malaria parasites, and the "266-GQYGHS (T)-271" short-peptide was present in PvLDH and PoLDH peptide chains, while "212-EEVEGIFDR-220" was only found in the PvLDH peptide chain, and "208-LISDAE-213" was only seen in the PfLDH peptide chain.
CONCLUSIONS
The gene polymorphism may be derived from the weak negative purification selection, while , and genes may maintain a relatively conservative state. There may be two B-cell epitopes "212-EEVEGIFDR-220" and "208-LISDAE-213" in the proximal region of the C terminal in the pLDH peptide chain, which is feasible to differentiate between and infections.
Topics: China; Epitopes, B-Lymphocyte; Humans; L-Lactate Dehydrogenase; Plasmodium; Plasmodium falciparum; Plasmodium vivax; Polymorphism, Genetic; Protozoan Proteins
PubMed: 35266354
DOI: 10.16250/j.32.1374.2021026