Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.     

Effect of adherence,cell morphology,and lipopolysaccharide on potassium conductance and passive membrane properties of murine macrophage J774.1 cells

Summary The effects of adherence, cell morphology, and lipopolysaccharide on electrical membrane properties and on the expression of the inwardly rectifying K conductance in J774.1 cells were investigated. Whole-cell inwardly rectifying K currents (K _i), membrane capacitance (C _m), and membrane potential (V _m) were measured using the patch-clamp technique. SpecificK _i conductance (G _K i, whole-cell Ki conductance corrected for leak and normalized to membrane capacitance) was measured as a function of time after adherence, and was found to increase almost twofold one day after plating. Membrane potential (V _m) also increased from –42±4 mV (n=32) to –58±2 mV (n=47) over the same time period.G _K i andV _m were correlated with each other;G _L (leak conductance normalized to membrane capacitance) andV _m were not. The magnitudes ofG _K i andV _m 15 min to 2 hr after adherence were unaffected by the presence of 100 m cycloheximide, but the increase inG _K iandV _m that normally occurred between 2 and 8 hr after adherence was abolished by cycloheximide treatment. Membrane properties were analyzed as a function of cell morphology, by dividing cells into three categories ranging from small round cells to large, extremely spread cells. The capacitance of spread cells increased more than twofold within one day after adherence, which indicates that spread cells inserted new membrane. Spread cells had more negative resting membrane potentials than round cells, butG _K i andG _L were not significantly different. Lipopolysaccharide-(LPS; 1 or 10 g/ml) treated cells showed increasedC _m compared to control cells plated for comparable times. In contrast to the effect of adherence, LPS-treated cells exhibited a significantly lowerG _K i than control cells, indicating that the additional membrane did not have as high a density of functionalG _K i channels. We conclude that both adherence and LPS treatment increase the total surface membrane area of J774 cells and change the density of Ki channels. In addition, this study demonstrates that membrane area and density of Ki channels can vary independently of one another.     

Effects of Chondroitin Sulfate and Its Oligosaccharides on Toll-Like Receptor-Mediated IL-6 Secretion by Macrophage-Like J774.1 Cells

The effects of two types of chondroitin sulphate (CS), CS-A and CS-C, their oligosaccharides (oligo-CSs), and disaccharides (Di-CSs) on toll-like receptor (TLR)-mediated secretion of interleukin (IL)-6 were compared using macrophage-like cell line J774.1. IL-6 secretion in the J774.1 cells was markedly increased by Pam3CS4, LPS, and CpG, the ligands to TLR1/2, 4, and 9 respectively. Among these three ligands, CpG-induced IL-6 was most clearly suppressed by CSs and their digests. Suppression of IL-6 secretion by smaller sized CS-A was stronger than that by intact CS-A, whereas no such size-dependent suppression was apparent for CS-C. Di-4S, the disaccharide unit of the CS-A digest, also showed much stronger suppression than Di-6S, the disaccharide unit of the CS-C digest, and the non-sulfated disaccharide unit, Di-0S. The suppressing activity of oligo-CSs, particularly Di-CSs, against TLR-mediated inflammation was dependent on the CS structure, including the sulfation site.     

Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.     

Lysosomal enzyme secretion by macrophages during intracellular storage of particles

Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase β-N-acetylglucosaminidase. Thus macrophage lysosomal enzyme secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.     

Rat lactate dehydrogenase A and B subunit concentrations are not regulated by mRNA abundance in liver and heart

RNA was isolated from rat liver and heart tissues at various times up to 12 weeks after birth, and probed on slot blots with lactate dehydrogenase A and B cDNA probes. Although the relative abundances of LDH A in liver and LDH B in heart increased substantially in the 12 weeks after birth, mRNAs for both isoenzymes remained remarkably stable in both tissues over the same period. The implications of these observations for the regulation of constitutive gene expression are discussed.     

An amperometric lactate biosensor based on lactate dehydrogenase immobilized onto graphene oxide nanoparticles‐modified pencil graphite electrode

A novel amperometric lactate biosensor was developed based on immobilization of lactate dehydrogenase onto graphene oxide nanoparticles‐decorated pencil graphite electrode. The enzyme electrode was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), and cyclic voltammetry at different stages of its construction. The biosensor showed optimum response within 5 s at pH 7.3 (0.1 M sodium phosphate buffer) and 35°C, when operated at 0.7 V. The biosensor exhibited excellent sensitivity (detection limit as low as 0.1 μM), fast response time (5 s), and wider linear range (5–50 mM). Analytical recovery of added lactic acid in serum was between 95.81–97.87% and within‐batch and between‐batch coefficients of variation were 5.04 and 5.40%, respectively. There was a good correlation between serum lactate values obtained by standard colorimetric method and the present biosensor (r = 0.99). The biosensor measured lactate levels in sera of apparently healthy subjects and persons suffering from lactate acidosis and other biological materials (milk, curd, yogurt, beer, white wine, and red wine). The enzyme electrode lost 25% of its initial activity after 60 days of its regular uses, when stored dry at 4°C.     

Determination of lactate dehydrogenase isoenzymes in single lymphocytes from normal and leukemia cell lines

This work demonstrates that our previously developed technique for single-erythrocyte analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be applied to study individual lymphocytes, with some modification in the cell lysing procedure. A tesla coil was shown to be capable of lysing the lymphocyte cells inside the capillary. The electromagnetic field induced by the tesla coil was believed to be responsible for breaking the cell membrane. The lactate dehydrogenase (LDH) isoenzyme activities and the relative ratios between different LDH isoenzymes were measured for normal lymphocytes as well as B-type and T-type acute lymphoblastic leukemia cells. Both the LDH activity and the isoenzyme ratios show large variations among individual cells. The former is expected due to variations in cell size. The latter implies that single-cell measurements are less useful than the average values over a cell population as markers for leukemia.     

The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase

Abstract The lactate dehydrogenase gene, ldh , of Alcaligenes eutrophus H16 was identified on a 14-kbp Eco RI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme. Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp Pst I subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions. One represented the ldh gene. The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E. coli sequence of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh , and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins. ORF3 was located upstream of and colinear to ldh , and its putative gene translational product represented a hydrophobic protein. A sequence, which resembled the A. eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.     

Studies on lactate dehydrogenase of Lactobacillus plantarum spp. involved in lactic acid biosynthesis using permeabilized cells

Lactate dehydrogenase (LDH, EC 1.1.1.27) catalyses the reduction of pyruvate to lactate in facultative anaerobes. Whole cells of Lactobacillus plantarum NCIM 2084 showed low levels of LDH activity but permeabilization of cells by treatment with organic solvents toluene, chloroform and diethyl ether increased the measurable LDH activities, ether treated cells showing the highest increase. The maximum intracellular activity was obtained upon treating the cells with ether (1%) at 28°C for 1 min. The LDH activity in permeabilized cells was nearly three-fold higher than that in the cell-free extract prepared by sonication. The kinetic properties of LDH in the permeabilized cells were comparable to that of cell-free extract, indicating that catalytically it functions similar to the isolated enzyme.     

Protein kinase activity on the cell surface of a macrophage-like cell line,J774.1 cells

Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [γ-³²P]ATP, a transfer of [³²P]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg²⁺-dependent but cAMP-independent, and Mg²⁺ could be substituted for by Mn²⁺. The reaction products were found to be proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonoester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phosphorylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.     

Potent effects of,and mechanisms for,modification of crosstalk between macrophages and adipocytes by lactobacilli

The murine macrophage‐like cell line J774.1 was treated with heat‐killed cells of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC 0356). Interleukin (IL)‐6, IL‐12, and tumor necrosis factor‐α were profiled from the J774.1 cells using enzyme‐linked immunosorbent assay methods. The conditioned medium from cultured J774.1 cells was transferred to the preadipocyte cell line 3T3‐L1 (which is a mouse embryonic fibroblast‐adipose‐like cell line). Growth and differentiation of 3T3‐L1 cells were monitored by analyzing lipid accumulation and expression of peroxisome proliferator‐activated receptor (PPAR)‐γ mRNA. The medium conditioned by 3T3‐L1 cells was added to J774.1 cells and the cytokines in the supernatant analyzed. Compared with that of cells exposed to a PBS‐conditioned medium, lipid accumulation in 3T3‐L1 cells was significantly suppressed in a dose‐dependent manner by each medium that had been conditioned with LGG and TMC0356. PPAR‐γ mRNA expression in 3T3‐L1 cells was also significantly downregulated (P < 0.01, P < 0.05, respectively). The conditioned medium of 3T3‐L1 adipose phenotype significantly stimulated production of IL‐6 and IL‐12 in J774.1 cells treated with LGG and TMC0356. These results suggest that lactobacilli may suppress differentiation of preadipocytes through macrophage activation and alter the immune responses of macrophages to adipose cells.     

Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD⁺), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H₂O₂) in the culture medium. Under oxidative stress, the NAD⁺ generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD⁺ reveals an intricate link between metabolism and the processing of genetic information.     

The role of an NAD-independent lactate dehydrogenase and acetate in the utilization of lactate byClostridium acetobutylicum strain P262

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min^–1 (mg protein)^–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05 h^–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H₂ + 1 CO₂. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (K_s = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K _m for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol. Received: 17 October 1994 / Accepted: 30 January 1995     

Characterization of lactate dehydrogenase enzyme in seminal plasma of Japanese quail (Coturnix coturnix japonica)

Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential.     

Identification of 2-amino-5-aryl-pyrazines as inhibitors of human lactate dehydrogenase

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure–activity relationships.     

Molecular dynamics simulation of lactate dehydrogenase adsorption onto pristine and carboxylic-functionalized graphene

ABSTRACT

Lactate dehydrogenase (LDH) is a tetrameric enzyme which is composed of two subunits known as LDHA and LDHB, which are encoded by the LDHA and LDHB genes respectively. LDH catalyses the last step in anaerobic glycolysis through the reversible conversion of pyruvate to lactate via coupled oxidation of NADH cofactor. The LDHA plays an important regulatory role in anaerobic glycolysis, by catalysing the final step of the process. Therefore, it is likely that increases in the expression level of LDHA in cancer cells could facilitate the efficiency of anaerobic glycolysis. Measuring the level of serum LDHA is a key step in the diagnosis of many cancer types. In this study, the adsorption, stability, and dynamics of LDHA on the surface of pristine graphene (PG) and carboxylated graphene (COOH-Graphene) were investigated using its molecular dynamics simulation. Variations in root mean square deviation, root mean square fluctuation, solvent accessible surface area and adsorption energy of the LDHA during the simulation were calculated to analyse the effect of PG and COOH-Graphene on the overall conformation of LDHA. Results showed that the adsorption of LDHA on COOH-Graphene is mostly mediated by electrostatic interactions, whereas on the PG, both Van der Waals and π-π interactions are prominent.     

Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme

The effects of pH, salt concentration and the presence of oxidized and reduced forms of coenzyme on the interaction of skeletal muscle lactate dehydrogenase with the liposomes derived from the total fraction of bovine erythrocyte lipids were investigated by ultracentrifugation and were compared with those results obtained using the heart-rate isoenzyme which we have previously studied. Liposomes are good adsorptive systems for both types of isoenzyme. In the presence of erythrocyte lipid liposomes, bovine muscle and heart lactate dehydrogenases form two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. Soluble protein-phospholipid complexes reveal different dependences of their stabilities on pH values and it seems that the nature of the binding site in either isozyme is different. In addition, absorption of the isoenzymes on the liposomes also reveals in difference in the effects of NAD and NADH. While the presence of NAD dissociates LDH-H₄ from the liposomes and NADH does not influence its adsorption, NAD promotes the binding of LDH-M₄, and NADH favors the dissociation.     

The physiological role of the isoenzymes of lactate dehydrogenase in potatoes

Four of the five isoenzymes of lactate dehydrogenase present in potato tubers have been isolated and their kinetic properties examined. The pyruvate-reductase activity of isoenzyme-4 is greatly reduced at low pH, the affinity for both pyruvate and NADH is reduced and ATP has a stronger inhibitory effect. If the design properties of an enzyme dictate a high affinity for substrates, then the Km values for lactate, glyoxylate and NAD are consistent with an oxidative role for isoenzyme-4. The same considerations do not permit a conclusion about the physiological role of isoenzymes-1 to-3. However, an overview of the kinetic properties of these isoenzymes indicates that isoenzyme-1 is best adapted for the role of pyruvate reductase. Consideration of the relationships between kinetic constants and electrophoretic mobilities of the isoenzymes, leads us to predict that isoenzyme-5 is well adapted for a role in the oxidation of lactate or glyoxylate. The lactate dehydrogenase of potato leaves appears to consist prodominantly of an isoenzyme with the same mobility as isoenzyme-2 of the tubers and the two isoenzymes are probably identical. The kinetic properties of this isoenzyme are consistent with roles in either oxidation or reduction.Abbreviation Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol