Electroacupuncture Improves Blood-Brain Barrier and Hippocampal Neuroinflammation in SAMP8 Mice by Inhibiting HMGB1/TLR4 and RAGE/NADPH Signaling Pathways / 中国结合医学杂志

OBJECTIVE@#To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo.@*METHODS@#Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- β (Aβ), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining.@*RESULTS@#Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aβ in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01).@*CONCLUSION@#EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A β deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

Exogenous niacin treatment increases NADPH oxidase in kiwifruit / O tratamento com niacina exógena NADPH oxidase em kiwis

Abstract Kiwifruit are a popular fruit worldwide; however, plant growth is threatened by abiotic stresses such as drought and high temperatures. Niacin treatment in plants has been shown to increase NADPH levels, thus enhancing abiotic stresses tolerance. Here, we evaluate the effect of niacin solution spray treatment on NADPH levels in the kiwifruit cultivars Hayward and Xuxiang. We found that spray treatment with niacin solution promoted NADPH and NADP+ levels and decreased both O2·- production and H2O2 contents in leaves during a short period. In fruit, NADPH contents increased during early development, but decreased later. However, no effect on NADP+ levels has been observed throughout fruit development. In summary, this report suggests that niacin may be used to increase NADPH oxidases, thus increasing stress-tolerance in kiwifruit during encounter of short-term stressful conditions.

Resumo Kiwis são uma fruta popular em todo o mundo; No entanto, o crescimento das plantas é ameaçado por estresses abióticos como a seca e as altas temperaturas. O tratamento com niacina em plantas mostrou aumentar os níveis de NADPH, aumentando assim a tolerância a stress abiótico. Aqui, avaliamos o efeito do tratamento com spray de solução de niacina sobre os níveis de NADPH nos cultivares de kiwis Hayward e Xuxiang. Descobrimos que o tratamento por spray com solução de niacina promoveu níveis de NADPH e NADP + e diminuiu a produção de O2·- e os teores de H2O2 nas folhas durante um curto período. Nos frutos, os teores de NADPH aumentaram durante o desenvolvimento precoce, mas diminuíram mais tarde. No entanto, não se observou qualquer efeito nos níveis de NADP + ao longo do desenvolvimento do fruto. Em resumo, este relatório sugere que a niacina pode ser utilizada para aumentar NADPH oxidases, aumentando assim a tolerância ao estresse em kiwis durante o encontro de condições estressantes de curto prazo.

Application of n-dodecane as an oxygen vector to enhance the activity of fumarase in recombinant Escherichia coli: role of intracellular microenvironment

Abstract The effect of the intracellular microenvironment in the presence of an oxygen vector during expression of a fusion protein in Escherichia coli was studied. Three organic solutions at different concentration were chosen as oxygen vectors for fumarase expression. The addition of n-dodecane did not induce a significant change in the expression of fumarase, while the activity of fumarase increased significantly to 124% at 2.5% n-dodecane added after 9 h induction. The concentration of ATP increased sharply during the first 6 h of induction, to a value 7600% higher than that in the absence of an oxygen-vector. NAD/NADH and NADP/NADPH ratios were positively correlated with fumarase activity. n-Dodecane can be used to increase the concentration of ATP and change the energy metabolic pathway, providing sufficient energy for fumarase folding.

Kinetic mechanism of glucose dehydrogenase from Halobacterium salinarum.

The kinetic mechanism of glucose dehydrogenase (EC 1.1.1.47) from Halobacterium salinarum was studied by initial velocity and product inhibition methods. The results suggest that both, in the forward and reverse direction, the reaction mechanism is of Bi Bi sequential ordered type involving formation of ternary complexes. NADP+ adds first and NADPH formed dissociates from the enzyme last. For the reverse direction, NADPH adds first and NADP+ leaves last. Product inhibition experiments indicate that (a), the coenzymes compete for the same site and form of the enzyme and (b), ternary abortive complexes of enzyme-NADP(+)-glucono-delta-lactone and enzyme-NADPH-glucose are formed. All the other inhibitions are noncompetitive.

Hepatic pyridine nucleotides content in rat--a better indicator for determining available niacin values of food.

After 10 days depletion period with niacin free diet, weanling rats were repleted with either reference diets containing niacin at four levels (2, 4, 8 and 12 mg/kg diet) or test diets containing test material at two levels as source of niacin. Gains in body weight and hepatic pyridine nucleotides content increased with increase in niacin intake. The correlation coefficient for hepatic pyridine nucleotides content and niacin intake was 0.98, whereas for weight gain and niacin intake was 0.89. Dried skim milk and pearl millet were taken as test materials. Dried skim milk with most of its niacin in free form showed 98% niacin equivalent to be bioavailable whereas in pearl millet niacin was in bound form and bioavailability equivalent was only 48 per cent.

Modification of maize NADP-malic enzyme by Woodward's reagent 'K'.

Maize leaf NADP-malic enzyme was rapidly inactivated by micromolar concentrations of Woodward's reagent K (WRK). The inactivation followed pseudo-first order reaction kinetics. The order of reaction with respect to WRK was 1, suggesting that inactivation was a consequence of the modification of a single residue per active site. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification and also exhibited altered surface charge as seen from the elution profile on "Mono Q" anion exchange column and the mobility on native polyacrylamide gel electrophoresis. Substrate NADP and NADP + Mg2+ strongly protected the enzyme against WRK inactivation indicating that the modified residue may be located at or near the active site. Binding affinity of NADPH to the malic enzyme was studied by the fluorescence technique. The native enzyme binds NADPH strongly resulting in enhancement of the fluorescence emission and also causes a blue shift in the emission maximum of NADPH from 465 nm to 450 nm, however, the modified enzyme neither exhibited the enhancement of fluorescence emission nor the blue shift, indicating loss of NADPH binding site on modification. The essential carboxyl group may be involved in NADPH binding during catalysis by the enzyme.

Fatty acid synthesis by isolated leucoplasts from developing Brassica seeds: role of glycolytic intermediates as the source of carbon and energy.

Fatty acid synthesis from Na [1-14C] acetate in leucoplasts isolated from developing seeds of Brassica campestris was completely dependent on exogenous supply of ATP. None of the intermediates of glycolysis or pentose phosphate pathway tested could replace ATP in the reaction mixture. In absence of exogenously supplied ATP, maximum activity was obtained with glu-6-P (68%) followed by fru-6-P (50%) and PEP (44%), respectively. With other intermediates as energy sources, the activity ranged from 1 to 38%. In complementary experiments (presence of ATP), none of the metabolites gave activity higher than the ATP control activity. Under optimum conditions for fatty acid synthesis from acetate, Brassica leucoplasts readily utilized labelled glucose as the substrate for fatty acid synthesis. Omission of NADH and NADPH individually from the reaction mixtures containing labelled glucose resulted only in 46 and 20% loss in activity, respectively, compared to the corresponding losses of 56 and 50%, when labelled acetate was used as the substrate. Similarly, deletion of ATP from the reaction mixture containing glucose as the substrate decreased the rate of fatty acid synthesis by about 65%, while the corresponding decrease with acetate as the substrate was 96%. Inclusion of 5 mM cold acetate, pyruvate, malate and glu-6-P in the reaction mixture containing glucose as the labelled substrate reduced label incorporation into fatty acids by 38 to 69%, maximum reduction being observed with pyruvate followed by glu-6-P, acetate and malate, respectively. With labelled acetate as the substrate, maximum reduction in label incorporation was obtained with cold glucose (5 mM) followed by glu-6-P, pyruvate and malate, respectively. The study demonstrated the operation of complete glycolytic pathway in Brassica leucoplasts, allowing the plastids to use glucose as a source of carbon, reducing power and energy for fatty acid synthesis.

Influence of Ca2+ on kinetics and thermodynamics of the NADPH-dependent microsomal lipid peroxidation.

The effect of Ca2+ on kinetics and thermodynamics of lipid peroxidation in microsomes prepared from liver of male Swiss albino mice (7-8 weeks old) was studied. Ca2+ was found to increase the Vmax in temperature dependent manner. Michaelis-Menten constant (Km) also increased with temperature. However, the linearity and extent of change in Km remained unaffected in presence of Ca2+, and was suggestive of non-competitive and mixed type of activation. The activation constant (Ka) obtained by the replotting of slopes of the Lineweaver-Burk plots against the reciprocal of Ca2+ concentration showed linear variation with temperature. The linear pattern of Arrhenius plots indicated non-involvement of parallel reactions of other intermediate species in the lipid peroxidation. Thermodynamic parameters delta H degree, delta S degree and delta G degree, associated with lipid peroxidation process were studied. The positive value of delta H degree is suggestive of the endothermic nature of the process. It appears that the NADPH induced lipid peroxidation is an entropy driven process.

Redox state of pyridine nucleotides, but not glutathione, regulate Ca2+ release by H2O2 from mitochondria of pulmonary smooth muscle.

Treatment of bovine pulmonary artery smooth muscle mitochondria with H2O2 stimulated oxidation of GSH and NAD(P)H along with an increase in Ca2+ release. Addition of oxaloacetate to mitochondrial suspension stimulated Ca2+ release and oxidation of NAD(P)H while GSH level remained unchanged. Subsequently, addition of beta-hydroxybutyrate which reduced mitochondrial pyridine nucleotides caused reuptake of the released Ca2+ without causing appreciable alteration of GSH level. Treatment of the mitochondria with 1,3-bis(2-dichloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, significantly decreased GSH level without producing discernible change in Ca2+ release and NAD(P)H oxidation.

Purification of two azoreductases from Escherichia coli K12.

Two azoreductases (I and II) were purified to homogeneity from extracts of E. coli K12. Azoreductase I was a dimer of two identical subunits of molecular weight 28000 whereas azoreductase II was a monomer of 12,000 molecular weight. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I and II used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Ponceau SX was the best substrate for both the enzymes. However, azoreductase II utilized tartrazine, amaranth and orange II less efficiently than azoreductase I.

Effect of tamoxifen, estradiol 17 beta on coenzymes NAD, NADPH and the metabolism of estradiol and estrone in rabbit uterus in vivo.

Biotransformation of estradiol (E2) and estrone (E1) and the concentrations of NAD, NADPH and 17 beta-estradiol dehydrogenase (E2DH) were measured in the uterus of rabbits treated with tamoxifen (Tam) in two doses; 100 micrograms/day, (Tam 100) and 500 micrograms/day, (Tam 500), E2 (10 micrograms/day) and combination of E2 + Tam 500 for 4 days. The concentration of NAD in Tam 500 treated group was significantly higher than E2, low dose Tam and E2 + Tam 500 treated groups (P < 0.01). The concentration of NAD in E2+ Tam 500 uteri was also significantly higher than E2 treated uteri. The concentration of NADPH was not significantly different from each other amongst the various treatment groups. The studies have shown that E2DH in E2 treated uteri was less than control and Tam 500 treated groups. A significant rise in the enzyme estradiol oxidoreductase (E2OR) activity (P < 0.02) was observed in E2 + Tam 500 treated uteri over control and other treated groups whereas high dose Tam decreased the E2OR activity significantly over the E2 treated group. The rate of conversion of E1 to E2 in Tam 500 treated group was significantly less than the other treatment groups except E2 + Tam 500 treated group (P < 0.04). This study showed that E2 decreases the uterine biosynthesis of NAD and E2DH and the biotransformation of E2 to E1, while high dose Tam increases uterine NAD, E2DH activity and E2 to E1 conversion.

Chronic granulomatous disease of childhood: differential diagnosis and prognosis

Of a total of 111 children with primary immunodeficiency, 20 had phagocytic disorders (18 per cent) and 10 of them (8 boys and 2 girls) were diagnosed as chronic granulomatous disease (CGD). The children presented with repeated infections already during the first months of life. The main clinical findings were: abscess (n=8), otitis (n=8), pneumonia (n=8), lymphadenitis and pyodermits (n=6) and septicemia (4), NBT reduction was almost absent in all the children, except one of them. Bactericidal activity against S. aureus and phagocytosis were impaired in CGD patients. Different patterns of laboratory tests and prognosis were observed and girls had a better evolution

Polyphosphate binding sites in Liophis miliaris hemoglobin: evidence with reduced nicotinamide adenine dinucleotide phosphate

The water-snake Liophis miliaris presentes hemoglobin which binds organic polyphosphate through a simple single-site per tetramer (Mol. Wt. 64500) as judged by titration curves of reduced nicotinamide adenine dinucleotide phosphate either in the presence or absence of inositol hexaphosphate. The site seems to have the same structural nature of that found on other hemoglobins and is able to strongly bind most of the known protein effectors such as inositol hexaphosphate, adenosine triphosphate or 2,3-disphosphoglicerate. The high association constant at pØ7 of reduced nicotinamide for the deoxy hemoglobin of about K (D) = 7 x 10***6 M***-1 comparaed to human hemoglobin (K(D) = 7 x 10***5 M***-1), and to that of adenosine triphosphate (its natural erythrocytic polyphosphate) still higher of about K(D) = 10***11 M ***-1, shows clearly the very high affinity of this snake hemoglobin for such allosteric effector. The results besides corroborating the dimer-tetramer transition mechanism proposed to describe the oxygen transport by the hemoglobin of Liophis miliaris - may explain the difficulties to obtain the oxy dimeric conformation of the protein by usual hemolysis and stripped off procedures

Prostaglandin catabolism and ulcerative colitis: effect of sulphasalazine, 5-aminosalicylic acid and other drugs

1. Evidence is presented for the occurrence of type 1 prostaglandin 15-hidroxydehydrogenase in human rectal mucosa. No evidence of the presence of type 2 dnzyme was found. 2. A 15-keto-prostaglandin reductase, responsible for the breakdown of 13, 14-dihydro 15-keto prostaglandins to 13, 14-dihydro prostaglandins, was also present in human rectal mucosa. 3. Ulcerative colitis patients catabolized prostaglandins to the same extent as the control group. PGE was catabolized significantly better than PGF2 alfa. 4. 5-Aminosalicylic acid and sulphapyridine did not affect prostaglandin catabolism. Sulphasalazine, methilsulphasalazine, indomethacin, flurbiprofen, disodium cromoglycate, sodium salicylate and carbenoxolone sodium inhibited prostaglandin catabolism to the same extent in both groups.Salicylazosulphadimidine was a more potent inhibitor of PGE1 catabolism than of PGF2alfa. 5. The increased prostaglandin synthesis reported for ulcerative colitis patients was not paralleled by increased catabolism, a fact possibly contributing to the accumulation of such compounds in the diseased state

Effect of alpha-tocopherol on the microsomal lipid peroxidation induced by doxorubicin: influence of ascorbic acid.

alpha-Tocopherol (40 mg/rat/day) was administered, orally, to doxorubicin treated rats (2 mg/kg, twice weekly, for 4 weeks) singly and also in combination with ascorbic acid (1 g/100 ml/day) in drinking water. The vitamin therapy was carried out for a period of 1 month. The microsomal lipid peroxide levels in liver and heart were found to be increased in doxorubicin treated rats. alpha-tocopherol and ascorbic acid treatment decreased the lipid peroxide level and also NADPH-dependent lipid peroxidation. A significant depletion of glutathione in liver and heart of doxorubicin treated animals was found to be ameliorated by vitamin therapy. Ascorbic acid was found to maintain the level of microsomal alpha-tocopherol. The activities of the detoxifying enzymes like catalase, superoxide dismutase and glutathione peroxidase were suppressed in doxorubicin treated rats and vitamins coadministration maintained the levels of these enzymes. Ascorbic acid was found to potentiate the antioxidant nature of alpha-tocopherol.

The effect of sorbose on NAD(P)ase production by Aspergillus nidulans

NAD(P)ase activity was stimulated when 1% sorbose was present in the culture medium of A. nidulans, and this effect was partially reversed by 1% glucose. 2. The level of extracellular NAD(P)ase was more affected by sorbose in the culture medium than the intracellular enzyme and no morphological changes were obtained. 3. The sorbose effect on NAD(P)ase activity appears to be specific sinle two other exoenzymes tested (ß-glucosidase and alkaline protease) show normal secretion patterns. 4. These findings suggest that the sorbose effect on NAD(P)ase production may be the consequence of metabolic disorders not necessarily linked with the morphological changes induced by the ketohexose

Fluorescence studies on the binding of reduced nicotinamide adenine dinucleotide phosphate to human hemoglobin A and its variant hemoglobin providence

The affinity constants for the binding of NADPH to human hemoglobin A were directly determined by fluorescence analyssis since nucleotide fluorescence is quenched on binding to the protein. The binding constants 6.1 x 10**5, 5.02 x 10**5 and 1.2 x 10**5 were found for deosyhemoglobin at pH 6.5, 7.0,respectively. Oxyhemoglobin does not bind NADPH significantly. These results are consistent with those found in oxygen-hemoglobin equilibrium experiments. The human hemoglobin variant, Providence-Asp, which has a marked decrease in 2,3 DPG affinity was also investigated. NADPH does not bind to the variant suggesting that the Lys B 82 residues is of fundamental importance to nucleotide binding and showing that the binding site is the same as that of 2,3 DPG or other organic polyphosphate, aloosteric modulators of hemoglobins. Experiments of inositol hexaphosphate (IHP)-NADPH site competition corroborate these results