[Study of chemical kinetics between pyrroloquinoline quinine and D-serine in body by ion-pair liquid chromatography].

As a new neurotransmitter present in the glial cells, D-serine (DSer) plays an important role in central nervous system diseases. Pyrroloquinoline quinine (PQQ) can promote the production of the nerve growth factor and has a protective effect on nerve injuries. The chemical kinetics of PQQ and DSer was studied by determining the free contents of PQQ using ion-pair liquid chromatography (LC), so it can provide important information for the mechanisms of PQQ in the regulation of neurotransmitter. The PQQ and the production of the incubation were separated on an Amethyst C18-P column using tetrabutylammonium bromide as ion-pair reagent. The average recoveries were between 94.2% and 99.3%, and the relative standard deviations were between 1.05% and 2.03%. The average rate constants (K) of PQQ with DSer were 0.032, 0.07 and 0.17 h(-1) at 25, 37 and 50 degrees C, respectively. The average activation energy (E(a)) was 54.7 kJ/mol. The values of half life (t1/2) were 22.0, 9.8 and 3.99 h at 25, 37 and 50 degrees C, respectively. The results showed that PQQ can regulate the balance of DSer in the brain. The method is simple and reliable.

Preliminary studies of (99m)Tc-memantine derivatives for NMDA receptor imaging.

INTRODUCTION: Novel technetium-labeled ligands, (99m)Tc-NCAM and (99m)Tc-NHAM were developed from the N-methyl-d-aspartate (NMDA) receptor agonist memantine as a lead compound by coupling with N(2)S(2). This study evaluated the binding affinity and specificity of the ligands for the NMDA receptor. METHODS: Ligand biodistribution and uptake specificity in the brain were investigated in mice. Binding affinity and specificity were determined by radioligand receptor binding assay. Three antagonists were used for competitive binding analysis. In addition, uptake of the complexes into SH-SY5Y nerve cells was evaluated. RESULTS: The radiochemical purity of (99m)Tc-labeled ligands was more than 95%. Analysis of brain regional uptake showed higher concentration in the frontal lobe and specific uptake in the hippocampus. (99m)Tc-NCAM reached a higher target to nontarget ratio than (99m)Tc-NHAM. The results indicated that (99m)Tc-NCAM bound to a single site on the NMDA receptor with a K(d) of 701.21 nmol/l and a B(max) of 62.47 nmol/mg. Specific inhibitors of the NMDA receptor, ketamine and dizocilpine, but not the dopamine D(2) and 5HT(1A) receptor partial agonist aripiprazole, inhibited specific binding of (99m)Tc-NCAM to the NMDA receptor. Cell physiology experiments showed that NCAM can increase the viability of SH-SY5Y cells after glutamate-induced injury. CONCLUSIONS: The new radioligand (99m)Tc-NCAM has good affinity for and specific binding to the NMDA receptor, and easily crosses the blood-brain barrier; suggesting that it might be a potentially useful tracer for NMDA receptor expression.

Generation of a liver targeting fusion interferon and its bioactivity analysis in vitro.

The objective of this study was to generate a liver targeting fusion interferon, galactosyl-human serum albumin-interferon alpha2b (G-HSA-IFN) and to evaluate its bioactivity in vitro on HepG2.2.15 cells which express hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). The cell proliferation was determined by Sulpho Rhodamine B (SRB) staining method and flow cytometry (FCM) assay. Hochest33342 and Propidium Iodide (PI) double staining and Western blot analysis of Bcl-2/Bax were also performed to evaluate cell lethality and apoptosis. The concentrations of HBsAg and HBeAg secreted in culture supernatant were detected using Enzyme-Linked Immunosorbent Assay (ELISA). The results demonstrated that G-HSA-IFN could inhibit the proliferation of HepG2.2.15 cells and the cell cycle was arrested at G0/G1 phase. Western blotting results showed that the expression of Bcl-2 was inhibited in a dose-dependent manner while the expression of Bax was enhanced. The expression of HBsAg was inhibited by G-HSA-IFN in a dose-dependent manner, while no significant inhibiting effect on the expression of HBeAg was observed. Conclusively, G-HSA-IFN could not only significantly inhibit the HBsAg expression and the proliferation of HepG2.2.15 cells, but also induce the apoptosis of the target cells, rendering it a promising drug candidate for hepatitis B.

Preparation of 99mTc-PQQ and preliminary biological evaluation for the NMDA receptor.

Pyrroloquinoline quinone (PQQ), an essential nutrient, antioxidant, redox modulator and nerve growth factor found in a class of enzymes called quinoproteins, was labeled with 99mTc by using stannous fluoride (SnF2) method. Radiolabeling qualification, quality control and characterization of 99mTc-PQQ and its biodistribution studies in mice were performed and discussed. Effects of pH values, temperature, time and reducing agents concentration on the radiolabeling yield were investigated. The quality control procedure of 99mTc-PQQ was determined by thin layer chromatography (TLC), radio high-performance liquid chromatography (RHPLC) and paper electrophoresis methods. The average radiolabeling yield was 94 ± 1% under optimum conditions of 0.99 mg of PQQ, 30 µg of SnF2, 0.5 mg of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) and 18.5 MBq of Na99mTcO4 at pH 6 and 25 °C with a response volume of 1 ± 0.1 mL. 99mTc-PQQ was stable and anionic. Lipid-water partition coefficient of 99mTc-PQQ was -1.49 ± 0.16. The pharmacokinetics parameters of 99mTc-PQQ were t1/2α = 18.16 min, t1/2ß = 100.45 min, K12 = 0.013 min-1, K21 = 0.017 min-1, Ke = 0.016 min-1, AUC (area under the curve) = 1040.78 ID% g-1 min and CL (plasma clearance) = 0.096 mL min-1. The dual-exponential equation was Y = 10.88e-0.038t  + 5.21e-0.0069t . The biodistribution of 99mTc-PQQ was studied in ICR (Institute for Cancer Research 7701 Burhelme Are., Fox Chase, Philadelphia, PA 1911 USA) mice. In vitro autoradiographic studies clearly showed that the 99mTc-PQQ radioactivity accumulated predominantly in the hippocampus and cortex, which had a high density of N-methyl-d-aspartate Receptor (NMDAR). The enrichment can be blocked by NMDAR redox modulatory site antagonists-ebselen (EB) and 99mTc-PQQ is therefore a promising candidate for the molecular imaging of NMDAR. To date, however, there have been no studies characterizing 99mTc-PQQ.

Neuroprotective effects of salidroside against beta-amyloid-induced oxidative stress in SH-SY5Y human neuroblastoma cells.

Beta-amyloid (Abeta) peptide, the hallmark of Alzheimer's disease (AD), invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. In this study, salidroside (Sald), an active compound isolated from a traditional Chinese medicinal plant, Rhodiola rosea L., was investigated to assess its protective effects and the underlying mechanisms against Abeta-induced oxidative stress in SH-SY5Y human neuroblastoma cells. Abeta(25-35)-induced neuronal toxicity was characterized by the decrease of cell viability, the release of lactate dehydrogenase (LDH), morphological alterations, neuronal DNA condensation, and the cleavage of poly(ADP-ribose) polymerase (PARP) by activated caspase-3. Pretreatment with salidroside markedly attenuated Abeta(25-35)-induced loss of cell viability and apoptosis in a dose-dependent manner. The mechanisms of salidroside protected neurons from oxidative stress included the induction of antioxidant enzymes, thioredoxin (Trx), heme oxygenase-1 (HO-1), and peroxiredoxin-I (PrxI); the downregulation of pro-apoptotic protein Bax and the upregulation of anti-apoptotic protein Bcl-X(L). Furthermore, salidroside dose-dependently restored Abeta(25-35)-induced loss of mitochondrial membrane potential (MMP) as well as suppressed the elevation of intracellular reactive oxygen species (ROS) level. It was also observed that Abeta(25-35) stimulated the phosphorylation of mitogen-activated protein (MAP) kinases, including c-Jun NH(2)-terminal kinase (JNK) and p38 MAP kinase, but not extracellular signal-regulated kinase1/2 (ERK1/2). Salidroside inhibited Abeta(25-35)-induced phosphorylation of JNK and p38 MAP kinase, but not ERK1/2. These results suggest that salidroside has protective effects against Abeta(25-35)-induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.

Preparation and biological evaluation of a glycosylated fusion interferon directed to hepatic receptors.

The antiviral activity and biodistribution of a glycosylated fusion interferon directed to hepatic receptors were evaluated to determine whether its pharmaceutical concentration in the liver could be improved. The novel glycosylated fusion interferon, galactosyl-human serum albumin-interferon-alpha2b (G-HSA-IFN) was obtained from a long-term recombinant fusion protein (HSA-IFN) by covalent coupling with a bifunctional reagent, 2-imino-2-ethyloxymethy1-1-thiogalactose. There are about 24 thiogalactose residues in each G-HSA-IFN molecular on average. The antiviral activities of IFNalpha2b, HSA-IFN, and G-HSA-IFN were compared in a cytopathic effect inhibition assay with the WISH/VSV system in vitro, and the modification had little effect on its antiviral activity. Both G-HSA-IFN and HSA-IFN were labeled with 125I and the radiochemical purity of 125I-G-HSA-IFN was greater than 96%. 125I-G-HSA-IFN bound to the asialoglycoprotein receptor (ASGP-R) on hepatic cells much more specifically than 125I-HSA-IFN, with specific binding rates of 89.53% and 6.66%, respectively (p < 0.01). Biodistribution research in mice showed that 125I-G-HSA-IFN could concentrate effectively in the liver (>45%/g) and suggested that it also could be a good imaging agent of hepatic receptors.

Expression, purification, and characterization of recombinant human beta-amyloid42 peptide in Escherichia coli.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Evidence indicates that abnormal processing and extracellular deposition of the beta-amyloid42 peptide, the longer form of proteolytic derivative of the transmembrane glycoprotein-amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Since it is convenient and economical to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify beta-amyloid42 using glutathione-S-transferase (GST) fusion system. beta-Amyloid42 gene was inserted into a vector pGEX-4T-1 to construct a GST-fusion protein. The fusion protein GST-beta-amyloid42, expressed in BL21 (DE3) strain, was purified with GSH-affinity chromatography followed by thrombin cleavage. The digested product was further purified with an additional GSH-affinity and a Benzamidine chromatography step. After cleavage and purification, the beta-amyloid42 moiety showed the expected size of 4.5 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blot. Moreover, the fibrillar recombinant beta-amyloid42 exhibited great aggregation activity and showed neurotoxicity on neuron cells in vitro. These results suggest that our method will be useful in obtaining a large quantity of recombinant beta-amyloid42 peptide for further physiological and biochemical studies.

Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells.

Oxidative stress plays an important role in Alzheimer's disease and other neurodegenerative disorders. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L, shows potent antioxidant property. In this paper, the neuroprotective effects of salidroside on hydrogen peroxide (H2O2)-induced apoptosis in SH-SY5Y cells were investigated. Pretreatment with salidroside markedly attenuated H2O2-induced cell viability loss and apoptotic cell death in a dose-dependent manner. The mechanisms by which salidroside protected neuron cells from oxidative stress included the induction of several antioxidant enzymes, thioredoxin, heme oxygenase-1, and peroxiredoxin-I; the downregulation of pro-apoptotic gene Bax and the upregulation of anti-apoptotic genes Bcl-2 and Bcl-X(L). Furthermore, salidroside dose-dependently restored H2O2-induced loss of mitochondrial membrane potential as well as the elevation of intracellular calcium level. These results suggest that salidroside has protective effects against oxidative stress-induced cell apoptosis, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases implicated with oxidative stress.

[Effect and mechanism of recombinant hirudin on fibrinolysis].

AIM: To study the effect of recombinant hirudin (rH) on tPA-induced fibrinolysis and the possible mechanism of its action. METHODS: The effect of rH on thrombin-fibrin complex (Th-Fn) was detected by 99mTc labeled rH. In the in vitro clot lysis, tPA as plasminogen activator, and recalcified plasma as plasminogen resource were used to study the influence of rH on fibrinolysis by detecting TAFIa, D-Dimer and FXIII. RESULTS: In a canine model of femoral artery thrombosis, a clear radioactivity strip was imaged in 30 - 60 min on a part image, and the femoral vein thrombosis developed at 30 min. rH efficiently inhibited clot regeneration. Addition of TM could inhibit clot lysis obviously, and CPI could shorten the delay of clot lysis which due to TAFIa. There was a dose-dependent relationship with TM concentration and TAFI activation. FXIII activation was inhibited by low concentration of rH ( < or = 0.2 u x mL(-1)), and the level of fibrinolysis product, D-Dimer, increased. CONCLUSION: rH could inhibit the thrombin binding to fibrin. rH inhibited the activation of TAFI and FXIII by combining with thrombin which resulted in enhancement of thrombolysis.

[In vitro inhibitory effect of 23-HBA on angiogenesis].

AIM: To study the inhibitory effect of 23-HBA on angiogenesis in vitro. METHODS: The effect of 23-hydroxy butulinic acid (23-HBA) on the in vitro proliferation of human microcapillary endothelial cells(HMECs) was examined by sulfonylrhodamine B (SRB) assay. The effect of 23-HBA on endothelial cell migration, and tubule formation on Matrigel was also observed. The CD31 expression in HMECs was dectected by immunohistochemical staining. RESULTS: The proliferation of HMECs was inhibited significantly by 23-HBA with IC(50) being 40.44 mg/L. 23-HBA inhibited endothelial cell migration and tubule formation in a dose-dependent manner. The expression of CD31 in HMECs was reduced after treatment with 10 mg/L 23-HBA. CONCLUSION: 23-HBA can inhibit angiogenesis in vitro, which would become a promising antiangiogenic drug.