Correlations of AQP4 expression and polymorphism with diabetic retinopathy.

OBJECTIVE: The aim of this study was to investigate the relationships of aquaporin 4 (AQP4) rs200498749, rs149465 and rs650217 polymorphisms and gene expression with diabetic retinopathy (DR). PATIENTS AND METHODS: A total of 400 patients with diabetes mellitus (DM) treated in our hospital were enrolled in this study. All subjects were divided into two groups, including DM group (n=200, without DR) and DR group (n=200, with DR). The polymorphisms rs200498749, rs149465 and rs650217 of AQP4 gene were analyzed in the two groups. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect gene expression, and statistical analysis was performed in combination with clinical data. RESULTS: The distribution of alleles of AQP4 rs650217 (p=0.015) in DR group was different from that in DM group, and the frequency of T allele was significantly higher in DR group than DM group. The distribution of genotypes of AQP4 rs149465 (p=0.000) and rs650217 (p=0.000) showed statistically significant difference between DR group and DM group. The frequency of AA genotype of polymorphism rs149465 and CT genotype of polymorphism rs650217 was significantly higher in DR group than DM group. Besides, there was a difference in the distribution of recessive models of AQP4 rs149465 (p=0.023) and rs650217 (p=0.014) between DR group and DM group. DR group exhibited remarkably lowered frequency of AA + AT recessive model of the polymorphism rs149465 and raised frequency of CT + TT recessive model of the polymorphism rs650217. Similarly, a difference was found in the distribution of haplotypes CAT (p=0.014) and CTC (p=0.003) of AQP4 rs200498749, rs149465 and rs650217 between DR group and DM group. The polymorphism rs200498749 of AQP4 gene was significantly correlated with AQP4 gene expression (p<0.05). Meanwhile, the expression of AQP4 gene was clearly higher in patients with CC genotype in DR group (p<0.05). AQP4 polymorphism rs200498749 was related to fasting blood glucose (p=0.000) and hemoglobin A1c (HbA1c) (p=0.000) in DR group, and polymorphism rs650217 had an association with serum creatinine level (p=0.034). AQP4 polymorphisms rs149465 (p=0.023) and rs650217 (p=0.042) were correlated with clinical stage in DR group. In addition, the proportion of patients with AA genotype of rs149465 at stage VI and with TT genotype of rs650217 at stage I rose significantly (p<0.05). CONCLUSIONS: AQP4 gene polymorphism has a potential relationship with the susceptibility and progression of DR.

Nigari (deep seawater concentrate) enhances the treadmill exercise performance of gerbils.

The beneficial effect of magnesium supplementation on exercise performance has been reported by many researchers. In the present study, the effect of nigari, a concentrate of deep seawater containing high magnesium levels, on exercise performance, was examined. Gerbils were given double-distilled water or nigari (18 mg · kg(-1), po) orally 30 min before exercise. All animals were subjected to forced exercise on a treadmill for 90 min at three successive speeds of 10, 15, and 20 m · min(-1). The retention numbers were recorded. The retention numbers were 85.0 ± 21.0, 46.0 ± 9.7, and 48.0 ± 14.2 in the control group, and 44.0 ± 10.9, 23.0 ± 8.4, and 13.0 ± 4.8 in the nigari-treated group at the three speeds, respectively. The retention numbers were significantly reduced at higher speeds (by 50% at 15 and 73% at 20 m · min(-1), respectively) in the nigari-treated group when compared to those of the control group, respectively. Thus, nigari administration appeared to reduce retention numbers and enhance exercise performance in gerbils.

Determination of hepatitis B surface antigen using magnetic immunoassays in a thin channel.

We report novel methods for detection of hepatitis B surface antigen (HBsAg) based on competitive and sandwiched magnetic immunoassays using functional magnetic nanoparticles in a thin channel. Magnetic nanoparticles labeled with hepatitis B antibody are flowed through a thin channel to form a predeposition layer for capturing HBsAg. Competitive and sandwiched magnetic immunoassays were studied and detection limit, linear range, and sample selectivity were compared. The detection limits of competitive and sandwiched magnetic immunoassays were found to be 0.26 and 0.25 pg/ml, respectively. The linear range of HBsAg concentration was 0.26 pg/ml-2.6 ng/ml for competitive magnetic immunoassay and was 0.89 pg/ml-8.9 ng/ml for sandwiched magnetic immunoassay. The advantages of these methods over ELISA and other methods for HBsAg detection are lower detection limits and wider linear ranges. The running time was less than 30 min. Competitive magnetic immunoassay was faster than sandwiched magnetic immunoassay for detection of HBsAg. The measurements of HBsAg in serum samples from these methods differed by about 10% from those of ELISA. These methods can provide simple, fast, and sensitive detections of biomarkers and other immunoassay-related samples.

The impact of a leptin gene SNP on beef calf weaning weights.

Prior research indicates that a SNP at position 305 of exon 2 in the leptin gene affects milk production in dairy cows. Dairy cows with at least one copy of the T allele have been shown to have higher milk production than CC cows. If that effect carries over to beef breeds, it is reasonable to expect that CT and TT beef cows will wean heavier calves than CC beef cows. We tested this hypothesis for a herd of mixed breed cows using anova. Results indicated that both crossbred CT and TT beef cows wean significantly heavier beef calves than CC crossbred beef cows. A lack of observations generally hinders detection of significance in other breeds. However, two other comparisons were found to be significant. The results suggest further investigation into the link between leptin genotype and calf weaning weights. Aside from interest to animal scientists, these results have the potential to alter mating and replacement selection decisions by cow-calf producers, given the importance of weaning weights on profitability.

Pharmacokinetic study of levofloxacin in rat blood and bile by microdialysis and high-performance liquid chromatography.

The aim of this study was to develop a rapid and sensitive method for the simultaneous determination of unbound levofloxacin in rat blood and bile using high-performance liquid chromatography coupled with microdialysis for further pharmacokinetic study. Microdialysis probes were simultaneously inserted into the jugular vein toward the right atrium and the bile duct of male Sprague-Dawley rats for biological fluid sampling after administration of levofloxacin 3 mg/kg through the femoral vein. Levofloxacin and dialysates were separated using a Merck LiChrospher reversed-phase C18 column maintained at ambient temperature. The mobile phase was comprised of acetonitrile-1 mM 1-octanesulfonic acid (40:60, v/v, pH 3.0 adjusted with orthophosphoric acid). The fluorescence response for levofloxacin was observed at excitation and emission wavelengths of 292 and 494 nm, respectively. The detection limit of levofloxacin was 50 ng/ml. Intra-day and inter-day precision and accuracy of levofloxacin measurements fell well within the predefined limits of acceptability. The disposition of levofloxacin in the blood and bile fluid suggests that there was rapid exchange and equilibration between the blood and hepatobiliary systems, and the plasma level of levofloxacin was greater than that of the bile. Thus, levofloxacin undergoes hepatobiliary excretion but might not be related to the P-glycoprotein transport system.

Determination and pharmacokinetic profile of omeprazole in rat blood, brain and bile by microdialysis and high-performance liquid chromatography.

The disposition and biliary excretion of omeprazole was investigated following i.v. administration to rats at 10 mg/kg. We used a microdialysis technique coupled to a validated microbore HPLC system to monitor the levels of protein-unbound omeprazole in rat blood, brain and bile, constructing the relationship of the time course of the presence of omeprazole. Microdialysis probes were simultaneously inserted into the jugular vein toward right atrium, the brain striatum and the bile duct of the male Sprague-Dawley rats for biological fluid sampling after the administration of omeprazole (10 mg/kg) through the femoral vein. The concentration-response relationship from the present method indicated linearity (r2>0.995) over a concentration range of 0.01-50 microg/ml for omeprazole. Intra-assay and inter-assay precision and accuracy of omeprazole fell well within the predefined limits of acceptability. Following omeprazole administration, the blood-to-brain coefficient of distribution was 0.15, which was calculated as the area under the concentration versus time curve (AUC) in the brain divided by the AUC in blood (k=AUCbrain/AUCblood). The blood-to-bile coefficient of distribution (k=AUCbile/AUCblood) was 0.58. The decline of unbound omeprazole in the brain striatum, blood and bile fluid suggests that there was rapid exchange and equilibration between the compartments of the peripheral and central nervous systems. In addition, the results indicated that omeprazole was able to penetrate the blood-brain barrier and undergo hepatobiliary excretion.

General pharmacological properties of Sho-seiryu-to (TJ-19) extracts.

The general pharmacological properties of TJ-19 extracts were orally investigated in various experimental animals. TJ-19 extracts showed no effect on general behavior and on central nervous system such as spontaneous locomotor activity, proconvulsant and anti-convulsant responses, analgesic activity, body temperature and hexobarbital sleeping time at all doses of 0.5, 1 and 2 g/kg in mice. Further, TJ-19 extracts showed no effect on contractile responses of isolated guinea pig ileum induced by acetylcholine, histamine and BaCl2 at concentrations of 10(-6), 10(-5), and 10(-4) g/ml. TJ-19 extracts, however, increased the respiratory rate, heart rate, blood pressure, systolic pressure, diastolic pressure, and decreased the blood flow in dogs at all doses of 0.5, 1 and 2 g/kg via duodenal administration. Further, TJ-19 extracts decreased the interval of PR and QT of EKG parameters in dogs at doses of 1 and 2 g/kg. TJ-19 extracts increased the intestinal transport of charcoal meal in rats at doses of 1 and 2 g/kg. TJ-19 increased the urinary Na+ excretion at all doses of 0.5, 1, and 2 g/kg, and increased the urinary K+ and Cl- excretion at 1 and 2 g/kg, although it showed no effect on urine volume output in rats. These data suggest that TJ-19 stimulates the sympathetic nervous system function at a pharmacological dose of under 0.5 g/kg, and has possibility to increase the intestinal peristalsis and urinary electrolyte excretion at higher doses.

Simultaneous monitoring of extracellular glucose, pyruvate, and lactate in rat testies during ischemia: a microdialysis study.

A dual-probe microdialysis technique was developed for the simultaneous monitoring of glucose and related metabolite levels during testicular ischemia in rats. The determinations of lactate and pyruvate were achieved by liquid chromatography within 5 min. Glucose was determined by a microdialysis analyzer. A unilateral ligation was produced by occlusion of the right artery for 2 or 4 h in anesthetized rats. Microdialysis probes were inserted in both sides of the testis to simultaneously monitor glucose, lactate and pyruvate during basal, ischemia and reperfusion periods. Dynamic and comparative changes in these analytes in ipsilateral and contralateral testes were demonstrated. The present technique can be used as a tool for exploring energy related metabolites and their relationships in testicular ischemia.

Synthesis of 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole analogues as novel antiplatelet agents.

1-Benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (28, YC-1) was selected as the lead compound for systemic structural modification. After screening for antiplatelet activity, SARs of YC-1 analogues were established. Among these potent active derivatives, compounds 29, 30, 31, 44, and 45 functioned as potent activators of sGC and inhibitors of PDE5 with potency comparable to that of YC-1. In addition, compound 58 was found to be a selective and potent inhibitor of protease-activated receptor type 4 (PAR4)-dependent platelet activation.

Systemic administration of a water-soluble hexasulfonated C(60) (FC(4)S) reduces cerebral ischemia-induced infarct volume in gerbils.

Buckminsterfullerence and its derivatives have recently been shown to exhibit considerable in vivo biological activities. A water-soluble hexasulfonated C(60) (FC(4)S) has been shown to protect against oxidative stress. Neuroprotective effects of FC(4)S were investigated in the present study. Focal cerebral ischemia was produced by a permanent occlusion of the right middle cerebral artery in gerbils. Infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride transcardiac perfusion 24 h after cerebral ischemia. Chronic pretreatment of FC(4)S (0.5 and 5.0 mg/kg/day, intraperitoneally for 2 weeks) significantly reduced the infarct volume (by 42% and 68%, respectively) when compared to that of the control group. Results revealed that chronic pretreatment of FC(4)S may protect the brain against focal cerebral ischemia.

On-line microdialysis coupled with microbore liquid chromatography with ultraviolet detection for continuous monitoring of free cefsulodin in rat blood.

A microdialysis method followed by a microbore liquid chromatographic ultraviolet detection procedure has been performed for the assay of unbound cefsulodin in rat blood. A microdialysis probe was inserted into the jugular vein for blood sampling. This method involves an on-line design for submitting dialysate into the liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol-100 mM monosodium phosphoric acid (10:90, v/v, pH 5.0) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. Detection wavelength was set at 265 nm. Microdialysis probes, being laboratory-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity as well as intra- and inter-day variabilities. The method was then applied to pharmacokinetics profiling of cefsulodin in the blood following intravenous administration of cefsulodin (20 mg/kg) in rats. Pharmacokinetics were calculated from the corrected data for dialysate concentrations of cefsulodin versus time. Based on pharmacokinetic calculation, cefsulodin best fitted to a two-exponential disposition. This study provided specific pharmacokinetic information for protein-unbound cefsulodin and demonstrated the applicability of this continuous sampling method for pharmacokinetic study.

Determination of pyruvate and lactate in primary liver cell culture medium during hypoxia by on-line microdialysis-liquid chromatography.

A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia. It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber. The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium. Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate. The detection limit of this assay was 0.2-2.0 microM with acceptable intra- and inter-assay reproducibilities. In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator. The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6+/-5.6 and 607+/-143 microM, respectively. These levels drastically changed during hypoxia and reperfusion. In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation.

Simultaneous monitoring of extracellular glucose, pyruvate, lactate and glutamate in gerbil cortex during focal cerebral ischemia by dual probe microdialysis.

The aim of this study was to monitor dynamic changes in energy-related metabolites in the cortex of gerbils subjected to cerebral ischemia by a dual probe microdialysis technique. Focal cerebral ischemia was produced in anesthetized gerbils by occlusion of the right common carotid artery and the right middle cerebral artery for 60 min. Two microdialysis probes were inserted into both sides of the cortex to simultaneously monitor extracellular glucose, lactate, pyruvate and glutamate. Dynamic and comparative changes in these analytes, on the ipsilateral and contralateral sides of the brain, were simultaneously monitored by liquid chromatography and a microdialysis analyzer. The present study demonstrated decreases in glucose and pyruvate, increases in lactate and glutamate on the ipsilateral side whereas all analytes remain constant on the contralateral side of cortex during cerebral ischemia. In vitro recovery of each microdialysis probe was performed to ensure the quality of experiments. The detection limits of pyruvate, glutamate, lactate and glucose were 0.2, 1.0, 2.0 and 20 microM, respectively. The intra- and inter-assay correlations were less than 5% in standard mixtures and pooled brain dialysates.

Determination of unbound cefamandole in rat blood by microdialysis and microbore liquid chromatography.

To analyze unbound cefamandole in rat blood, a method combing microdialysis with microbore liquid chromatography has been developed. A microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats to examine the unbound cefamandole level in the rat blood following cefamandole administration (50 mg/kg, i.v.). The dialysates were directly submitted to a liquid chromatographic system. Samples were eluted with a mobile phase containing acetonitrile-methanol-100 mM monosodium phosphate (pH 5.0; 15:20:65, v/v). The UV wavelength was set at 270 nm for monitoring the analyte. Using the retrograde method, at infusion concentrations of 1 microg/mL of cefamandole, the in vivo microdialysis recoveries were 55.44% for the rat blood (n = 6). Intra- and inter-assay accuracy and precision of the analyses were < or = 10% in the range of 0.1-10 microg/mL. Pharmacokinetic parameters were calculated from the recovery-corrected dialysate concentrations of cefamandole vs time data. The elimination half-life (t1/2,beta) was 21.6 +/- 1.6 min. The results suggest that the pharmacokinetics of unbound cefamandole in blood following cefamandole administration (50 mg/kg, i.v., n = 5) fit best to the two-compartmental model.

A dual-probe microdialysis study in simultaneously monitoring extracellular pyruvate, lactate, and biogenic amines in gerbil striata during unilateral cerebral ischemia.

A dual-probe microdialysis technique coupled with liquid chromatographic assays was developed for the simultaneous monitoring of neurochemicals in gerbil striata during cerebral ischemia. Isocratic separation of lactate and pyruvate was achieved within 5 min whereas the separation of biogenic amines was completed within 30 min. An unilateral ligation was produced by occlusion of the right common carotid artery for 30 mins in anesthetized gerbils to perform a typical focal cerebral ischemia. Microdialysis probes were inserted in both sides of the striata to simultaneously monitor biogenic amines, lactate and pyruvate during cerebral ischemia. Dynamic and comparative changes of these analytes in ipsilateral and contralateral sides of the brain can be simultaneously measured by the assay. The present assay can be used as a research tool to explore neurochemical substances and their relationships during cerebral ischemia.

Differential alteration of catecholamine release during chemical hypoxia is correlated with cell toxicity and is blocked by protein kinase C inhibitors in PC12 cells.

Release of neurotransmitters, including dopamine and glutamate, has been implicated in hypoxia/ischemia-induced alterations in neuronal function and in subsequent tissue damage. Although extensive studies have been done on the mechanism underlying the changes in glutamate release, few have examined the mechanism that is responsible for the changes in catecholamines. Rat pheochromocytoma-12 (PC12) cells synthesize, store, and release catecholamines including DA and NE. Therefore, we used HPLC and ED to evaluate extracellular DA and NE concentrations in a medium during chemical hypoxia in PC12 cells. Chemical hypoxia produced by KCN induced differential release of DA and NE. Under normal glucose conditions, KCN induced release of NE, but not DA. Under glucose-free conditions, KCN-induced release of DA was elevated transiently, whereas the release of NE increased progressively. Under parallel conditions, KCN biphasically elevated the level of cytosolic free calcium ([CA(2+)](i)) in glucose-free DMEM, peaking at 95 +/- 18 nM at 1,107 +/- 151 s, followed by a new plateau level at 249 +/- 24 nM sustained from 4,243 +/- 466 to 5,263 +/- 440 s. Cell toxicity, as measured by LDH release, was increased significantly by KCN in glucose-free DMEM but was diminished in the presence of glucose, and was correlated with DA release by chemical hypoxia. The protein kinase C (PKC) inhibitor GO6976 or staurosporine inhibited KCN-induced LDH release as well as the release of NE and DA. Taken together, selective activation of DA but not NE was correlated with the LDH release by chemical hypoxia, and was diminished with GO6976 or staurosporine. These results suggest that selective activation of PKC isoforms is involved in the chemical hypoxia-induced DA release, which may lead to neuronal cell toxicity.

Simultaneous determination of unbound cefoperazone in rat blood and brain using microdialysis.

A sensitive microbore HPLC method was developed for the simultaneous determination of unbound cefoperazone in rat blood and brain using microdialysis. Two microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats. Cefoperazone (50 mgkg(-1), i.v.) was then administered via the femoral vein. Blood and brain dialysates were collected and eluted with a mobile phase containing methanol-100 mM monosodium phosphoric acid (30:70, v/v, pH 5.5). The wavelength of the UV detector was set at 254 nm. The detection limit of cefoperazone was 20 ng mL(-1). Isocratic separation of cefoperazone was achieved within 10 min. The intra- and inter-assay accuracy and precision of the analyses were < or =10% in the range of 0.05-10 microg mL(-1). The ratio of the area under the concentration curve of cefoperazone in rat brain and blood was estimated to be about 7-8%. It is concluded that cefoperazone is capable of penetrating the blood-brain barrier.

Effects of naloxone on lactate, pyruvate metabolism and antioxidant enzyme activity in rat cerebral ischemia/reperfusion.

Whether naloxone may modulate energy metabolism and endogenous antioxidant enzyme activities in ischemic cortex was studied. Cerebral ischemia/reperfusion (I/R) was produced by occluding two common carotid arteries and the right middle cerebral artery for 90 min followed by reperfusion in anesthetized Sprague-Dawley rats. Both pre-treatment (0.03 or 0.3 mg) and post-treatment (0.3 mg) of naloxone by intracerebroventricular infusion significantly reduced cortical infarct volumes. Pre-treatment with 0.03 mg reduced ischemia-induced suppression of extracellular pyruvate level and enhancement of lactate/pyruvate ratio as well as cerebral I/R-induced increases of endogenous catalase, glutathione peroxidase, and manganese superoxide dismutase activities. In conclusion, neuroprotective effects of naloxone in terms of reducing brain infarction involve attenuation of the disturbance of cellular functions following cerebral I/R via restoration of mitochondrial activities or energy metabolism.

Determination of sunscreen agents in cosmetic products using microwave-assisted extraction and liquid chromatography.

Microwave-assisted extraction (MAE), was used to extract sunscreen agents from cosmetic products. The extracts were analyzed by liquid chromatography (LC). The present method allows the determination of three sunscreen agents, Eusolex 2292, 4360 and 6300. The precision of the assay at 40 microg/ml of sunscreen agents ranged from 1.5 to 2.2%, and the detection limits were 2.0-4.0 ng/ml.

Monitoring of extracellular pyruvate, lactate, and ascorbic acid during cerebral ischemia: a microdialysis study in awake gerbils.

In vivo microdialysis coupled with liquid chromatography was developed for the continuous monitoring of brain neurochemicals during cerebral ischemia in awake, free moving gerbils. The dead volume of the microdialysis system was estimated to be less than 30 microl. The detection limits of the present assay were 0.2 to 2.0 microM for analytes at a signal to noise ratio of five. To validate this assay, a focal cerebral ischemia was produced by occlusion of one common carotid artery for 60 min and then reperfusion for additional 3 h in awake gerbils. A microdialysis probe was inserted into the striatum of the gerbil. Dialysates were autoinjected and analyzed extracellular pyruvate, lactate, and ascorbic acid by liquid chromatography with a UV detector during cerebral ischemia. Significant changes of pyruvate and the lactate/pyruvate ratio were observed. Biphasic and dynamic changes in ascorbic acid and lactate were proposed to correlate a secondary damage. This assay can be used as a tool to study dynamic changes of brain neurochemicals in awake animals.