Annexin A5 ; biosynthesis ; isolation & purification ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVE: To establish an LC-MS/MS method for the determination of isoniazid and moxifloxacin in human plasma and pleural effusion. METHODS: The theophylline was used as internal. The protein of samples were precipitated with acetonitrile. The supernatant was separated on an Agilent ZORBAX SB-Aq (4.6 mm×150 mm, 5 μm). The mobile phase was consisted of acetonitrile and 5 mmol·L-1 ammonium acetate(0.1% formic acid solution). Electrospray ionization (ESI) source was applied and operated in the positive multiple reaction monitoring (MRM) mode. The MS/MS ion transition of m/z was 138.1/121.1 for isoniazid, 402.1→384.0 for moxifloxacin and 181.1/124.0 for theophylline. RESULTS: Chromatograms showed no endogenous interfering peaks with blank samples.The calibration curves of isoniazid and moxifloxacin were both linear over the concentration range of 0.1-10 μg·mL-1. Both inter-and intra-day relative standard deviations were less than 7.56%. CONCLUSION: The method is specific, sensitive and accurate, and proved to be suitable for the determination of isoniazid and moxifloxacin in human plasma and pleural effusion samples.

OBJECTIVETo investigate the role of endoplasmic reticulum stress in the apoptosis of testicular germ cells in hyperlipidemic rats.

METHODSWe randomly assigned 42 four-week-old male Wistar rats into a normal control group (n = 12) and a high-fat group (n = 30) to be fed on a normal diet and a high-fat diet, respectively, for 10 weeks. Then we measured the concentrations of triglyceride (TG) and total cholesterol (TC) in the serum using an automatic biochemistry analyzer, detected the apoptosis of testicular germ cells by TUNEL staining, and determined the protein and mRNA expressions of GRP78 and. caspase-12 in the testis tissue by immunohistochemistry and RT-PCR, respectively.

RESULTSThe concentrations of TG and TC were significantly increased in the animals of the high-fat group ([3.00 ± 0.92] and [3.04 ± 0.39] mmol/L) as compared with the control rats ([1.43 ± 0.41] and [1.55 ± 0.23] mmol/L) (P < 0.01), and so was the apoptosis index of the testicular germ cells ([37.17 ± 2.74]% vs [5.16 ± 0.81]%, P < 0.01). The high-fat group, in comparison with the control, also showed remarkably upregulated protein and mRNA expressions of GRP78 (0.32 ± 0.03 and 0.86 ± 0.05 vs 0.19 ± 0.01 and 0.37 ± 0.03, P < 0.01) and caspase-12 (0.34 ± 0.02 and 0.87 ± 0.01 vs 0.12 ± 0.01 and 0.34 ± 0.03, P < 0.01) in the testis tissue.

CONCLUSIONThe apoptosis of testicular germ cells is increased in hyperlipidemic rats, which may be attributed to endoplasmic reticulum stress.

Animals ; Apoptosis ; physiology ; Caspase 12 ; metabolism ; Cholesterol ; blood ; Diet, High-Fat ; adverse effects ; Endoplasmic Reticulum Stress ; physiology ; Heat-Shock Proteins ; metabolism ; In Situ Nick-End Labeling ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Spermatozoa ; pathology ; Staining and Labeling ; Testis ; metabolism ; Transcriptional Activation ; Triglycerides ; blood ; Up-Regulation

Humans ; Infant ; Infant, Low Birth Weight ; Infant, Newborn ; Male ; Tyrosinemias ; diagnosis ; therapy

Objective To evaluate the feasibility of monitoring the gene expression of VEGF165 via the diglycylcysteine (GGC) reporter gene system by reporter probe of 99Tcm-GH. Methods DNA fragments encoding GGC binding motifs were prepared by PCR and positioned at the C end of VEGF165 gene after the linearization of pcDNA3-VEGF165 plasmid. A replication-defective adenovirus vector Ad5-VEGF165GGC motif-internal ribosomal entry site(IRES) -enhanced green fluorescent protein (EGFP) (Ad5-VIE)was constructed, with a cytomegalovirus (CMV) early promoter driving the expression of VEGF165 gene,GGC motif and EGFP, under the aid of an IRFS. A replication-defective adenovirus carrying the Ad5-EGFP was used as the control. Mensenchymal stem cells (MSC) were infected with the recombinant adenovirus at a multiplicity of infection (MOI) from 0 to 100 infectious units (0,10,25,50,100). The cellular uptake of 99Tcm-GH in infected MSC were then studied at 30, 60, 90 and 120 min. VEGF165 was detected by quantitative reverse transcriptase real-time PCR (RT-PCR), Western-blot, and immunohistochemisty. EGFP was observed by RT-PCR and fluorescence microscopy. The correlation analysis was studied between the cellular uptake of 99Tcm-GH and the expression of VEGF165. SPSS 13.0 was applied for statistical analysis. Independent samples t-test, q-test and Pearson correlation coefficient were used. Results After infected with different viral titer of Ad-VIE, the cellular uptake of 99Tcm-GH increased with the increasing virus titer(r2 =0.86, P ＜0.05), with the peak rate (7.94 ±0.75) % at MOI = 100. In time-dependent uptake study, the cellular uptake rates increased rapidly with the time extension, and the highest uptake occurred at 120 min with the peak uptake rate (7.72 ±0.22)%. The uptake rates of 99Tcm-GH in Ad5-VIE-infected cells were significantly higher than those of Ad5 -EGFP-transfected cells at all time points (t = 15.10- 54.92, all P ＜0.05). The VEGF165 and EGFP mRNA levels increased with increasing virus titer, and the VEGF165 mRNA correlated well with the EGFP mRNA(r2 = 0. 99, P ＜ 0.05). After infected with different MOI of Ad5-VIE, good relationship was found between the cellular uptake of 99Tcm-GH and the expression of VEGF165protein in MSC(r2 =0.90, P ＜0.05). Inmunohistochemisty showed VEGF165 protein expressed obviously at Ad5-VIE-infected MSC, and the EGFP was observed by fluorescence microscopy. Conclusions The cellular uptake of 99Tcm-GH in Ad5-VIE-infected MSC are well correlated with the expression of VEGF165 in vitro. The expression of therapeutic gene VEGF165 can be monitored by the GGC peptide expression.

The cloning of promoter is important for studying the genetic engineering and the regulation of gene expression in plants. Two promoters Os772 and Os359, which are predicted to be highly expressed in the endosperm of rice from the EST database were cloned. After construction of the Os772∶∶GUS and Os359∶∶GUS expression vectors, they were transformed into rice. X-Gluc staining of transgenic plants showed that Os772 and Os359 can promote GUS gene expression in matured endosperm but not in root, stem, leaf and flower. This result indicates Os772 and Os359 are two rice endosperm-specific promoters.

Objective To evaluate the roles of magnetic resonance imaging and proton magnetic resonance spectroscopy(1H-MRS) in the diagnosis of meningiomas. Methods 98 patients with meningiomas underwent conventional pre-contrast MR and contrast MR. Among them, 28 cases had two dimensional single voxel or multi voxel 1 H-MRS simultaneously both in the lesion's region and the contralateral side. Results On precontrast MR images of 98 cases, T1 WI showed 58.1% (61/105) isointensities, 31.4% (33/105) faintly low intensities and 10. 5% (11/105) mixed intensities; T2WI showed 40. 0% (42/105) isointensities, 41.0%(43/105) hyperintensities, 10.5% (11/105) faintly low intensities and 8.5% (9/105) mixed intensities. After administration of Gd-DTPA, the solid part of the tumors exhibited various enhancement in all the 98 cases.28 cases of MRS exhibited specific different spectral peaks, including increased of choline-containing compounds(Cho), absent or decreased of acetylaspartate(NAA), and the unchanged of creatine(Cr). The value of NAA, Cr, Cho, NAA/Cr, Cho/Cr, NAA/Cho in the tumor center of meningioma were 0. 09 ± 0.06,0.31 ± 0. 22, 0.46 ± 0. 16, 0.33 ± 0. 42, 1.50 ± 0. 68, 0. 15 ± 0.08, compared with the contralateral normal region, Cr has no significant difference (P ＞ 0. 05), NAA, Cho, NAA/Cr, Cho/Cr, NAA/Cho had significantly differences(P ＜ 0.05). Conclusion Conventional pre-contrast MR and contrast MR is the most important dignostic means for meningiomas, 1H-MRS combined with MRI can improve the diagnostic accuracy of meningiomas.

Objective The expression and impaired function of ion channels might be one of the pathophysiological mecha -nisms responsible for diarrhea in inflammatory bowel disease ( IBD) .Proper animal model is the key to explore detailed pathophysiolog-ical process.The purpose of this study was to build a rat model of acute colitis induced by dextran sodium sulfate (DSS) in C57BL/6 mice and evaluate diarrhea-associated clinical , histological , pathological parameters and expressions of ion channel protein . Methods C57BL/6J mice of model group were treated with 4%DSS solution for 7 days to induce acute colitis.Mice body weight, stool moisture, stool consistency and the degree of hematochezia were recorded .The histopathological changes of mice colon specimens were observed visually and microcosmically, and the ion channel SLC26A3 protein was detected by Western Blot . Results All experimental mice survived.In the experiment, compared with control group , bloody diarrhea and weight lose occurred in model group , along with increased stool moisture ([73.30 ±8.31]% after experiment vs [44.32 ±6.42]% before experiment, P=0.004), and rapidly in-creased disease activity index (DAI) of acute colitis ([3.50 ±0.87] after experiment vs [1.0 ±0.00] before experiment, P=0.000).At the end of this experiment , compared with control group , the model group resulted in higher colonic damage score and pathological inflammation score (P=0.00, P=0.002), significantly shortened co-lon (P=0.00) and decreased expression of SLC26A3. Conclusion The intestinal mucosal injury and phenotypic features of 4%DSS-induced acute colitis are very similar to those of human ulcerative colitis .Impaired expression of intestinal ion transporter SLC26 A3 coexists with diarrhea in model group mice , and this model can support the research on mechanism of functional changes of ion channels in inflammatory diarrhea .

BACKGROUND:Preliminary studies of our research group mainly focus on the role of nucleus of solitary tract in gastric noxious stimulation and acupuncture point, while dorsal nucleus of vagus nerve, nucleus of solitary tract and area postrema are the necessary component for vagus nerve complex, and dorsal nucleus of vagus nerve plays a crucial role in the regulation of gastric functions. OBJECTIVE:To observe the effect of acupuncture at Zusanli, Neiguan, Pishu and Shenshu acupoints on the gastric-related neurons in dorsal motor nucleus of the vagus nerve. METHODS:The gastric related neurons in dorsal motor nucleus of the vagus nerve of gastric distension rats were identified using extracellular recording technique, according to neuroelectrophysiology method. Acupuncture stimulation was given to Zusanli, Neiguan, Pishu and Shenshu for 15 seconds. The effect of body stimulation on the gastric distension related neurons in dorsal motor nucleus of the vagus nerve of rats was recorded. RESULTS AND CONCLUSION:Among the involved 24 rats, 90 neurons at paraventricular hypothalamic necleus exhibited discharge. Among the discharged neurons, 54 neurons were related to gastric distension. After acupuncture stimulation at Zusanli, Neiguan, Pishu and Shenshu, there were 44, 39, 38, 27 neurons giving a response, the reaction rate was 82%, 72%, 70%and 50%, respectively. Acupunture at different nerve segment acupoints can activate the dorsal motor nucleus of the vagus nerve to different degrees, which is involved in the regulation of gastric motility.

BACKGROUND:Previous studies concerning neuromechanism of acupuncture for regulating gastric function mainly focused on peripheral nerve, involving in central nervous system. In particular, there are few studies addressing higher central nervous system.
OBJECTIVE:To explore the effects of acupuncture at Neiguan (PC6) and Zusanli (ST36) on gastric-related neurons in the hypothalamic paraventricular nucleus.
METHODS:Using microelectrode extracellular recording technique, gastric related neurons in the paraventricular nucleus were found in gastric distension rats. Stimulation was conducted for 30 seconds by hand acupuncture at Neiguan and Zusanli. The effects of acupuncture on gastric-related neurons in the paraventricular nucleus were observed.
RESULTS AND CONCLUSION:The discharge of 109 neurons in the hypothalamic paraventricular nucleus of 60 rats was recorded. A total of 56 gastric-related neurons of the 109 neurons were observed. The number of neurons response to acupunctures at Zusanli, Neiguan, Pishu (BL20) and Weishu (BL21) were respectively 44, 47, 29, and 33, with the reaction proportion of 78.57%, 83.47%, 51.79%and 58.93%. Results suggested that there exist gastric distension stimulation and acupuncture stimulations from neurons soma to visceral reaction in the hypothalamic paraventricular nucleus, which simultaneously participates in the regulation of acupuncture on the gastric function.