[In vitro co-cultivation of Toxoplasma gondii tachyzoites with rat brain astrocytes].

Purified astrocytes were cultured in plates. When astrocytes grew over 80% of the plate, tachyzoites of Toxoplasma gondii RH strain were added for co-culture. In the period of 0-72 h, change of the astrocytes and tachyzoites was observed after Giemsa staining. In 0-48 h, monodansylcadaverine (MDC) was used to study the action of autophagy in the process of tachyzoites invading astrocytes. At 1 h co-culture, tachyzoites had entered in astrocytes and the autophagosomes appeared. At 4 h, the autophagosomes increased pronouncedly. However, after 12 h, number of autophagosomes considerably decreased and damage of the cells occurred. 48 h later, autophagosomes disappeared and more astrocytes were destroyed. At 72 h most cells destroyed and tachyzoites were released. The result showed that autophagy is inhibited when the astrocytes were in vitro infected by tachyzoites.

Aqueous extracts of Tribulus terrestris protects against oxidized low-density lipoprotein-induced endothelial dysfunction.

OBJECTIVE: To investigate the role of aqueous extracts of Tribulus terrestris (TT) against oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) dysfunction in vitro. METHODS: HUVECs were pre-incubated for 60 min with TT (30 and 3 µg/mL respectively) or 10(-5) mol/L valsartan (as positive controls) and then the injured endothelium model was established by applying 100 µg/mL ox-LDL for 24 h. Cell viability of HUVECs was observed by real-time cell electronic sensing assay and apoptosis rate by Annexin V/PI staining. The cell migration assay was performed with a transwell insert system. Cytoskeleton remodeling was observed by immunofluorescence assay. The content of endothelial nitric oxide synthase (eNOS) was measured by enzyme-linked immunosorbent assay. Intracellular reactive oxygen species (ROS) generation was assessed by immunofluorescence and flow cytometer. Key genes associated with the metabolism of ox-LDL were chosen for quantitative real-time polymerase chain reaction to explore the possible mechanism of TT against oxidized LDL-induced endothelial dysfunction. RESULTS: TT suppressed ox-LDL-induced HUVEC proliferation and apoptosis rates significantly (41.1% and 43.5% after treatment for 3 and 38 h, respectively; P<0.05). It also prolonged the HUVEC survival time and postponed the cell's decaying stage (from the 69th h to over 100 h). According to the immunofluorescence and transwell insert system assay, TT improved the endothelial cytoskeletal network, and vinculin expression and increased cell migration. Additionally, TT regulated of the synthesis of endothelial nitric oxide synthase and generation of intracellular reactive oxygen species (P<0.05). Both 30 and 3 µg/mL TT demonstrated similar efficacy to valsartan. TT normalized the increased mRNA expression of PI3Kα and Socs3. It also decreased mRNA expression of Akt1, AMPKα1, JAK2, LepR and STAT3 induced by ox-LDL. The most notable changes were JAK2, LepR, PI3Kα, Socs3 and STAT3. CONCLUSIONS: TT demonstrated potential lowering lipid benefits, anti-hypertension and endothelial protective effects. It also suggested that the JAK2/STAT3 and/or PI3K/AKT pathway might be a very important pathway which was involved in the pharmacological mechanism of TT as the vascular protective agent.

[Y-chromosome-specific microsatellite variation in Li ethnic groups of Hainan Island, China].

OBJECTIVE: The study was conducted to reveal the distribution of genetic polymorphism of four Y chromosome specific short tandem repeat (Y-specific STR) loci in Li ethnic groups in Hainan Island, China. METHODS: Four tetranucleotide STR loci were simultaneously amplified with fluorescently labeled primers, and genotypes were determined with an automated DNA sequencer. RESULTS: Among 230 unrelated males, the alleles at the four Y-specific STR loci were composed of some complex repeat structure. 4,5,4,5 alleles were observed in loci DYS3891, DYS390, DYS391, DYS393 respectively. A set of human allele ladders for the typing of the four Y-specific STRs was obtained in Li ethnic population. Gene diversity index (D) and haplotype diversity data were estimated for the four Y-STRs. CONCLUSION: The preliminary study indicates a reference population for detecting male migration events and should be useful in population genetics and forensic applications.

[Polymorphism analysis for DYS19 and DYS287 of three Li populations in Haina Island, China].

The distribution of Y-chromosome specific microsatellite DYS19, in 289 males of three ethnic populations in Hainan Island was studied with the method of PCR followed by denatured polyacrylamide gel electrophoresis and silver staining. The three ethnic populations are Bendi Li, Qi Li and Ha Li. The results showed that four alleles (190bp, 194bp, 198bp, 202bp designated as alleles B, C, D and E respectively), were observed in DYS19, the frequency ranged from 0.660-0.854, and was predominant in three Li subgroups. The pairwise comparison with Fisher's Exact test reveals that there exists significant difference (P<0.01) in DYS19 phenotype distribution between Qi Li and Ha Li populations. The polymorphism of the Y-chromosome specific Alu insert sequence DYS287 was investigated also in three Li populations. The results demonstrated that with 150 bp product in all Bendi-Li, Qi-li and Ha-li, YAP element is absent. The preliminary study provides reference populations for detecting male migration events and for reconstructing paternal history.

A colloidal gold probe-based silver enhancement immunochromatographic assay for the rapid detection of abrin-a.

High-affinity anti-abrin-a monoclonal and polyclonal antibodies were used to develop a sandwich immunochromatographic assay and silver enhancement technology was used to further increase the sensitivity. Using a matrix of double distilled water or soybean milk with added abrin-a, the visual detection limit was found to be 10 ng mL(-1). The detection limit was 0.1 ng mL(-1) for abrin-a, an increase in sensitivity of 100-fold when the silver enhancement technology was employed. The assay was portable and very simple to perform and the detection was completed within 20 min without complicated handling procedures. There was no significant cross-reactivity with several homologous toxins and associated agglutinin. The assay reagents could be stored for 12 weeks at 4°C without significant loss of activity. These characteristics make the strip assay to be an ideal candidate for the development of a rapid toxin detection kit.

Preparation and identification of monoclonal antibody against abrin-a.

BALB/c mice were immunized four times with formalin-prepared abrin-a. Using the polyethylene glycol method, immunized splenocytes were isolated and fused with SP2/0 cells. An indirect ELISA was established and used to detect positive clones secreting monoclonal antibodies (mAbs) against abrin-a. After analysis, three hybridoma clones secreting IgG-subtype mAbs were obtained. The antibodies were purified from the hybridoma growth medium using protein A or G affinity chromatography. Western blot analysis was used to analyze the antigenic epitopes on abrin-a recognized by the mAbs. The mAbs were specific for abrin-a, with no detectable cross-reactivity with several homologous toxins and associated agglutinins. Sandwich ELISA was then developed using these mAbs, which had a detection limit for abrin-a of 7.8 ng/mL.