AIM:To ascertain the species status of the Anopheles dirus from Hainan Province,Chi- na.METHODS:The nucleotide sequence of the second internal transcribed spacer(ITS2 ) of PCR- amplified r DNA was determined for the An.dirusspecimens which included 2 individu- als of the species A colony (AFRIMS) from Thailand and 5 individuals from Hainan Province.RESUL TS:A841 bp fragment was amplified from single mosquito.The fragment included the ITS2 and small portions of flanking 5 .8S (96 bp) and 2 8S (2 9bp) genes.The ITS2 was71 6 bp in length,the sequence was identical for all7individuals from both Hainan Province and Thailand.No evidence of intraspecies or intrapopulation variation was detected in the ITS2 and flanking regions.CONCL USION:The result suggests the existence of An. dirus A in Hainan Province,which was in agreement with previous studies from cytogenetic analysis and egg characteristics determined by scanning electron microscopy.

Mitotic chromosomal karyotypes and their heterochromatin banding of thirteen anopheline mosquitoes in China were observed. Five species belonging to subgenus Cellia were Anopheles maculatus, An. dints, An. kochi, An. splendidus and An. minimus. Eight species belonging to subgenus Anopheles were An. barbirostris, An. messeae, An. crawfordi, An. kunmingensis, An. kweiyangensis, An, hyrcanus, An. sinensis and An. anthropophagus. The results show that the interspecific differences of sex chromosomes and heterochromatin differences in autosomes are useful in anopheline sibling species identification. Two types of completely different chromosomal karyotypes of An. maculatus were found from Yunnan and Sichuan Provinces, and two types of obviously different heterochromatin banding of An. dims were found from Hainan and Yunnan Provinces. Thus, An. maculatus and An. dirus are respectively sibling species complex in China. The salivary gland polytene chromosomes of An. hyrcanus, An. sinensis and An. anthropophagus were also studied. The authors find that the main differ ences of polytene chromosomes of the three species are fixed paracentric inversions in arm 2L.

Objective To determine sequence of sporogony stage-specific (S type) 18S ribosomal RNA gene of Plasmodium yoelii (P.y) By265 strain, and by using it to detect the malaria parasites within vector mosquito. Methods A pair of conserved DNA primers, universe primer (Pu) and reverse transcription one (Pr), was designed and synthesized according to sequence of the 18S rRNA gene of Plasmodium berghei (P.b). The segment of the S type 18S rDNA of P.y was amplified by reverse transcript-polymerase chain reaction (RT-PCR) from dissected midguts of Anopheles stephensi infected with P.y on the 7th day after infective blood-meal, and its sequence was then determined. One P.y sporogony stage-specific primer (Pys) was selected according to the sequence. Using this primer and Pr, the parasites within mosquitoes were semi-quantitatively detected through RT-PCR between 1-7 d post-infection. Results The length of the amplified segment was 920 bp. Alignment in match region of the 18S rDNA among S type of P.y (PyS), S type of P.b (PbS) and asexual blood stage-specific one of P.y (PyA) revealed that the similarity between the former and the latter two reached 95\^3% and 94\^0% respectively. The density of amplified band was significantly concordance with the intensity of oocyst in the midgut. Sensitivity of RT-PCR method was higher than that of the traditional dissection and oocyst observation also. The assay could detect the 18S rRNA molecule of the parasites on the third day post-infection while their oocysts were difficult to be recognized under an optical microscope at that time. Conclusion This S type 18S rDNA sequence in P.y species was first reported (AF266261). As a molecular marker, it could be applied to monitoring the parasite development in its vector at an earlier stage semi-quantitatively with an adequate sensitivity and specificity.

Two isolates of Plasmodium jalcipanm from Hainan Province were cloned by limiting dilution method and eight clones were established. Some characteristics including drug sensitivity and antigenicity of these clones were observed. The results showed that the established clones were different from each other in chloroquine sensitivity and antigenicity. The ID50 of chloroquin3e against 6 clones from isolate Fcc-7801 varied between 60.60 and 13.08 nmol/L The ID50 of chloroquine against 2 clones from isolate Fcc-1 were 93.63 and 49.64 nmol/L, respectively. According to the indirect fluorescent antibody (IFA) Teactivity of these clones with a panal of murine anti-gpl95 McAbs, 8 clones could be divided into 5-serotypes, 3 of which were consistent with the Ⅴ , Ⅵ, Ⅶ serotypes devided by J. S. McBride (1985).

Objective To isolate and identify genes related to malaria parasite infection in vector mosquito, and to explore the mechanisms. Methods Anopheles stephensi infected with Plasmodium yoelii was used as tester (T) group, while uninfected but normal blood fed as driver (D) one. Engorged female mosquitoes of two groups were collected separately at 24 hours after biting. An enriched subtractive cDNA pool was generated through the course of suppression subtractive hybridization (SSH) and selective PCR amplification. The subtracted library was screened by hybridization using T and D cDNA mixture as probes, respectively. The positive clones, which produced stronger signal when probed with T than with D, were sequenced and their sequence homologues in GenBank database were searched with BLAST by internet. Results The analysis of subtraction efficiency showed that the differentially expressed genes in T comparing to in D were enriched significantly. In dot blot screening, 24 of 58 randomly selected clones (41.4%) were shown up regulation in malaria infected mosquitoes. The BLAST search of 23 genes revealed that 12 were homologous to functionally known genes, 4 were homologous to functionally unknown entries, and 7 were novel without any relatives. Nine of the 23 genes (39.1%) also hit homologous sequences in the An. gambiae EST database generated from an immune competent cell line treated with lipopolysaccharide (LPS). Conclusion An enriched cDNA pool of the mosquito genes which up regulated responsively at the early stage of malaria parasite infection was obtained. Expression screening against the pool indicated that various biochemical processes and mechanisms might be involved in the response of mosquito to parasite infection, especially those related with the innate immune system and energy metabolism.

Objective To investigate the correlation between expression of carcinoembryonic antigen (CEA) mRNA in preoperative peritoneal lavage fluid and prognosis of operation in patients with gastric cancer.Methods The expression levels of CEA mRNA in preoperative peritoneal lavage fluid of 68 patients with gastric cancer and 30 patients with gastric benign tumor were measured by reverse transcription polymerase chain reaction.The correlation between the expression level of CEA mRNA in preoperative peritoneal lavage fluid and clinicopathological features,prognosis of postoperative were analyzed.Results The expression level of CEA mRNA in preoperative peritoneal lavage fluid of patients with gastric cancre was 1.74 ± 0.25 and with gastric benign tumor was 0.19 ± 0.04,and there was statistical difference (P ＜ 0.05).The expression level of CEA mRNA in preoperative peritoneal lavage fluid had a correlation with the tumor infiltration depth,tumor differentiation,lymph node metastasis,distant metastasis and TNM stage (P ＜ 0.05).There was statistical difference in the recurrent / metastasis rate and survival rate 1 year after operation between low (34 cases) and higher (34 cases) expression level of CEA mRNA in preoperative peritoneal lavage fluid of gastric cancer patients:47.06％ (16/34) vs.82.35％ (28/34) and 79.41％ (27/34) vs.55.88％ (19/34),P ＜ 0.05.Conclusion The expression of CEA mRNA in preoperative peritoneal lavage fluid has close correlation with the clinicopathologic features,and the high expression of CEA mRNA indicats poor prognosis,indicating that CEA mRNA in preoperative peritoneal lavage fluid could be used as a prognostic marker in patients with gastric cancer.

Using scanning electromicroscope, seven species of anopheline mosquito eggs were studied. Among them, the microstructure of exochorion of Anopheles liangshanensis. An. kweiyangensis An. kunmingensis, An. hyrcanus and An. messeae were not reported before. The results showed that ultrastructure of plastron network, frill and tubercles of deck were useful in distinguishing sibling species of anopheline eggs. Microstructure of float had little difference between species. The micropylar area and lobed tubercles presented obvious intraspecific variations, so, it should be careful for using in classification.

BACKGROUND:Bismth-doped iron nanoparticles modified by hyaluronic acid (HA-BiIOPs) not only act as an effective MRI contrast agent, but also as a radiotherapy sensitizer.OBJECTIVE:To fabricate the HA-BiIOPs and to observe its effect to enhance the radiosensitivity of glioblastoma cells U87MG under X-ray radiation.METHODS:HA-BiIOPs were synthesized using hydrothermal polyol method. (1) Cytotoxicity: A cytotoxicity test was carried out on U87MG cells and rat vascular smooth muscle cells (VSMCs). Cell proliferation rate of two kinds of cells cultured with different concentrations of HA-BiIOPs (0, 12.5, 25, 50, 100, 200, 400 mg/L) at 24 hours after culture were determined by cell counting kit-8 assay. (2) Histological analysis: ICR mice were sacrificed after intravenous injection of HA-BiIOPs, and pathological changes of mouse visceral organs were observed under an optical microscope. (3) Cellular uptake: The HA-BiIOPs after entered into the cytoplasm were observed by Prussian blue staining. (4) Radiosensitization test: U87MG cells at Logarithmic growth stage were cultured in culture medium as control group, subjected to X-ray irradiation (0, 3, 6, 9 Gy) as radiotherapy group, cultured in HA-BiIOPs (0, 12.5, 25, 50, 100, 200 and 400 mg/L) as HA-BiIOPs group or subjected to HA-BiIOPs culture plus X-ray irradiation as combined therapy group. Then, the cell proliferation rate and cloning efficiency were measured at 24 hours after treatment.RESULTS AND CONCLUSION:(1) The HA-BiIOPs at different concentrations were non-cytotoxic for VSMC and U87MG cells. (2) After intravenous injection of HA-BiIOPs, there was no obvious toxicity to the mouse susceptible organs. (3) After 6 hours of culture, the HA-BiIOPs could be internalized by U87MG cells. (4) The proliferation rate of U87 cells was negatively correlated with the concentration of HA-BiIOPs (0-200 mg/L) and X-ray dose (0-9 Gy). Especialy, the combination of 6 Gy X-ray irradiation with 200 mg/L HA-BiIOPs dramatically decreased the cell viability that was decreased to (41±7)%. In the combined therapy group with 6 Gy X-ray and 100 mg/L HA-BiIOPs, the cells proliferation rate was significantly lower than that in the control and radiotherapy groups (P < 0.05). These results indicate that HA-BiIOPs have a radiosensitizative effect on glioblastoma cells U87MG.

AIM: To explore the effect of aspirin on inducible nitric oxide synthesis and gene expression under inflammation in endothelial cells. METHODS: Using NADPH, Griess methods and RT-PCR, the activity of isozymes of NO synthase (NOS), nitric oxide (NO) level, and iNOS mRNA expression were examined respectively. Also, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content and cell viability were measured. RESULTS: Aspirin (3 mmol/L) reduced inducible NO production and NOS activity(P

AIM: To explore the regulatory effects of ferritin expression and intracellular iron change on aspirin resistance to oxidative damage in endothelial cells. METHODS: Using ELISA to measure the levels of ferritin expression under different aspirin concentrations, in the presence of iron cheltor desferioxamine and add to FeCl 3. Then using RNA-protein bandshift assay and RT-PCR to examine the activation of IRP and the expression of IRP 2 mRNA onaspirin induced ferritin formation. RESULTS: Aspirin at low concentration (0.1mmol/L) induced significant increase in ferritin expression in a concentration-dependent fashion up to 25% over basal levels. Aspirin induced cytoprotection from H 2O 2 damage increased significantly following ferritin formation in endothelial cells.However, in the presence of iron chelator desferrioxamine, aspirin enhanced ferritin synthesis was abrogated with a 3 fold increase in the activity of IRP and significant increase in IRP 2 mRNA level. In contrast, FeCl 3 and aspirin both increased the level of induced ferritin synthesis with significant decrease in IRP activity and IRP 2 mRNA level. CONCLUSION: The effect of aspirin induced ferritin synthesis on resistance to oxidative damage in endothelium was operated through down-regulating IRP activation and IRP 2 mRNA level.