An aquaporin and an aquaglyceroporin have roles in low temperature adaptation of mosquitoes (Anopheles sinensis).

Mosquitoes (Anopheles sinensis), widely geographically distributed in Asia including China, are the primary vector of the malaria parasite Plasmodium vivax and other parasitic diseases such as Malayan filariasis. An. sinensis can survive through low winter temperatures. Aquaporin channels are found in all life forms, where they facilitate environmental adaptation by allowing rapid trans-cellular movement of water (classical aquaporins) or water and solutes such as glycerol (aquaglyceroporins). Here, we identified and characterized 2 aquaporin (AQP) homologs in An. sinensis: AsAQP2 (An. sinensis aquaglyceroporin) and AsAQP4 (An. sinensis aquaporin). When expressed in frog (Xenopus laevis) oocytes, AsAQP2 transported water, glycerol, and urea; AsAQP4 transported only water. Water permeation through AsAQP2 and AsAQP4 was inhibited by mercuric chloride. AsAQP2 expression was slightly higher in adult female mosquitoes than in males, and AsAQP4 expression was significantly higher in adult males. The 2 AsAQPs were highly expressed in Malpighian tubules and midgut. AsAQP2 and AsAQP4 expression was up-regulated by blood feeding compared with sugar feeding. At freezing point (0 °C), the AsAQP4 expression level increased and An. sinensis survival time reduced compared with those at normal temperature (26 °C). At low temperature (8 °C), the AsAQP2 and AsAQP4 expression levels decreased and survival time was significantly longer compared with those at 26 °C. These results suggest that AsAQP2 and AsAQP4 have roles in water homeostasis during blood digestion and in low temperature adaptation of A. sinensis. Together, our results show that the 2 AQPs are important for mosquito diuresis after blood feeding and when exposed to low temperatures.

[Proteomic study of the hippocampus tissue from rats with chronic Toxoplasma gondii infection].

OBJECTIVE: To observe the proteome changes in the hippocampus tissue of rats with chronic Toxoplasma gondii infection. METHODS: Six male SD rats were randomly divided into control group and infection group. Each rat in infection group was intraperitoneally injected with 4 x 10(7) purified T. gondii tachyzoites. Rats in the control group received equivalent volumes of sterile normal saline. At the fifth day post-infection, blood samples were taken from the lateral tail vein and Ciemsa staining of blood cells was performed to find Toxoplasma gondii. Rats were dissected at the 10th week post-infection, total protein in the hippocampus was separated by using two-dimensional gel electrophoresis (2-DE). After Coomassie blue staining, the Image Analysis software was used to select and separate proteins on the gel. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for peptide mass fingerprint PMF). Proteins were identified by using Mascot software to search the MSDB and SwissProt databases. RESULTS: Microscopy examination of blood smears confirmed that the rats in infection group were all infected by 11 gondii. The number of protein spots of rats from infection group and control group was 311 +/- 19 and 327 +/- 13 respectively. Compared with the control group, 5 protein spots disappeared, 4 protein spots were up-regulated and 7 were down-regulated in the infection group. The 9 differentially expressed protein spots were identified by MALDL-TOF-MS: phosphoglycerate kinase 1, similar to alpha-enolase, glutamine synthetase, creatine kinase, creatine kinase B-type, ATP synthase, aconitase 2, mitochondrial precursor, actin and an unnamed protein. The first three proteins were up-regulated and the other five proteins were down-regulated in infection group. CONCLUSION: Nine differential expression proteins are found from the hippocampus tissue in rats chronically infected with T. gondii and normal SD rats.

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice.

It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland-specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat ß-casein, bovine αs1-casein, or goat ß-lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken ß-globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17-8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7-40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland-specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary-specific expression of recombinant hLF in a transgenic mouse model.

Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells.

To investigate the role of ROS in the helicobacter pylori (Hp) induced mtDNA mutations, AGS cells were treated by extracts of Hp11638 or Hp11638M. The ROS levels, cytochrome C reductions, and intracellular ATP levels were measured. The coding region and the D-Loop region were amplified and sequenced. Results showed the ROS levels, cytochrome C reduction and mtDNA mutations were markedly increased and cell viability decreased after treatment with both Hp extracts, and 616 mutations were detected in D-Loop region and 3 heteroplasmic point mutations in the Cytb gene. No mutations were found in the coding region. The mutation rates of mtDNA D-Loop region were positively correlated with the ROS levels and negatively to the ATP levels.

Biologic characteristics of the side population of human small cell lung cancer cell line H446.

BACKGROUND AND OBJECTIVE: Recently, the theory of cancer stem cells (CSCs) has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population (SP) cells in small cell lung cancer (SCLC) cell line H446, which lays the foundation for the isolation and purification of CSCs. METHODS: Fluorescence-activated cell sorting (FACS) was used to sort SP and non-SP (NSP) cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to evaluate the expression levels of the mRNA of CD133, ABCG2, and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice. RESULTS: The percent of Hoechst 33342 negative cells was about (5.1 +/- 0.2)% in H446 by fluorescence microscopy. The percent of SP cells was (6.3 +/- 0.1)% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2, CD133, and nucleostemin in SP cells were 21.60 +/- 0.26, 7.10 +/- 0.14, and 1.02 +/- 0.08 folds higher than that in NSP cells (P < 0.01, P < 0.01, and P > 0.05, respectively). In vivo, SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells, but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors. CONCLUSION: The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.

[Serological investigation on Toxoplasma gondii infection in patients with unknown central nervous system diseases].

Totally 207 patients with unknown central nervous system diseases and 203 healthy persons were investigated for serum IgG of anti-Toxoplasma antibody assessed by ELISA. The serum IgG positive rate in 207 patients with unknown central nervous system diseases was 19.81%, and that in 203 health people was 5.42%, and there was a significant difference between them (P < 0.01). The IgG positive rates in different types of central nervous system diseases were different, which were 22.81%, 24.32%, 16.05%, and 18.75%, respectively in encephalopathy, epilepsy, mental disorder and neurasthenia. The IgG positive rate in different types of central nervous system diseases were significantly higher than that in healthy population (P < 0.01). The IgG positive rates in patients who contacted or did not contact cats or dogs were 32.97% and 9.48% respectively (P < 0.01). In conclusion, the infection rate in patients with unknown central nervous system diseases is higher than that in healthy persons; therefore, it is necessary to assay the serum IgG in them.

[Preliminary study on serum proteomics in mice with acute toxoplasma gondii infection by using functionalized magnetic beads and MALDI-TOF-MS technique].

OBJECTIVE: To detect and analyze the serum protein biomarkers in mice with acute Toxoplasma gondii infection. METHODS: The serum samples from 8 C57BL/6J mice with acute Toxoplasma gondii infection and 8 normal healthy paired mice were prepared with WCX magnetic beads, and then analyzed on PBS II -C mass spectrometer reader. The protein spectra of the serum samples were normalized by the Ciphergen Protein Chip software. The peak labeling was performed by the Biomarker Wizard software. The specific protein biomarkers were screened by the Biomarker Pattern software to construct a diagnostic model for acute Toxoplasma gondii infection. RESULTS: A total of 13 distinguished proteomic peaks were detected. Nine peaks were of up-regulated expressions including m/z values of 1 932.76, 1 976.85, 2 090.53, 5 004.5, 5 776.01, 5 803.05, 5 847.99, 5 877.51 and 7 501.58, respectively; and four peaks were of down-regulated expressions including m/z values of 1 866.40,4 063.71, 8 120.31 and 8 203.83, respectively. CONCLUSION: The potential protein biomarkers for acute Toxoplasma gondii infection are discovered in mouse serum by MALDI-TOF-MS combined with WCX magnetic beads.

[Expression of Toll-like receptor 4 in brain tissue of chronic Toxoplasma gondii infection rats and its effect on brain injury].

OBJECTIVE: To explore the expression of Toll-like receptor 4 (TLR4) in brain tissue of chronic Toxoplasma infection rats and its effect on brain injury. METHODS: Ten male SD rats were randomly divided into 2 groups, namely control and infection groups. Each rat in the infection group was intraperitoneal injected with Toxoplasma gondii tachyzoites 10(7)/ml x 2 ml, and that in the control group was injected with 2 ml sterile normal sodium. After 10 weeks, the expression of TLR4 mRNA in the brain was determined by RT-PCR, and the levels of IL-1beta and IL-4 in peripheral blood sera were detected by ELISA. RESULTS: Compared with the control group, the expression of TLR4 gene and the peripheral blood serum level of IL-1beta of rats in the Toxoplasma gondii infection group were both significantly increased, with all P values were less than 0.05, and the level of IL-4 was also increased, but the difference had no statistically significance (P > 0.05). CONCLUSION: TLR4 might be involved in inflammatory reactions of brain injury for chronic Toxoplasma gondii infection rats.

Analysis of diferentially expressed protein from primary and recurrent ovarian cancer serum.

OBJECTIVE: To study the value of the differentially expressed proteins from primary and recurrent ovarian cancer serum for early diagnosis of primary and recurrent ovarian cancer. METHODS: WCX kit (Bruker Daltonics GraBH) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology were used to detect serum samples from 49 patients with primary ovarian cancer and 21 patients with recurrent disease. RESULTS: In the mass range (Mr) from 1,000 to 12,000 Da, eight differentially expressed protein peaks were screened from primary ovarian cancer serum. Among them, four protein peaks with Mr 1,457, 1,857, 2,202, 7,761 were lowly expressed and the others with Mr 2,946, 5,333, 5,859, 5,901 were highly expressed. Ten diferentially expressed protein peaks were screened from recurrent ovarian cancer serum. Among them, 1,944, 1,980, 2,080, 2,661, 2,993, 4,450, 4,659, 5,359 Da protein expressions were increased significantly, and 1897, 7868 Da protein expressions were decreased significantly. The pattern of primary ovarian cancer was applied to 8 early-stage ovarian cancer serum samples, and 7 serum samples were successfully predicted with the accuracy of 87.5%. The pattern of recurrent ovarian cancer was applied to 9 without pelvic or abdominal mass recurrent ovarian cancer serum samples, and 8 serum samples were successfully predicted with the accuracy of 88.9%. CONCLUSION: Combination of MALDI-TOF-MS and WCX kit technology can directly screen the diferrential expressed protein from primary and recurrent ovarian cancer serum. They have clinical significance for enhancement of sensitivity and specificity of ovarian cancer diagnosis.