Association of plasma metallothionein 3 and its polymorphisms with childhood autism / 中华实用儿科临床杂志

Objective To investigate the association of plasma metallothionein 3 (MT3) and its polymorphisms with childhood autism,in order to provide the objective evidence for autistic etiology and molecular diagnosis.Methods A total of 132 autistic children were recruited from several special autism training schools in Wuhan and the Hubei Maternal and Child Health Hospital between January 2011 and November 2014.Three hundred and sixteen healthy children from the out-patients of Zhongnan Hospital of Wuhan University during the same period were enrolled as healthy controls.Enzyme linked immunosorbent assay was utilized to measure plasma MT3 protein levels in a dataset of 81 cases and 80 controls,while eight single nucleotide polymorphisms (SNP) located in MT3 gene were genotyped in another greater dataset that included 132 cases and 236 controls by the matrix-assisted laser desorption/ionization time of flight mass spectrometry within the Sequenom platform.Results Plasma MT3 protein level was significantly lower in autistic group compared to healthy controls [(740.0 ± 327.4) ng/L vs (1 007.1 ± 554.3) ng/L,P ＜ 0.001],particularly for boys when stratified by gender (P =0.005).No difference existed in any allele or genotype frequencies between the 2 groups (all P ＞ 0.05).Conclusions The selected autistic children harbored abnormal expression profiles of plasma MT3 protein,which may have no connection with its gene polymorphisms.

Preparation and stability research of all-trans-retinoic acid liposomes / 第三军医大学学报

Objective To prepare and optimize formulation of ATRA liposomes. Methods ATRA liposomes was prepared by reverse evaporating method and to determine the concentration and the entrapment efficiency of ATRA liposomes by HPLC. Results The entrapment efficiency of the three ATRA liposomes formulations reached 33.98%, 42.11%, 70.34% respectively. The liposome was stable. The ATRA content of three formulations almost showed no change during the observing days. Conclusion The technique of preparing the ATRA liposomes is feasible and the method of quality control is simple and accurate.

Construction and screening of anti-tumor molecule based on a proliferation-inducing ligand / 第三军医大学学报

Objective To construct, express, purify and screen immunosuppressive molecule against human soluble APRIL (a proliferation-inducing ligand). Methods The cDNA of soluble APRIL (sAPRIL) was mutated and TEL Th epitope was added. Mutants was expressed by pQE-80L/DH5? system, identified by SDS-PAGE and Western blotting, purified by Ni-NTA resin. Their activity on stimulating the proliferation of Raji cell was detected. Results Four sAPRIL mutants were constructed and expressed as follows: HEL Th epitope was added at C end (Ⅰ); HEL Th epitope was added at N end (Ⅱ); the last 45-bp DNA was deleted at C end and HEL Th epitope was added (Ⅲ); the first 45-bp DNA was deleted at N end and HEL Th epitope was added (Ⅳ). Specific protein bands according to mutant Ⅰ-Ⅳ were detected by SDS-PAGE and Western blotting. Mutant protein was purified by Ni-NTA successfully. Mutants Ⅰand Ⅱ promoted cell proliferation remarkably, and mutant Ⅲand Ⅳnot. Conclusion Four sAPRIL mutants was constructed and expressed successfully. Immunosuppressive molecule against sAPRIL that can not promote cell proliferation was screened out, and it laid a foundation for further study on their immunosuppressive function.

Design and construction of cDNA mutant of human sAPRIL / 第三军医大学学报

Objective To construct 6 kinds of human soluble proliferation-inducing ligand (sAPRIL) cDNA mutants. Methods Six pair primers according to the cDNA sequence of human sAPRIL for different mutants were designed as follows: HEL Th epitope was added at C or N end, the last or first 45 bp DNA was deleted and HEL Th epitope was added, the last or first 90 bp DNA was deleted and HEL Th epitope was added. Different enzyme site was designed at 5′ end of 12 primers, BamH Ⅰ or SalⅠ or HandⅢ. At the same time, 2 pairs of HEL Th epitope sequence were designed and synthesized, and different incision enzyme sticky end sequence was added at the 5′ or 3′ end of each epitope DNA. Mutant sequences were amplified by 6 pairs primers with sAPRIL cloning plasmid as template. The PCR fragment was digested and then ligated with the HEL Th epitope DNA that had been reannealed. The ligation fragments of the head were ligated with digested pQE80 vector fragment again, and were transformed into DH5? competent cell. Recombinant plasmids were identified by digestion and sequencing. Results Six sAPRIL cDNA mutant DNA sequences were obtained by PCR, and the expression plasmids of sAPRIL cDNA mutants including HEL Th epitope sequence were constructed successfully. The result of sequencing proved that mutations generated as designed fully. Conclusion Six sAPRIL cDNA mutant expression plasmids were constructed successfully.

Construction of cDNA library from adults of Anopheles dirus / 第三军医大学学报

Objective To construct cDNA library from adults of Anopheles dirus for cloning the immune genes or related genes for malaria parasites development. Methods The mRNA of adult Anopheles dirus was isolated. The library was constructed by using the Zap Express vector(Stratagene) and the quality was evaluated. Results The efficiency of the library was 2.1?10 6 pfu/ml with 98% clones positive. The average length of the insert fragment was over 1 kb. Conclusion cDNA library of adult Anopheles dirus with high efficiency can be constructed by using the Zap Express library construction Kit (Stratagene).

Effect of blood-feeding and plasmodium yoelii infection on the transcript abundance of ribosomal protein S7 from Anopheles dirus hemocyte / 第三军医大学学报

Objective To observe the effect of blood feeding and plasmodium yoelii infection on the transcript abundance of ribosomal protein S7 from Anopheles dirus hemocyte Methods Anopheles dirus of 3～5 days old were divided into normal group (N), blood feeding group(B) and Plasmodium yoelii infection group(I) Hemocytes of 50 Anopheles dirus from each group were collected by centrifuge method on days 1, 2, 3, 4, 7 and 11 after blood feeding, respectively Then, all hemocyte samples were used for total RNA isolation and analyzed by RT PCR Finally, the same volume(10 ?l) of all the PCR products from each group was used for agarose gel electrophoresis and the data obtained were analyzed statistically Results There was no significant difference in ribosomal protein S7 signal between the three groups Conclusion Similar to Anopheles gambiae and human rpS7, Anopheles dirus rpS7 might be also used as an internal control for the studies of the role of Anopheles dirus related immune factors in attacking Plasmodium infection

SDS-PAGE analysis of differentially expressed proteins from Anopheles stephensi hemolymph produced during the Plasmodium yoelii oocysts melanization / 第三军医大学学报

Objective To explore primarily the differentially expressed proteins in the hemolymph from adult female Anopheles stephensi ( An stephensi ) infected with Plasmodium yoelii ( P yoelii ) after being fed with sucrose solution containing nitroquine or not at different time points Methods Hemolymph of 2 groups of adult female An stephensi was collected with the expulsion method from the first day to the fifth day after the feeding Hemolymph samples were examined with SDS PAGE The protein gels were visualized by either Coomassie brilliant blue or silver staining, scanned and automatically analyzed by the BioRad1000 gel image analysis system for differential proteins bands Results On the second day of feeding with nitroquine, a few oocysts were partially melanized Furthermore, during the period from the fifth day to the ninth day, the number of mosquitoes with malanized oocysts and the number of melanized oocysts gradually increased The number of hemolymph protein binds in the treatment group was markedly more than that in the control Many different bands, mainly located at the molecular weight of (20～40)?10 3 and (60～80)?10 3, were visualized in the 2 groups The number of protein bands stained by the silver staining was more than that by the Coomassie brilliant blue staining Conclusion There are differentially expressed proteins in the hemolymph in An stephensi infected with P yoelii after being fed with sucrose solution containing nitroquine These differential proteins may be the melanization engaging proteins

Relationship between hemolymph phenol oxidase and melanization of Plasmodium yoelii oocysts in Anopheles dirus / 第三军医大学学报

Objective　To explore the relationship between hemolymph phenol oxidase and the melanization of Plasmodium yoelii oocysts in Anopheles dirus. Methods　An Anopheles dirus-Plasmodium yoelii system was used Anopheles dirus were divided into 3 groups, that is, non-blood-fedding (N), normal-blood-fedding (B) and infected-blood-fedding (I). The activities of MPO and o-DPO in hemolymph from 3 groups were determined with native polyacrylamide gel electrophoresis (PAGE) and density scanning at 5, 7, 11 and 15 d after blood feeding. Results　Both MPO and o-DPO activity were significantly higher in group I than group N and B (P<0.05). But with the melanization of Plasmodium yoelii oocysts, both MPO and o-DPO activity in group I were decreased in comparison with group N, especially on the 15 th day after infected-blood feeding. MPO and o-DPO activity in group B were significantly stronger than those of group N. Conclusion　Blood feeding and infection of Plasmodium yoelii both can activate the cascade. The heamolymph phenol oxidase may play an important role in the melanization of Plasmodium yoelii oocysts in Anopheles dirus.

Cloning, Location and Differential Analysis of Transcription Factor Relish Gene from Anopheles stephensi under Different Feeding Conditions / 中国寄生虫学与寄生虫病杂志

Objective To clone, locate and differentially analyze the transcription factor Relish gene from Anopheles stephensi, and to examine its signals-modulating action on prophenoloxidase cascade and melanization of Plasmodium yoelii oocysts. Methods Relish cDNA of total mosquitoes was amplified by RT-PCR with degenerated primers. Target PCR product was purified,cloned,sequenced and identified. Special Relish gene was amplified with specific primers from hemocytes or midgut,respectively. Semi-quantitative analysis was made under different feeding conditions. Relish message ribonucleic acid was identified with hybridization in situ. Results One cDNA segment of Relish similar to An.gambiae was acquired from An. stephensi. The same Relish gene was also manifested in the hemocytes and midgut. Marked up-regulation expression of Relish was observed at 6,12,24 or 48 h of Plasmodium yoelii infection and at 12 and 24 h after sucking nitroquine-acetate sucrose solution,that was before inducible oocyst melanization. Relish was also expressed in the hemocytes and midguts by ISH. Conclusion Transcription factor Relish of An. stephensi might play a role in signal modulation of Plasmodium yoelii infection and oocyst melanization.

The Internal Control Role of Ribosomal Protein S7 in the Defense of Anopheles dirus Against Plasmodium Infection / 中国寄生虫学与寄生虫病杂志

Objective To investigate the role of ribosomal protein S7(rpS7) in the defense of Anopheles dirus against infection. Methods rpS7 was amplified from Anopheles dirus hemocytes with degenerated primers designed according to the conservative region of S7, rpS7 was then cloned using T/A cloning kit and the inserted fragment was sequenced. The difference of the transcript abundance of rpS7 from Anopheles dirus hemocyte among non-blood-fed (N), normal-blood-fed (B) and Plasmodium yoelii infected groups (I) was also analyzed by RT-PCR and gel scanning system at d1, d2, d3, d4, d7 and d11 after blood feeding. Results There is no significant difference of rpS7 signal between the three groups. Conclusion Anopheles dirus S7 can be used as an internal control to study the role of Anopheles dirus related immune factors in Plasmodium infection.

IN VITRO CULTIVATION OF THE EXOERYTHROCYTIC STAGE OF PLASMODIUM YOELII AND AFFECTING FACTORSINVOLVED / 中国寄生虫学与寄生虫病杂志

AIM:To cultivate the exoerythrocytic stage of Plasmodium yoelii in vitro and to study some involved affecting factors.METHOD:In vitro cultivation.RESULTS:The monolayer hepatocytes grown in 1 6 - mm plastic cell culture dishes were inoculated with sporozoite sus- pension prepared from Anopheles stephensi mosquitoes for48hours.At a final density of2? 1 0 4 cells per well,the infection rate of hepatocyte,cultured in medium supplemented wit15 % bovine serum,was 0 .0 35? 0 .0 1 3% ,the diameter of the nearly mature EEF of Plas- modium yoelii was up to40 .3? 31 .6 ?m,and contained more than1 0 0 nuclei,the number of EEF might be4- 1 0 /cm2 .An intraperitoneal inoculation of the EE schizonts to mice could induce parasitemia.At a final density of0 .5? 1 0 4 or4? 1 0 4 cells per well or the hepatocytes cultured in medium supplemented with1 0 % bovine serum,no EEF could be observed.CON- CL USION:The density of hepatocytes and culture medium are important for the cultivation of the EE stage of Plasmodium yoelii.This procedure will lay foundation for the further studies of the sporozoite invasion,the development of EEF and the affecting factors in- volved.

ANALYSIS OF PAGUMOGONIMUS SKRJABINI ANTIGEN AND ITS APPLICATION IN SERODIAGNOSIS / 中国寄生虫学与寄生虫病杂志

Objective] To analyse the soluble antigens of different developmental stages of Pagumogonimus skrjabini and deve lop a specific and sensitive serodiagnostic method for pagumogonimiasis. [Methods] The soluble antigens of P.skrjabini of various stages were separated by SDS PAGE. The specific antigen of the adult fluke was recognized immunologically by immunoblot assay. The protein bands between 10～30 kDa purified by SDS PAGE and electrophoretic elution were used in dot ELISA. [Results] Using dot ELISA, the soluble antigens of adult were recognized by sera infected with P skrjabini . More reactive bands appeared at 10～30 kDa, but major protein bands were at 22、24 and 26 kDa. However, using sera from patients infected with other trematodes including schistosome and Clonorchis , cross reaction bands appeared within 60 to 90 kDa. When compared with ELISA of crude adult antigens for detecting 28 suspected patients, there was no significant difference between the two methods. The sera of 38 patients with other diseases were also detected by the two tests. No cross reaction occurred with the purified adult antigen dot ELISA while 13 2%(5/38) of the sera cross reacted in ELISA of crude adult antigens. [Conclusion] Dot ELISA using 10～30 kDa antigen might be a specific and sensitive serodiagnostic method for diagnoing pagumogonimiasis.

Enhanced transcript abundance of AdsP1 and AdsP3 from Anopheles dirus haemocytes after Plasmodium yoelii infection / 第三军医大学学报

Objective To investigate the expression patterns of serine proteases Adsp1 and Adsp3 in the main tissue of Anopheles dirus and the effects of blood feeding and Plasmodium infection on the transcript abundance of Adsp1 and Adsp3 from Anopheles dirus haemocytes. Methods Haemocytes were collected by centrifugation. The midguts and salivary glands were dissected from 50 adult female Anopheles dirus of 3-5 d old for the extraction of total RNA for amplification by RT PCR. Anopheles dirus of 3-5 d old were divided into normal group (N), blood feeding group (B), and Plasmodium yoelii infection group (I). Haemocytes of 50 adult female Anopheles dirus from each group after blood feeding were also collected, and the total RNA was isolated at 1, 2, 3, 4, 7, and 11 d, respectively. Then, the same volume (10 ?l) of total RNA was analyzed by semi quantitative RT PCR with Ads7 as the internal control. Agarose gel analysis was performed on PCR products from each group. Results Expression of both Adsp1 and Adsp3 mRNA was found in the haemocytes and salivary glands, but not in the midguts. Semi quantitative RT PCR indicated that transcript abundance of AdsP1 and AdsP3 from Anopheles dirus haemocytes was enhanced after blood feeding and Plasmodium yoelii infection, especially during melanotic encapsulation of Plasmodium yoelii . Conclusion AdsP1 and AdsP3 may be involved in the melanotic encapsulation response of Plasmodium yoelii by Anopheles dirus , and may be the prophenoloxidase activating enzyme of Anopheles dirus .

Correlation of Anopheles TEP1 Gene with Melanization Induced by Nitroquine / 中国寄生虫学与寄生虫病杂志

Objective To analyze the relationship between the TEP1 gene of Anopheles stephensi and melanotic encapsulation of Plasmodium yoelii induced by anti-malaria drug nitroquine. Methods Haemolymph samples from three groups of An. stephensi fed with sucrose solution, Plasmodium-infected blood and nitroquine, respectively, were collected at the 1st, 2nd, 3rd and 4th day after drug adminstration. Degenerate primers were designed according to the conserved amino acid sequence within TEPs of the mosquitoes. Fluorescent quantitation PCR was used to detect the variation of TEP1 gene transcript induced by nitroquine. The melanization of oocysts was observed by light microcopy. Results TEP1 gene was cloned, the predicted amino acid residues harbored a highly conserved canonical thioester motif GCGEQ. The fluorescent quantitation PCR revealed that nitroquine induced an up-regulation of TEP1 activity. The transcription of TEP1 gene in nitroquine treated group (2.423) was significantly higher than that of the infected blood-fed group(1.036) at the 3rd day after nitroquine treatment (P

Inhibition of CpG Oligodeoxynucleotide on the Development of Pre-erythrocytic Stage of Plasmodium / 中国寄生虫学与寄生虫病杂志

Objective To study the role of cytidine-phosphate-guanosine oligodeoxynucleotide(CpG ODN) on the development of Plasmodium liver stage.Methods Plasmodium yoelii BY265 18S rRNA was cloned,and the TaqMan real-time PCR was established on P.yoelii BY265 18S rRNA and mouse GAPDH as quantitative analysis model.The model was tested by the level of liver Plasmodium load with the liver cDNA in BALB/c mice infected by salivary gland sporozoites for 42 hours.Twelve BALB/c mice were randomly divided into CpG group,CpG control group and PBS control group which were injected respectively by ODN1826 30 ?g,ODN1826 control 30 ?g and 0.01 mol/L PBS 200 ?l via vena caudalis.Twenty-four hours later,each mouse was inoculated with 100 sporozoites.Mice were sacrificed in 42 hours after infection,and the liver load of Plasmodium was analyzed by TaqMan real-time PCR.Results The cloned Py BY265 18S rRNA gene showed 98% similarity to Py 17XNL.The quantitative analysis model consisted by 18S rRNA and GAPDH showed positive correlation between the level of liver Plasmodium load and the sporozoite inoculation dose to mice.The Plasmodium load in CpG ODN pre-treated mice was reduced to one fifth of the control group(0.28/1.33)(P

EFFECTS OF CYCLOPHOSPHAMIDE ON THE DEVELOPMENT OF EXO-ERYTHROCYTIC FORMS OF PLASMODIVM YOELII IN RATS / 中国寄生虫学与寄生虫病杂志

The percentage of sporozoites developed in exoerythrocytic forms in the rats treated intravenously with cyclophosphamide 24 hrs before inoculation of sporozoites was higher than that in untreated controls, so was the mean size of exoerythrocytic form. Cyclophosp-hamide could decrease the sensitivity of hepatocytes to the vector's tissues inoculated together with sporozoites, thus decreasing the incidence of cloudy swelling of hepatocytes induced by the vector's tissues and inhibiting the white blood cells infiltration induced by the ruptured mature exoerythrocytic form.The fact that cyclophosphamide might enhance the invasion of sporozoites into the hepatocytes indicates that the phagocytic activity of Kupffer's cells to destroy the sporozoites might be inhibited by cyclophosphamide.

The effects of hemocytes on normal and degenerated oocysts of P.yoelii in mosquitoes and on melanization of oocysts / 第三军医大学学报

Anopheles Stephensi,infected with Plasmodium yoelii.was fed on 10% sucrose solution containing 1%.difluoromethylornothine(DFMO)to induce the degeneration of its oocysts.On the 11th day after infection,the effects of hemocytes on the normal and degenerated oocysts were observed wtih transmission electron microscopy.In the control group.no hemacytes could be found around the normally-developed oocysts except those degenerated ones.In the DFMO group,all the oocysts underwent degeneration in various degrees and some of them were melanized.All the oocysts were attached by one or more hemocytes which belonged to the category of granulo-cytes morphologically.There were many granules with microtubular construction in the cytoplasm of the hemocytes and in the spaces between a hemocyte and an oocyst.The findings indicate that the degeneration of oocysts can exert a taxic effect on hemocytes and the latter may release the granules and possibly other substances to result in the encapsulation and melanization of the oocysts.

Observation of Nitroquine Effect on Sporogonic Development of Plasmodium yoelii / 第三军医大学学报

The effects of nitroquine on the sporogonic development of Plasmo-dium yoelii were observed under electron microscopy. The female mosquitoes were fed directly with 10% sucrose solution containing 0.1%Nitroquine.It was found that the oocysts were smaller and markedly degenerated as compared with that of the control. The surface of the oocysts was rough and uneven. Under a transmission electron microscope, the cytoplasm of the affected oocysts contained vacuoles; the membane of mitochondria and uncleus was damaged; and the number of residual bodies increased.No sporoblast formation was seen in most of the affectes oocysts. The nuclear membrane of the degenerated sporozoites was thickened and the density of nuclear matrix decreased markedly as compared with that of the control. These results indicate that the nucleus and the membrane are mainly affected during the sporogonic development of P. yoelii by nitroquine.

A Study on Laboratory Rearing of Anopheles dirus / 第三军医大学学报

Anopheles dims Peyton et Harrison, 1979 is an important malaria vector in Southeast Asia and China particularly in forested hilly zones. To establisha a laboratory mating colony for this mosquito is one of the subjects to be solved urgently in malaria research.This paper is to report our experience on breeding this mosquito. Ecological environment is created to simulate the natural conditions. The room-temperature of the insectary is 25?-28℃ and its relative humidity is around 80%.Mosquitoes are kept in cages with the si2e of 50 ? 50 ? 100cm. The larvae are fed with a mixture of de-fatted pig liver powder and yeast with a ratio of 1:3. The adults are fed with a mixture syrup containing 4% of orange juice, 10% of sucrose and other materials. The insectary is illuminated with a 3o w fluorescent light in the day time and a 15 w blue light in the night continuously to induce mating.The mating rate of the fourteenth generation reached 68% and the descendants bred can be used in research work now.

A Preliminary Observation on the Susceptibility of Anopheles dims to plasmodium yoelii / 第三军医大学学报

Anopheles dirus was fed with the mice of Kunming strain infected by plas-modium yoelii yoelii (By265 strain) for three or four days. Then the midgut and the salivary glands of the mosquitoes were examined. It was found that the infection rate of the malaria parasite of the midgut was 78.57% (44/56), but that of the salivary glands was only 4.88% (2/41) . After two passages of the malaria parasite through the mice and the mosquitoes, the infection rate of the salivery glands could be increased up to 13.96% (6/43).