Genotyping of Toxoplasma gondii from Rats (Rattus rattus) in Riyadh,Saudi Arabia

Toxoplasma 3 main clonal lineages are designated as type I, II, and III; however, atypical and mixed genotypes were also reported. This study was conducted for detection of Toxoplasma gondii genotypes in rats (Rattus rattus) in Riyadh region, Saudi Arabia. PCR test on T. gondii B1 gene was conducted on ELISA IgM positive samples for confirmation of the infection. However, genetic analysis of the SAG2 locus was performed to determine T. gondii genotypes using PCR-RFLP technique. PCR test on T. gondii B1gene showed that 22 (81.5%) out of the 27 ELISA IgM positive samples have T. gondii DNA. Genotypic analysis shows that, of the total 22 PCR positive samples, only 13 (59.1%) were of type II, 7 (31.8%) were of type III, and 2 (9.1%) were of an unknown genotype. It is obvious that the prevalence of both type II and III is high in rats. No reports have been available on T. gondii genotypes among rats in Riyadh region, and only little is known about its seroprevalence in rats. Future studies on T. gondii genotypes in rats using multi-locus markers is needed in Riyadh region, Saudi Arabia for better understanding of T. gondii pathogenesis and treatment in humans and animals.     

The C1B domains of novel PKCε and PKCη have a higher membrane binding affinity than those of the also novel PKCδ and PKCθ

The C1 domains of novel PKCs mediate the diacylglycerol-dependent translocation of these enzymes. The four different C1B domains of novel PKCs (δ, ε, θ and η) were studied, together with different lipid mixtures containing acidic phospholipids and diacylglycerol or phorbol ester. The results show that either in the presence or in the absence of diacylglycerol, C1Bε and C1Bη exhibit a substantially higher propensity to bind to vesicles containing negatively charged phospholipids than C1Bδ and C1Bθ. The observed differences between the C1B domains of novel PKCs (in two groups of two each) were also evident in RBL-2H3 cells and it was found that, as with model membranes, in which C1Bε and C1Bη could be translocated to membranes by the addition of a soluble phosphatidic acid without diacylglycerol or phorbol ester, C1Bδ and C1Bθ were not translocated when soluble phosphatidic acid was added, and diacylglycerol was required to achieve a detectable binding to cell membranes. It is concluded that two different subfamilies of novel PKCs can be established with respect to their propensity to bind to the cell membrane and that these peculiarities in recognizing lipids may explain why these isoenzymes are specialized in responding to different triggering signals and bind to different cell membranes.     

PSAG12-ipt嵌合基因转化马铃薯体系的研究

以根癌农杆菌介导法将PSAG12-ipt嵌合基因导入马铃薯栽培品种,对影响马铃薯遗传转化的多种因素进行系统研究.结果表明:马铃薯茎段分化效率高于叶片,马铃薯愈伤诱导和芽分化最适培养基为MS+6-BA 0.25mg/L+NAA 0.25mg/L+2,4-D 0.25mg/L,添加1%Na2SO3能有效防止褐化;茎段愈伤诱导和分化苗生根最适的Kan浓度分别为50mg/L和75mg/L;外植体预培养2d,OD600为0.2～0.5的农杆菌浓度侵染8min、共培养3d后进行选择培养能有效地提高植株再生能力.用PSAG12和ipt双重PCR检测再生植株,阳性转化率为65.8%.Southern blotting结果表明,转基因植株多以单拷贝形式整合进马铃薯基因组中.     

Toxoplasma gondii: Protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice

Infections by the intracellular protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals which can cause severe or lethal toxoplasmosis. So the development of a more effective vaccine is needed urgently. A multiantigenic vaccine against toxoplasmosis was constructed in the present study, which contains two T. gondii antigens, SAG1 and MIC4 on the basis of previous immunological and immunization studies. The eukaryotic plasmid pcDNA3.1-SAG1-MIC4, pcDNA3.1-SAG1, pcDNA3.1-MIC4 were constructed first, which can express surface protein SAG1 and microneme protein MIC4 from different stages of T. gondii life cycle, and the expression ability of these DNA vaccine in HeLa cells were examined by Western blot. The efficacy of these plasmids with or without co-administration of a plasmid encoding cholera toxin A2/B as a genetic adjuvant by mucosal way to protect BALB/c mice against toxoplasmosis was evaluated. We found these vaccines were able to elicit a significant humoral and cellular immune response in vaccinated mice and they can increase survival rate and prolong the life of mice that were infected by T. gondii especially in the pcDNA3.1-SAG1-MIC4 group. Co-delivery of cholera toxin A2/B further enhanced the potency of multiantigenic DNA vaccine by intranasal route. These results encourage further research towards achieving vaccinal protection against the T. gondii in animals and humans.     

Recent advances in polysaccharide bio-based flocculants

Natural polysaccharides, derived from biomass feedstocks, marine resources, and microorganisms, have been attracting considerable attention as benign and environmentally friendly substitutes for synthetic polymeric products. Besides many other applications, these biopolymers are rapidly emerging as viable alternatives to harmful synthetic flocculating agents for the removal of contaminants from water and wastewater. In recent years, a great deal of effort has been devoted to improve the production and performance of polysaccharide bio-based flocculants. In this review, current trends in preparation and chemical modification of polysaccharide bio-based flocculants and their flocculation performance are discussed. Aspects including mechanisms of flocculation, biosynthesis, classification, purification and characterization, chemical modification, the effect of physicochemical factors on flocculating activity, and recent applications of polysaccharide bio-based flocculants are summarized and presented.     

弓形虫膜表面抗原SAG2蛋白的高效表达及免疫活性分析

在大肠杆菌中以可溶性形式高效表达弓形虫膜表面抗原SAG2蛋白,并对其免疫活性进行分析。应用PCR技术从刚地弓形虫RH株的基因组DNA中扩增编码SAG2的基因片段,亚克隆至原核表达载体pET32a(+),在大肠埃希菌(E.coli)BL21内表达,并对其表达条件进行优化,Western blotting和ELISA分析纯化蛋白的免疫原性;纯化的重组蛋白免疫小鼠制备多抗,用间接免疫荧光试验(IFA)分析表达蛋白的免疫反应性。成功构建重组质粒pET32a(+)-tSAG2,所表达的融合蛋白大小约为38kD。在IPTG终浓度为0.1mmol/L、诱导时间4-6h和培养温度32℃条件下,重组SAG2蛋白主要以可溶性形式在大肠杆菌中高效表达,每升培养菌液约获得可溶性重组SAG2蛋白16mg。Western blotting及ELISA结果显示纯化蛋白具有良好的免疫原性。IFA显示重组蛋白的抗血清能够识别刚地弓形虫表面的SAG2天然蛋白,所表达蛋白具有良好的免疫反应性。截断的SAG2基因在大肠杆菌中得到了高效表达,重组蛋白保持了天然蛋白的免疫活性,为进一步利用该重组蛋白进行弓形虫病免疫诊断及基因工程亚单位疫苗的研制奠定基础。     

Molecular evidence for multiple Toxoplasma gondii infections in individual patients in England and Wales: public health implications

We sought to determine the SAG2 genotypes of Toxoplasma gondii associated with cases of acute human toxoplasmosis in England and Wales. The samples examined were collected from a wide range of cases including congenital infections, AIDS and immunosuppressed patients and were derived from a number of different tissues. Parasite DNA was detected by PCR amplification without the need for prior template purification, and SAG2 genotype was determined by both restriction enzyme analysis and direct DNA sequencing of the PCR amplification products. Parasites of both SAG2 type I and type II genotypes were seen with approximately equal frequency amongst the samples examined. Neither of these genotypes was found to be more frequently associated with a particular clinical presentation or sample tissue. Unexpectedly, we found clear evidence of mixed (SAG2 type I+type II) infections in approximately the same number of samples as were seen to be associated with either type I or II alone. Our use of direct DNA sequencing rather than simple restriction analysis was essential for the detection of mixed infections since incomplete restriction digestion of samples containing a single parasite type was occasionally observed. It is possible that the presence of more than one type of parasite in single samples might be related to our recent demonstration that mixtures of SAG2 type I and type II parasites are present in a significant proportion of commercial meat preparations. Moreover, the presence of mixed infections in single patients might offer a direct molecular method of assessing risk factors for infection.     

Analysis of the SAG5 locus reveals a distinct genomic organisation in virulent and avirulent strains of Toxoplasma gondii

We have recently characterised, in the virulent strain RH of Toxoplasma gondii, three glycosylphosphatidylinositol-anchored surface antigens related to SAG1 (p30) and encoded by highly homologous, tandemly arrayed genes named SAG5A, SAG5B and SAG5C. In the present study, we compared the genomic organisation of the SAG5 locus in strains belonging to the three major genotypes of T. gondii. Southern blot analysis using a SAG5-specific probe produced two related but distinct hybridisation patterns, one exclusive of genotype I virulent strains, the other shared by avirulent strains of either genotype II or genotype III. To understand the molecular bases of this intergenotypic heterogeneity, we cloned and sequenced the SAG5 locus in the genotype II strain Me49. We found that in this isolate the SAG5B gene is missing, with SAG5A and SAG5C laying contiguously. This genomic arrangement explains the hybridisation profiles observed for all the avirulent strains examined and indicates that the presence of SAG5B is a distinctive trait of genotype I. Furthermore, we identified two novel SAG1-related genes, SAG5D and SAG5E, mapping respectively 1.8 and 4.0 kb upstream of SAG5A. SAG5D is transcribed in tachyzoites and encodes a polypeptide of 362 amino acids sharing 50% identity with SAG5A-C, whereas SAG5E is a transcribed pseudogene. We also evaluated polymorphisms at the SAG5 locus by comparing the coding regions of SAG5A-E from strains representative of the three archetypal genotypes. In agreement with the strict allelic dimorphism of T. gondii, we identified two alleles for SAG5D, whereas SAG5A, SAG5C and SAG5E were found to be three distinct nucleotide variants. The higher intergenotypic polymorphism of SAG5A, SAG5C and SAG5E suggests that these genes underwent a more rapid genetic drift than the other members of the SAG1 family. Finally, we developed a new PCR–restriction fragment length polymorphism method based on the SAG5C gene that is able to discriminate between strains of genotype I, II and III by a single endonuclease digestion.