采用磁微粒分离酶联免疫法构建基于KGN细胞的雌激素生物合成筛选模型

雌激素在机体生长发育中发挥着重要作用,其合成代谢紊乱会导致乳腺癌和骨质疏松等疾病发生.目前,基于细胞的雌激素合成筛选模型需用到放射性底物,对环境污染大,成本较高,限制了具有组织特异性调控雌激素合成的药物筛选.我们以高表达芳香化酶的KGN细胞为检测对象,比较基于聚苯乙烯酶联免疫法和磁微粒分离酶联免疫法的雌二醇ELISA试剂盒的交叉反应和灵敏度,发现相对于聚苯乙烯酶联免疫法,磁微粒分离酶联免疫法能够稳定高效的检测雌激素合成.进一步比较培养基中酚红和底物睾酮对雌二醇检测的影响,成功建立通过磁微粒酶联免疫法检测KGN细胞雌二醇合成的筛选模型.     

Cbp1, a fungal virulence factor under positive selection,forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.     

Biosynthesis of recombinant human hepatitis B M-HBsAg in silkworm larvae

The middle surface antigen (M-HBsAg) of human hepatitis B virus is virus envelope protein. It's used as a basis for development of vaccine and test-system for detecting of hepatitis B virus. The cDNA of M-HBsAg was inserted into transfer vector pBK273 under the polyhedron promoter with obtaining of recombinant plasmid DNA pBHep-2. As a result of cotransfection pBHep-2 with wild type BmNPV the recombinant baculovirus rBmNPVHep which included the cDNA of M-HBsAg under the polyhedron promoter was obtained. Infection of silkworm larvae Bombyx mori with recombinant virus resulted in expression of foreign gene and accumulation of middle surface antigen of human hepatitis B virus mostly (>90%) in fat bodies of silkworm larvae.     

Expression of a foreign gene by cysteine proteinase null recombinant baculovirus

Baculovirus expression vector systems (BEVSs) are broadly used for producing foreign proteins in lepidopteran cells. Most commercial BEVSs are engineered to insert foreign genes into the polyhedrin (polh) locus. They lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. To avoid this, expression cassettes can be inserted in other parts of the virus genome. The preS2-S gene, coding for the recombinant middle surface antigen of the human hepatitis B virus (M-HBsAg), was expressed from the baculovirus construct rBmNPV-Δv-cath-M-HBsAg, inserting the foreign gene into the v-cath locus of Bombyx mori nucleopolyhedrovirus (BmNPV) so that v-cath was deleted and native polh was retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced till very late stages of infection. Infection of larvae with a mixture of the recombinant and wild-type baculoviruses was followed by degradation of the bulk of the produced M-HBsAg as early as 96 h after inoculation.     

Expression of foreign gene by cysteine proteinase null recombinant baculovirus

The baculovirus expression vector systems (BEVS) are broadly used for producing foreign proteins in lepidopteran larvae. Most commercial BEVS are engineered to insert foreign genes into the polyhedrin (polh) locus and lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. Current knowledge in baculovirus genomics makes it possible to engineer BEVS into other parts of the virus genome. In our work, we have expressed recombinant M-HBsAg (middle surface antigen of human hepatitis B) in the baculovirus construct, rBmNPV-Deltav-cath-M-HBsAg, inserting foreign gene into the v-cath locus of the Bombyx mori nucleopolyhedrovirus (BmNPV) such that the v-cath gene is deleted and the native polh gene is retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced at a very late stage of infection.     

Amyloid Peptide Channels

At least 16 distinct clinical syndromes including Alzheimer’s disease (AD), Parkinson’s disease (PD), rheumatoid arthritis, type II diabetes mellitus (DM), and spongiform encephelopathies (prion diseases), are characterized by the deposition of amorphous, Congo red-staining deposits known as amyloid. These “misfolded” proteins adopt β-sheet structures and aggregate spontaneously into similar extended fibrils despite their widely divergent primary sequences. Many, if not all, of these peptides are capable of forming ion-permeable channels in vitro and possibly in vivo. Common channel properties include irreversible, spontaneous insertion into membranes, relatively large, heterogeneous single-channel conductances, inhibition of channel formation by Congo red, and blockade of inserted channels by Zn²⁺. Physiologic effects of amyloid, including Ca²⁺ dysregulation, membrane depolarization, mitochondrial dysfunction, inhibition of long-term potentiation (LTP), and cytotoxicity, suggest that channel formation in plasma and intracellular membranes may play a key role in the pathophysiology of the amyloidoses.     

Investigation of the Gelation of Galactomannan Isolated from the Seeds of Styphnolobium japonicum (Fabaceae)

Russian Journal of Bioorganic Chemistry - This article is devoted to the study of the structuring process of galactomannan isolated from Japanese Sophora seeds with borate ions. Based on...     

Effects of pesticide fluometuron (cotoran) on template synthesis of RNA]

The experiments on the investigation of pesticide fluometuron (cotoran) influence on nuclease sensitivity and template activity of rat liver chromatin were carried out. Cotoran was found to bind specifically with non-histone proteins of chromatin. It was shown that this pesticide considerably decreases template activity of chromatin and its sensitivity to the action of nucleases. It suggests, that certain conformation changes occur in chromatin upon the action of cotoran.     

Use of radioactive—Labeled ABA for rapid determination of leaf abscission periods in cotton seedlings

A simple and rapid method is described for determination of cotton leaf abscission periods using radioactive-labeled ABA in cotton seedlings. A step-by-step protocol for cotton aged 5–6 days is provided.