Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

Small vs. Large Library Docking for Positive Allosteric Modulators of the Calcium Sensing Receptor.

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism.

B56δ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan's syndrome mutations.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

Disease mutations and phosphorylation alter the allosteric pathways involved in autoinhibition of protein phosphatase 2A.

Mutations in protein phosphatase 2A (PP2A) are connected to intellectual disability and cancer. It has been hypothesized that these mutations might disrupt the autoinhibition and phosphorylation-induced activation of PP2A. Since they are located far from both the active and substrate binding sites, it is unclear how they exert their effect. We performed allosteric pathway analysis based on molecular dynamics simulations and combined it with biochemical experiments to investigate the autoinhibition of PP2A. In the wild type (WT), the C-arm of the regulatory subunit B56δ obstructs the active and substrate binding sites exerting a dual autoinhibition effect. We find that the disease mutant, E198K, severely weakens the allosteric pathways that stabilize the C-arm in the WT. Instead, the strongest allosteric pathways in E198K take a different route that promotes exposure of the substrate binding site. To facilitate the allosteric pathway analysis, we introduce a path clustering algorithm for lumping pathways into channels. We reveal remarkable similarities between the allosteric channels of E198K and those in phosphorylation-activated WT, suggesting that the autoinhibition can be alleviated through a conserved mechanism. In contrast, we find that another disease mutant, E200K, which is in spatial proximity of E198, does not repartition the allosteric pathways leading to the substrate binding site; however, it may still induce exposure of the active site. This finding agrees with our biochemical data, allowing us to predict the activity of PP2A with the phosphorylated B56δ and provide insight into how disease mutations in spatial proximity alter the enzymatic activity in surprisingly different mechanisms.

Extended regulation interface coupled to the allosteric network and disease mutations in the PP2A-B56δ holoenzyme.

An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56δ (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56δ holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56δ termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.

Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition.

Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.

Small-molecule inhibitors that disrupt the MTDH-SND1 complex suppress breast cancer progression and metastasis.

Metastatic breast cancer is a leading health burden worldwide. Previous studies have shown that metadherin (MTDH) promotes breast cancer initiation, metastasis and therapy resistance; however, the therapeutic potential of targeting MTDH remains largely unexplored. Here, we used genetically modified mice and demonstrate that genetic ablation of Mtdh inhibits breast cancer development through disrupting the interaction with staphylococcal nuclease domain-containing 1 (SND1), which is required to sustain breast cancer progression in established tumors. We performed a small-molecule compound screening to identify a class of specific inhibitors that disrupts the protein-protein interaction (PPI) between MTDH and SND1 and show that our lead candidate compounds C26-A2 and C26-A6 suppressed tumor growth and metastasis and enhanced chemotherapy sensitivity in preclinical models of triple-negative breast cancer (TNBC). Our results demonstrate a significant therapeutic potential in targeting the MTDH-SND1 complex and identify a new class of therapeutic agents for metastatic breast cancer.

Roles of constitutive and signal-dependent protein phosphatase 2A docking motifs in burst attenuation of the cyclic AMP response element-binding protein.

The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.

Methylation-regulated decommissioning of multimeric PP2A complexes.

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivation of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.

PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis.

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B'-family PP2A regulatory subunits and holoenzymes. The B'-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B'α-binding motifs serve as common binding sites for B' subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B'-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B' holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B' that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B' holoenzymes in various cellular processes.

Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.

Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.

Monitoring the transmission of Schistosoma japonicum in potential risk regions of China, 2008 - 2012.

Schistosomiasis japonica, caused by Schistosoma japonicum infection, remains a major public health concern in China, and the geographical distribution of this neglected tropical disease is limited to regions where Oncomelania hupensis, the intermediate host of the causative parasite, is detected. The purpose of this study was to monitor the transmission of S. japonicum in potential risk regions of China during the period from 2008 through 2012. To monitor the transmission, 10 fixed surveillance sites and 30 mobile sentinel sites were selected in 10 counties of four provinces, namely Anhui, Jiangsu, Chongqing and Hubei. There were 8, 9, 6, 2 and 3 cases infected with S. japonicum detected in the 30 mobile sentinel sites during the 5-year study period, while 27 subjects were positive for the antibody-based serum test in the 10 fixed sentinel sites; however, no infection was found. In addition, neither local nor imported livestock were found to be infected. No O. hupensis snails were detected in either the fixed surveillance or the mobile sentinel sites; however, the snail host was found to survive and reproduce at Chaohu Lake, inferring the potential of transmission of the disease. It is suggested that the continuous surveillance of schistosomiasis japonica should be carried out in both the endemic foci and potential risk regions of China, and an active, sensitive system to respond the potential risk of transmission seems justified.

Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease.

The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

[Risk evaluation of Schistosomiasis japonica in potential endemic areas in China].

OBJECTIVE: To analyze the impact of large hydraulic projects on schistosomiasis transmission and evaluate the transmission risk in potential endemic areas. METHODS: During 2008-2010, surveillance on risk factors related to schistosomiasis transmission and risk assessment were carried out in potential endemic sites in counties of Xuyu, Hongze, Jinhu, and Gaoyou in Jiangsu Province, Weishan County in Shangdong, Qianjiang City and Yiling District of Yichang City in Hubei, Juchao District of Chaohu City in Anhui Province, Wanzhou and Kaixian in Chongqing in Three Gorges Dam region or passed by South-to-North Water Diversion Project and Zhangjiagang City in Jiangsu Province located in lower reach of Yangtze River. At least one fixed and three temporary monitoring sites were set in each county (city or district). Local inhabitants aged 6-65 years old were screened by indirect haemagglutination assay (IHA) or ELISA, and the sero-positives were tested by Kato-Katz or miracidium hatching techniques to investigate possible infection in 2008. The endemic status of schistosomiasis in mobile population was surveyed every year during 2008-2010. Infection status in livestocks was surveyed in Juchao, Qianjiang, Gaoyou and Wanzhou Counties in 2008-2010. Oncomelania hupensis distribution was investigated in risky and suspicious areas. Snail spreading investigation was conducted through salvaging floater and snails-inducing by straw curtains in rivers connected with Yangtze River. RESULTS: 8 256 local inhabitants were investigated by serological tests with a positive rate of 0.7% (60/8 256). Among the 60 serologically positive subjects, 55 individuals were examined by stool examination but none of them was egg positive. The antibody prevalence rate of migrating population in 2008-2010 was 2.0%, 1.4%, and 1.7%, respectively, with no significant difference (chi2 = 3.57, P > 0.05). Among the serologically positive subjects, egg-positive cases were found in migrating population in Juchao District each year and one case was found in Jinhu County in 2010. Oncomelania snails were only found in in Jinhu and Gaoyou without infected ones. A lot of aquatic shell-fish and snails were collected in the water floater and straw curtain without Oncomelania snails. CONCLUSION: Schistosomiasis is not endemic in the original nonendemic areas in Three Gorges Dam region and areas passed by South-to-North Water Diversion Project until now, but potential risk of transmission exists. Long term surveillance scheme on schistosomiasis should be established with varied monitoring factors in different category areas.

[Influence factors of Enterobius vermicularis infection among pupils in Chongqing City].

OBJECTIVE: To investigate Enterobius vermicularis infection among primary school students and its influence factors in Chongqing. METHODS: Beibei and Changshou were selected as investigated points from October to December 2011. One primary school was randomly chosen from each of the 5 different directions in every investigated point. Adhesive cellophane anal swab was used to examine pinworm eggs for 3 consecutive days. Information on children's family, hygiene habits and school environment was collected by questionnaire. RESULTS: The total infection rate of E. vermicularis was 6.7% (71/1 071). The infection rate in rural schools (7.9%, 60/755) was higher than that of urban schools (3.8%, 12/316) (chi2 = 6.1169, P < 0.05). The rate in males and females was 6.3% (34/536) and 7.1% (38/535), respectively (chi2 = 0.2463, P > 0.05). Among the investigated children aged 6-12 years, the infection rate in 6-year-old children (16.03%) was highest. There was a statistical significance among age groups (chi2 = 29.1492, P < 0.01). With the increase of age, the rate decreased. Multivariate logistic regression analysis showed that location (OR = 0.411), age groups (OR = 0.714), education level of mothers (OR = 0.568), materials of classroom-ground (OR = 0.116) and types of boarding (OR = 0.272) were the influence factors on E. vermicularis infection in primary schools (P < 0.05). CONCLUSIONS: Pinworm control should more focused on rural children, younger group, mothers with lower education, classroom with cement ground and lodging schools in Chongqing City.

[Prevalence of Enterobius vermicularis infection of children and its influencing factors in Chongqing City].

OBJECTIVE: To understand the infection status of Enterobius vermicularis of children aged 3-12 years and its influencing factors in Chongqing City. METHODS: One rural county and 1 urban county were selected as investigation areas. The children aged 3-12 years of 5 towns from 5 different directions of each county were investigated. About 150 children were investigated every town. The Enterobius vermicularis eggs of children were detected by the adhesive cellophane anal swab method. The children's parent education levels and personal circumstances of sanitation were investigated by the questionnaire survey. RESULTS: Among 1 592 children investigated, the total infection rate of Enterobius vermicularis was 6.85%. The infection rates of boys and girls were 6.29% and 7.40%, respectively. The infection rates of rural and urban areas were 12.13% and 2.14%, respectively. The infection rate in children aged 5-7 years was the highest (13.56%) among all the child aged groups. There were significant differences for the infection rates of Enterobius vermicularis among children of different aged groups, different areas, different education levels and occupations of parents, washing hand before meal or not, washing hand after WC or not, different classroom grounds, and lodging or not (P < 0.01 or P < 0.05). The multivariate logistic analysis indicated that aged group, different areas, classroom ground and lodging children were independent risk factors for Enterobius vermicularis infection. CONCLUSIONS: The infection rate of Enterobius vermicularis in rural area is higher than that in urban area. The control emphases should be the children of low group, rural area, poor classroom and lodging.

[Conditions of schistosomiasis laboratories at county level].

OBJECTIVE: To understand the conditions of schistosomiasis laboratories at county level, so as to supply the information for diagnostic capacity building. METHODS: A questionnaire survey was conducted at 40 random selected counties which were in charge of national surveillance for schistosomiasis. The information of staffs, areas of laboratories, equipment configuration and diagnostic methods from each laboratory were collected. The serum specimens and Kato-Katz thick smears from the national surveillance sites were randomly selected and retested by provincial staffs to evaluate the testing ability of the personnel. RESULTS: The average age of laboratory staffs was 40.93 +/- 9.56 years old, 69.39% of staffs were older than 35 years, 86.22% of them had education background below bachelor degree. Except missed professional titles of 4 persons, the staffs with primary, middle and high professional titles accounted for 56.63%, 39.29% and 2.04%, respectively. The laboratory areas varied greatly while independent schistosomiasis stations had the lowest areas with 52.81 +/- 40.08 m2, and the equipment configurations of laboratories were in a low level. The consistency rates of primary test and reexamination for serum specimens and thick smear slides were 95.89% and 99.53%, respectively, with the Kappa value over 0.90. Nine immunodiagnostic kits were used in these laboratories, and Kato-Katz technique and miracidium hatching technique were the main parasitological methods. CONCLUSIONS: The personnel structure of laboratories at primary prevention and treatment facilities for schistosomiasis is unreasonable, while the basic infrastructure of laboratories is backward and the use of diagnostic assays/methods is disordered. The diagnostic capacity building should be strengthened, and the construction and management of schistosomiasis laboratories should be standardized.

[Construction and expression of recombinant plasmid pET28alpha-Sj26GST of Schistosoma japonicum in Escherichia coli BL21].

OBJECTIVE: To construct and express recombinant plasmid pET28a-Sj26GST of Schistosoma japonicum (Sj) in Escherichia coli BL21 (DE3). METHODS: The total RNA was extracted from Sj adult worms by ultrasound-breaking. The Sj26GST antigen gene was amplified by RT-PCR from the total RNA, and then cloned into prokaryotic expression plasmid pET28alpha and transformed into E. coli BL2 (DE3). The BL21(pET28a-Sj26GST) was induced with isopropyl-beta-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified with SDS-PAGE and Western blot. RESULTS: The 676 bp Sj26GST gene was successfully amplified by RT-PCR and cloned into pET28alpha. The recombinant plasmid pET28a-Sj26GST was successfully constructed, with a relative molecular weight of expressed recombinant protein at approximately 36 x 10(3) as determined by SDS-PAGE. The amount of the expressed protein comprised 26% of the total bacterial proteins. The fusion protein could be recognized by the sera of rabbits infected with Sj. CONCLUSION: The recombinant plasmid pET28alpha-Sj26GST is successfully constructed and highly expressed in E. coli in a fused form with His-tag. The expressed fusion protein shows specific antigenicity.

[Impact of people's social behavior on schistosomiasis transmission in the Three Gorges Reservoir area].

OBJECTIVE: To investigate the changes in natural ecology and social environment after the construction of the dam in the Three Gorges Reservoir area, and to understand the social behavior status and education backgrounds of the local residents, and to analyze the potential impact of these factors on schistosomiasis spreading. METHODS: Data of nature and social economy after the construction of the dam in the Three Gorge area were collected, and a cross-sectional study with questionnaire survey on information including social demographic characteristics, people's production and life style, knowledge about schistosomiasis was conducted among natives, emigrations. RESULTS: After the construction of the dam, as the stream became slower, the water-level fluctuating zone on the bank of the reservoir was formed. The main source of drinking water and daily life activity are mainly depends on the river supplying (accounting about 68.7% and 75.8%, respectively); due to the needs of farming, washing cloths and vegetables, 45.6% of resident had a close contact with river water. People with away-from-home employment among emigrations were increased from 5.7% to 18.7%. The proportion of using hygienic lavatory was 88.6%. The main source of water for drinking and daily life among emigrations was tap water (85.4% and 87.0%). The residents had no awareness about the health hazard for schistosomiasis, with less health information CONCLUSION: The natural environment of reservoir areas and the life style are risk factors for the spread of schistosomiasis. The improved sanitation condition and the economy construction after the construction of the dam is propitious to keeping away from the introduction of schistosomiasis. However, people with away-from-home employment among emigrations are still at risk of schistosomiasis.