A Time Point Proteomic Analysis Reveals Protein Dynamics of Plasmodium Oocysts.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

Label-free quantitative detection and comparative analysis of lysine acetylation during the different life stages of Eimeria tenella.

Proteome-wide lysine acetylation has been documented in apicomplexan parasite Toxoplasma gondii and Plasmodium falciparum. Here, we conducted the first lysine acetylome in unsporulated oocysts (USO), sporulated 7 h oocysts (SO 7h), sporulated oocysts (SO), sporozoites (S), and the second generation merozoites (SMG) of Eimeria tenella through a 4D label-free quantitative technique. Altogether, 8532 lysine acetylation sites on 2325 proteins were identified in E. tenella, among which 5445 sites on 1493 proteins were quantified. In addition, 557, 339, 478, 248, 241, and 424 differentially expressed proteins were identified in the comparisons SO7h vs USO, SO vs SO7h, SO vs USO, S vs SO, SMG vs S, and USO vs SMG, respectively. The bioinformatics analysis of the acetylome showed that the lysine acetylation is widespread on proteins of diverse functions. Moreover, the dynamic changes of lysine acetylome among E. tenella different life stages revealed significant regulation during the whole process of E. tenella growth and stage conversion. This study provides a beginning for the investigation of the regulate role of lysine acetylation in E. tenella and may provide new strategies for anticoccidiosis drug and vaccine development. Raw data are publicly available at iProX with the data set identifier PXD040368.

Characterization of CpCaM, a protein potentially involved in the growth of Cryptosporidium parvum.

Cryptosporidium parvum is an important apicomplexan parasite causing severe diarrhea in both humans and animals. Calmodulin (CaM), a multifunctional and universal calcium-binding protein, contributes to the growth and development of apicomplexan parasites, but the role of CaM in C. parvum remains unknown. In this study, the CaM of C. parvum encoded by the cgd2_810 gene was expressed in Escherichia coli, and the biological functions of CpCaM were preliminarily investigated. The transcriptional level of the cgd2_810 gene peaked at 36 h post infection (pi), and the CpCaM protein was mainly located around the nucleus of the whole oocysts, in the middle of sporozoites and around the nucleus of merozoites. Anti-CpCaM antibody reduced the invasion of C. parvum sporozoites by 30.69%. The present study indicates that CpCaM is potentially involved in the growth of C. parvum. Results of the study expand our knowledge on the interaction between host and Cryptosporidium.

An apicoplast-resident folate transporter is essential for sporogony of malaria parasites.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

Molecular characterization and immune protection by cystathionine ß-synthase from Eimeria tenella.

Eimeria tenella is an obligate intracellular apicomplexan parasite that causes avian coccidiosis and leads to severe economic losses in the global poultry industry. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL) act together to generate H2S in the reverse transsulfuration pathway. In this study, E. tenella Cystathionine ß-synthase (EtCBS) was cloned using rapid amplification of cDNA 5'-ends (5'RACE) and characterized, and its immunoprotective effects were evaluated. The recombinant EtCBS protein (rEtCBS) was expressed and successfully recognized by anti-sporozoites (Spz) protein rabbit serum. EtCBS mRNA levels were highest in Spz by qPCR, and the protein expression levels were higher in unsporulated oocysts (UO) than in other stages by Western blot. Indirect immunofluorescence showed that EtCBS protein was found on the surface of Spz and second-generation merozoites (Mrz). The invasion inhibition assays showed that rabbit anti-rEtCBS polyclonal antibodies effectively inhibited parasite invasion host cells. Chickens immunized with rEtCBS protein showed prominently increased weight gains and decreased oocyst output compared to nonimmunized and infected control group. The results suggest that EtCBS could be a potential vaccine candidate against E. tenella.

Characterization of the Eimeria tenella rhoptry protein with a nuclear localization sequence (EtROP30).

Rhoptry proteins (ROPs), secreted by specific rhoptry organelles of apicomplexan parasites, are determinants of parasite pathogenesis and sources of vaccine candidates. Twenty-eight ROPs of Eimeria tenella have been predicted by genomic approaches, and in the present study, E. tenella rhoptry protein 30 (EtROP30) was characterized. Subcellular localizations of EtROP30 in sporozoites and merozoites were in the apical complex and rhoptry-like bulb, suggesting that EtROP30 is a member of ROPs in E. tenella. Sequence analysis showed that EtROP30 contained an N-terminal secretory signal, a protein kinase domain with eight E. tenella-specific rhoptry kinase 1 subfamily (ROPK-Eten1) motifs, and a C-terminal nuclear localization sequence (NLS), making EtROP30 the only ROP that contains both a secretory signal and an NLS in E. tenella. Subsequent experiments showed that EtROP30 was a secreted protein in the sporozoite stage, relying on NLS for migration to the host nucleus. In addition, EtROP30 showed significantly higher expression levels in the parasite merozoite stage, indicating that EtROP30 plays a critical role during parasite reinvasion and development and may be a viable option as a vaccine candidate for anti-parasitic infection. The immunization protection efficacies of EtROP30 were evaluated. Significant improvements in mean body weight gain, reduction of cecum lesion score, and number of oocysts excreted were observed, indicating that EtROP30 has good immunogenicity against E. tenella. In the present study, a ROP of E. tenella with secretory and nuclear localization characteristics has been identified, and proved to be an effective vaccine candidate against this parasite.

Comparative Phosphoproteomic Analysis of Sporulated Oocysts and Tachyzoites of Toxoplasma gondii Reveals Stage-Specific Patterns.

Toxoplasma gondii is an obligate intracellular protozoan of severe threat to humans and livestock, whose life history harbors both gamic and apogamic stages. Chinese 1 (ToxoDB#9) was a preponderant genotype epidemic in food-derived animals and humans in China, with a different pathogenesis from the strains from the other nations of the world. Posttranslational modifications (PTMs) of proteins were critical mediators of the biology, developmental transforms, and pathogenesis of protozoan parasites. The phosphoprotein profiling and the difference between the developmental phases of T. gondii, contributing to development and infectivity, remain unknown. A quantitative phosphoproteomic approach using IBT integrated with TiO2 affinity chromatography was applied to identify and analyze the difference in the phosphoproteomes between the sporulated oocysts and the tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii. A total of 4058 differential phosphopeptides, consisting of 2597 upregulated and 1461 downregulated phosphopeptides, were characterized between sporulated the oocysts and tachyzoites. Twenty-one motifs extracted from the upregulated phosphopeptides contained 19 serine motifs and 2 threonine motifs (GxxTP and TP), whereas 16 motifs identified from downregulated phosphopeptides included 13 serine motifs and 3 threonine motifs (KxxT, RxxT, and TP). Beyond the traditional kinases, some infrequent classes of kinases, including Ab1, EGFR, INSR, Jak, Src and Syk, were found to be corresponding to motifs from the upregulated and downregulated phosphopeptides. Remarkable functional properties of the differentially expressed phosphoproteins were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. S8GFS8 (DNMT1-RFD domain-containing protein) and S8F5G5 (Histone kinase SNF1) were the two most connected peptides in the kinase-associated network. Out of these, phosphorylated modifications in histone kinase SNF1 have functioned in mitosis and interphase of T. gondii, as well as in the regulation of gene expression relevant to differentiation. Our study discovered a remarkable difference in the abundance of phosphopeptides between the sporulated oocysts and tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii, which may provide a new resource for understanding stage-specific differences in PTMs and may enhance the illustration of the regulatory mechanisms contributing to the development and infectivity of T. gondii.

CRISPR/Cas9 -mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection.

Plasmodium relies on numerous agonists during its journey through the mosquito vector, and these agonists represent potent targets for transmission-blocking by either inhibiting or interfering with them pre- or post-transcriptionally. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities for the study of agonist function and for developing malaria control strategies through gene deletion to achieve complete agonist inactivation. Here we have established a modified CRISPR/Cas9 gene editing procedure for the malaria vector Anopheles gambiae, and studied the effect of inactivating the fibrinogen-related protein 1 (FREP1) gene on the mosquito's susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants developed into adult mosquitoes that showed profound suppression of infection with both human and rodent malaria parasites at the oocyst and sporozoite stages. FREP1 inactivation, however, resulted in fitness costs including a significantly lower blood-feeding propensity, fecundity and egg hatching rate, a retarded pupation time, and reduced longevity after a blood meal.

Molecular characterization and immune protection of an AN1-like zinc finger protein of Eimeria tenella.

Coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria. Among them, Eimeria tenella is frequently considered to be the most pathogenic. Zinc finger proteins (ZnFPs) are a type of protein containing zinc finger domains. In the present study, a putative Eimeria tenella AN1-like ZnFP (E. tenella AN1-like zinc finger domain-containing protein, putative partial mRNA, EtAN1-ZnFP) was cloned and characterized, and its immune protective effects were evaluated. The 798-bp ORF sequence of EtAN1-ZnFP that encoded a protein of approximately 27.0 kDa was obtained. The recombinant EtAN1-ZnFP protein (rEtAN1-ZnFP) was expressed in Escherichia coli. Western blot analysis showed that the recombinant protein was recognized by the anti-GST monoclonal antibody and anti-sporozoite protein rabbit serum. qPCR analysis revealed that EtAN1-ZnFP was highly expressed in unsporulated oocysts and sporozoites. Immunostaining with an anti-rEtAN1-ZnFP antibody indicated that EtAN1-ZnFP was uniformly distributed in the cytoplasm of sporozoites, except for the refractive body; furthermore, this protein was evenly distributed in the cytoplasm of immature schizonts but seldom distributed in mature schizonts. The results of the in vitro invasion inhibition assay indicated that the antibodies against rEtAN1-ZnFP efficiently reduced the ability of E. tenella sporozoites to invade host cells. Animal challenge experiments demonstrated that the chickens immunized with rEtAN1-ZnFP protein significantly decreased mean lesion scores and fecal oocyst output compared with challenged control group. The results suggest that EtAN1-ZnFP can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.

Correlative light and electron microscopy of wall formation in Eimeria nieschulzi.

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII).

Therapeutic Efficacy of Bumped Kinase Inhibitor 1369 in a Pig Model of Acute Diarrhea Caused by Cryptosporidium hominis.

Recent reports highlighting the global significance of cryptosporidiosis among children have renewed efforts to develop control measures. We evaluated the efficacy of bumped kinase inhibitor (BKI) 1369 in the gnotobiotic piglet model of acute diarrhea caused by Cryptosporidium hominis, the species responsible for most human cases. Five-day treatment with BKI 1369 reduced signs of disease early during treatment compared to those of untreated animals. Piglets treated with BKI 1369 exhibited significant reductions of oocyst excretion, mucosal colonization by C. hominis, and mucosal lesions, which resulted in considerable symptomatic improvement. BKI 1369 reduced the parasite burden and disease severity in the gnotobiotic pig model. Together these data suggest that a BKI-mediated therapeutic may be an effective treatment against cryptosporidiosis.

Distribution and Processing of Eimeria nieschulzi OWP13, a New Protein of the COWP Family.

Eimeria species are important veterinary coccidian parasites and are transmitted between hosts via oocysts. The infectious sporozoites are protected by the oocyst and sporocyst wall. Tyrosine-rich proteins are well-known components of the Eimeria oocyst wall. Recently, cysteine motif containing proteins (COWP family), as described in Toxoplasma gondii and Cryptosporidium spp., have also been characterized in Eimeria. Here, we identified a novel COWP-related protein, EnOWP13, and tracked it via transfection technology in Eimeria nieschulzi. The subsequent analysis suggests that the mCherry-tagged EnOWP13 localizes to the wall-forming bodies I and the outer wall. Immunohistochemical analysis confirmed the distribution of wall-forming bodies similar to avian Eimeria species and revealed that the wall-forming bodies I show peroxidase activity. The EnOWP13 amino acid composition and FITC-cadaverine-positive wall-forming bodies I suggest a participation of an enzyme with transglutaminase activity. This is the first description and characterization of this novel outer oocyst wall protein, which is also orthologous to other Eimeria species and Toxoplasma gondii, suggesting a new potential cross-linking mechanism of wall-forming proteins via isopeptide bonds.

Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor.

Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers.

Dietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phosphatidylcholines (PCs) and sphingomyelins (SMs), affect cryopreservation success. The aim of the present study was to evaluate embryonic developmental rates after the IVF of oocytes retrieved from Nellore heifers fed for approximately 90 days with rumen-protected PUFAs rich in LA. In addition, we evaluated embryo cryotolerance and the membrane structure lipid composition using matrix-assisted laser desorption ionisation mass spectrometry of fresh and vitrified embryos. Embryo development to the blastocyst stage (mean 43.2%) and embryo survival after vitrification and warming (mean 79.3%) were unaffected by diet. The relative abundance of one lipid species (PC ether (PCe; 38:2, which means that this lipid has 38 carbon atoms and 2 double bonds in the fatty acyl residues) was increased after PUFAs supplementation. However, 10 ions were affected by cryopreservation; ions consistent with PC 32:0, PC 34:1, SM 24:1, PC 40:6 or PC 42:9, PC plasmalogen (PCp) 44:10 or PC 42:7, triacylglycerol (TAG) 54:9 and a not assigned ion (m/z 833.2) were lower in blastocysts that survived to the cryopreservation process compared with fresh blastocysts, whereas the abundance of the ions PC 36:3 or PC 34:0, PCe 38:2 or PC 36:6 and PC 36:5 or PCe 38:1 were increased after cryopreservation. Thus, the results demonstrate that the mass spectrometry profiles of PC, SM and TAG species differ significantly in bovine blastocysts upon cryopreservation. Because the lipid ion abundances of fresh and vitrified-warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.

Experimental Toxoplasma gondii and Eimeria tenella co-infection in chickens.

The widespread apicomplexan parasites Toxoplasma gondii (T. gondii) and Eimeria tenella (E. tenella) are important pathogens with high prevalence in poultry. The aim of our study was the investigation of mutual influences in co-infected chickens, focusing on immune response and course of infection. Two separate trials were performed using in total 96 1-day-old chickens, divided into four study groups: group NC (negative control, uninfected), group PC-T (oral or intramuscular infection with T. gondii oocysts (trial 1) or tachyzoites (trial 2), respectively), group PC-E (oral infection with E. tenella (trial 1) or E. tenella and Eimeria acervulina (trial 2)), and group TE (co-infection). T. gondii and Eimeria infections were validated by different parameters, and cytokine expression in the gut and spleen was investigated. T. gondii-specific antibodies were detected earliest 4 days post infection (p.i.) by immunoblot and direct DNA detection was possible in 22.1% of all tissue samples from infected chickens. Eimeria spp. merogony seemed to be enhanced by co-infection with T. gondii, interestingly without marked differences in oocyst excretion between co-infected and Eimeria spp. mono-infected chickens. An increase of messenger RNA (mRNA) expression of Th1- (IFN-γ, IL-12, TNF-α) and Th2-related cytokines (IL-10) mainly in groups PC-E and TE was observed, however, without statistically significant differences between co-infection and single infection with Eimeria. In conclusion, most of the measurable immune response could be attributed to Eimeria infection. To the best of our knowledge, this is the first report on co-infection experiments of T. gondii with Eimeria spp. in chickens.

Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts.

The surfaces of aged (10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labelled with fluorescein isothiocyanate while aged oocysts did not. In contrast, the walls of aged oocysts bound a lectin (wheat germ agglutinin, WGA), but not the walls of fresh oocysts. Exposure of oocysts to detergent solutions or trypsin did not affect the binding properties of the walls of the oocysts. However, exposure of fresh oocysts to acidified pepsin enabled labelling of the walls with WGA, presumably due to the relevant moieties on the oocyst walls becoming exposed. WGA binding, but not mAb binding, was partially abrogated with periodate exposure. These findings reveal a significant difference in the binding properties of oocyst walls from "aged" and "fresh" oocysts. The results are of relevance when considering technologies for isolating or detecting T. gondii oocysts in environmental samples based on oocyst surface properties, as used for other protozoan parasites. Our results suggest the possibility of developing a WGA-based separation procedure for isolating Toxoplasma oocysts from environmental matrices, in which pepsin pre-treatment would be included to ensure that both fresh and aged oocysts were isolated.

Expression of the Plasmodium berghei actin II gene is controlled by elements in a long genomic region.

Plasmodium parasites have two actin isoforms. Actin I is ubiquitously expressed, while the second actin isoform is expressed in the sexual stages and ookinetes. Reverse genetic analysis revealed two phenotypes in parasites lacking the protein: a block in male gametogenesis (exflagellation) and a second phenotype in oocyst development, dependent upon the expression of the gene in female gametocytes. Here, we report that the genetic complementation of two independent mutants lacking actin II does not fully restore wild-type function. Constructs were integrated in the c-rrna locus, previously used for expression of transgenes, in order to determine the dependence of expression on actin II flanking genomic regions. Partial restoration of male gametogenesis was achieved when the transgene contained, in addition to the coding region, 1.2 kb upstream of the actin II open reading frame. Another transgene, which comprised 2.7 kb of actin II 5' flanking regions and the cognate 3' downstream sequence, fully restored exflagellation. However, in both complemented strains, oocyst development was severely impaired compared to the WT. These data suggest that male gametocyte expression of actin II is dependent upon extensive flanking regions, while female expression requires even longer genomic sequences for correct expression of the gene.

Occurrence and first multilocus microsatellite genotyping of Neospora caninum from naturally infected dogs in dairy farms in Henan, Central China.

Neospora caninum is one of the important causes of abortion in dairy cattle worldwide. The dog is known as a definitive host of N. caninum and can transmit the parasite to cattle by shedding oocysts. The aim of the present study is to detect the presence of N. caninum in feces of dairy farm dogs and determine the genetic characteristics of N. caninum in Central China. A total of 78 fecal samples were collected from dogs in dairy farms from May to November 2014 and examined by microscopy and nested PCR based on Nc5 gene. Neospora-like oocysts were microscopically detected in two fecal samples, of which only one (Nc-LY1) was confirmed to be N. caninum by nested PCR. Seven out of 78 fecal samples (9.0 %) were N. caninum DNA positive, of which Neospora-like oocysts were simultaneously microscopically detected only in one sample (Nc-LY1). No statistical associations were found between the positive rates and age or sex of dogs (P > 0.05). The N. caninum-positive DNA samples were further analyzed by multilocus microsatellite (MS) genotyping for MS4, MS5, MS6A, MS7, MS8, MS10, MS12, and Cont-14. Only the fecal sample in which oocysts were detected was successfully genotyped at all genetic loci, and a new genotype was identified. To our knowledge, this study is the first report of genetic characterization of N. caninum isolates from naturally infected dogs based on multilocus microsatellites in China.

[REPRODUCTION OF SCHISTOSOMA MANSONI MOTHER SPOROCYST].

The development of generative elements of Schistosoma mansoni mother sporocysts (MS) was examined by histological methods. About 20 large cells, on average, determined as germinal cells (GC) were found in the miracidium. These cells formed a C-shape cellular aggregation (a band) beginning in the caudal part of the larva, and reaching the nerve ganglion in the anterior part. At the level of the 3d tier of epithelial plates of the miracidium, this band shifted to the external body wall, bypassing the zone of excretory channels. Apparently, this shift resulted in the subdivision of a single pool of GC into two structurally associated groups. A group of several undifferentiated cells (UC) was also revealed in the caudal part of the body. After the metamorphosis of the miracidium into sporocysts, GC had increased in size and on the 3d day started to divide, forming first embryos of daughter sporocysts. During the same time, germinal masses were being formed in the subtegumental area of the MS body. Since this time point, proliferation of UC occured only in germinal masses. A part of UC also differentiated there into GC. These cells formed sporocystoid embryos, developing as far as the germinal ball, and then came out into the sporocyst schizocoel (approximately in 10 days p. i.). Thus, in S. mansoni, the formation of generative elements into MS occurs in two stages. Primary GC are formed during the development of the miracidium into the egg, whereas secondary GC develop in germinal masses of the sporocyst.

Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) ß3 subunits in enhanced functional expression of mouse α6ß2ß3- and α6ß4ß3-nAChRs.

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mß4hß3- (∼ 5-8-fold) or WT mα6mß4mß3-nAChRs (∼ 2-fold) yielded higher function than mα6mß4-nAChRs. Function was not detected when mα6 and mß2 subunits were expressed together or in the additional presence of hß3 or mß3 subunits. However, function emerged upon expression of mα6mß2mß3(V9'S)-nAChRs containing ß3 subunits having gain-of-function V9'S (valine to serine at the 9'-position) mutations in transmembrane domain II and was further elevated 9-fold when hß3(V9'S) subunits were substituted for mß3(V9'S) subunits. Studies involving WT or gain-of-function chimeric mouse/human ß3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hß3 subunits as templates for site-directed mutagenesis studies, substitution with mß3 subunit residues in extracellular N-terminal domain loops "C" (Glu(221) and Phe(223)), "E" (Ser(144) and Ser(148)), and "ß2-ß3" (Gln(94) and Glu(101)) increased function of mα6mß2*- (∼ 2-3-fold) or mα6mß4* (∼ 2-4-fold)-nAChRs. EC50 values for nicotine acting at mα6mß4*-nAChR were unaffected by ß3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops ß2-ß3 and E) interfaces of ß3 subunits are some of the molecular impediments for functional expression of mα6mß2ß3- or mα6mß4ß3-nAChRs.