Control of Seed Germination by Abscisic Acid : III. Effect on Embryo Growth Potential (Minimum Turgor Pressure) and Growth Coefficient (Cell Wall Extensibility) in Brassica napus L

The physical mechanism of seed germination and its inhibition by abscisic acid (ABA) in Brassica napus L. was investigated, using volumetric growth (= water uptake) rate (dV/dt), water conductance (L), cell wall extensibility coefficient (m), osmotic pressure (_i), water potential (Ψ_i), turgor pressure (P), and minimum turgor for cell expansion (Y) of the intact embryo as experimental parameters. dV/dt, _i, and Ψ_i were measured directly, while m, P, and Y were derived by calculation. Based on the general equation of hydraulic cell growth [dV/dt = Lm/(L + m) (Δ - Y), where Δ = _i - of the external medium], the terms (Lm/(L + m) and _i - Y were defined as growth coefficient (k_G) and growth potential (GP), respectively. Both k_G and GP were estimated from curves relating dV/dt (steady state) to of osmotic test solutions (polyethylene glycol 6000).

During the imbibition phase (0-12 hours after sowing), k_G remains very small while GP approaches a stable level of about 10 bar. During the subsequent growth phase of the embryo, k_G increases about 10-fold. ABA, added before the onset of the growth phase, prevents the rise of k_G and lowers GP. These effects are rapidly abolished when germination is induced by removal of ABA. Neither L (as judged from the kinetics of osmotic water efflux) nor the amount of extractable solutes are affected by these changes. _i and Ψ_i remain at a high level in the ABA-treated seed but drop upon induction of germination, and this adds up to a large decrease of P, indicating that water uptake of the germinating embryo is controlled by cell wall loosening rather than by changes of _i or L. ABA inhibits water uptake by preventing cell wall loosening. By calculating Y and m from the growth equation, it is further shown that cell wall loosening during germination comprises both a decrease of Y from about 10 to 0 bar and an at least 10-fold increase of m. ABA-mediated embryo dormancy is caused by a reversible inhibition of both of these changes in cell wall stability.

     

MAGE-C1/CT7 and MAGE-C2/CT10 are frequently expressed in multiple myeloma and can be explored in combined immunotherapy for this malignancy

The exact function of MAGE-C1/CT7 and MAGE-C2/CT10 is not yet understood in multiple myeloma (MM). However, the homologs MAGE-C1/CT7 and MAGE-C2/CT10 genes encode highly immunogeneic cancer/testis antigens (CTAs) and can be potential targets for T cell-based immunotherapy. MAGE-C1/CT7 and MAGE-C2/CT10 mRNA expression were investigated in MM patients, solitary plasmacytomas, monoclonal gammopathies of undetermined significance (MGUS) and bone marrow (BM) aspirates from healthy donors by RT-PCR. MAGE-C1/CT7 and MAGE-C1/CT10 were expressed in 67 and 59 % of the 46 MM analyzed patients. At least one of the genes was expressed in 76 % of MM cases. Solitary plasmacytoma also showed MAGE-C1/CT7 and MAGE-C2/CT10 expression. MAGE-C1/CT7 and MAGE-C2/CT10 were not expressed in normal BM samples, showing restricted expression of these CTA genes in MM, solitary plasmacytoma and MGUS. In the present study, we found high expression of the homologs MAGE-C1/CT7 and MAGE-C2/CT10 in monoclonal gammopathies and speculate whether these genes might represent a valuable therapeutic option for myeloma, in particular for combined immunotherapy.     

A new species of Stenoloba Staudinger, 1892 from China (Lepidoptera,Noctuidae, Bryophilinae)

A new species of Stenoloba from the olivacea species group, Stenoloba solaris, sp. n. (Lepidoptera, Noctuidae), is described from Yunnan, China. Illustrations of the male holotype and its genitalia are provided. A diagnostic comparison is made with Stenoloba albistriata Kononenko & Ronkay, 2000, Stenoloba olivacea (Wileman, 1914), and Stenoloba benedeki Ronkay, 2001 (Fig. 4).Open in a separate windowFigures 1–5.Stenoloba spp. adults and biotope. 1 Stenoloba solaris, sp. n., male, holotypus, Yunnan, China (GBG/ZSM) 2 Stenoloba albistriata, male, paratypus, N. Vietnam (ZFMK) 3 Stenoloba olivacea, male, Taiwan (HNHM) 4 Stenoloba benedeki, male, paratypus, N. Vietnam (HNHM) 5 Type locality of Stenoloba solaris, sp. n. China, NW Yunnan, Lijiang/Zhongdian near Tuguancum, 27°29''700"N, 99°53''700"E.     

Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay

Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.     

Germline mutations and variants in the succinate dehydrogenase genes in Cowden and Cowden-like syndromes

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.     

Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007–2014

The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it reinforces the relevance of using commercially available microbiological methods to assess antifungal susceptibility. These data improve our knowledge of the national distribution of species of the psilosis group, as there are very few studies of these species in Italy.     

Identification of Lactococcus lactis Genes Required for Bacteriophage Adsorption

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that 645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and 645 that showed no homology in the C-terminal part.     

Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae

DNA polymerase (Pol) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS–PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Pol. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Δdpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Δdpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Δdpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Pol in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S.cerevisiae Pol is conserved in eukaryotes.     

Interactive effects of haloclines and food patches on the vertical distribution of 3 species of temperate invertebrate larvae

In laboratory experiments, we examined the effect of haloclines and determined whether the presence of food patches overrides this effect on larval vertical distribution of the sea star Asterias rubens, the sea urchin Strongylocentrotus droebachiensis and the mussel Mytilus edulis. We experimentally constructed haloclines in which the salinity of the bottom water layer was 35 and that of the top layer was 21, 24, 27, and 30 (21/35, 24/35, 27/35, and 30/35) for A. rubens and S. droebachiensis, and 24, 27, 30 and 32 (24/35, 27/35, 30/35, and 32/35) for M. edulis. For each species and stage, additional halocline treatments (A. rubens: 24/32 and 27/32; 4-arm S. droebachiensis: 21/29 and 24/32; 6-arm S. droebachiensis: 24/29 and 24/32; M. edulis: 27/32 and 30/32) were used to determine whether the larval response to inhibitory salinity gradients was due to the absolute salinity of the top layer or the relative salinity difference between the two layers. Also, we measured the density of A. rubens and M. edulis to determine whether the specific gravity of larvae can explain the observed vertical distributions. Larvae aggregated at and below the halocline and these aggregations were more pronounced with increasing strength of the vertical salinity gradient. Threshold salinities in the top layer which inhibited ~ 100% of the larvae from crossing the halocline were 24 for A. rubens and M. edulis, and 21 for S. droebachiensis. These distributional patterns were not the result of larval density, which was greater than all treatment water densities for M. edulis and S. droebachiensis and lower for A. rubens. The effect of the presence of a food patch at inhibitory haloclines (A. rubens: 24/35 and 27/35; 4-arm S. droebachiensis: 21/34 and 24/34; M. edulis: 27/35) was determined by using three algal densities: 0, 5000 or 10 000 cells ml^- 1Thalassiosira pseudonana in either the top or the bottom water layer. For both A. rubens and M. edulis, the number of larvae at the halocline increased in the presence of a food patch, but this effect did not depend on algal density in the patch. For 4-arm S. droebachiensis, there was no effect of the presence of a food patch on larval vertical distribution. Our results suggest that low salinity may act as a barrier to vertical movement and that the presence of food patches above the halocline may strengthen the larval aggregation response to inhibitory haloclines.     

Diversity of Cryptosporidium spp. in Apodemus spp. in Europe

The genetic diversity of Cryptosporidium spp. in Apodemus spp. (striped field mouse, yellow-necked mouse and wood mouse) from 16 European countries was examined by PCR/sequencing of isolates from 437 animals. Overall, 13.7% (60/437) of animals were positive for Cryptosporidium by PCR. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences showed the presence of Cryptosporidium ditrichi (22/60), Cryptosporidium apodemi (13/60), Cryptosporidium apodemus genotype I (8/60), Cryptosporidium apodemus genotype II (9/60), Cryptosporidium parvum (2/60), Cryptosporidium microti (2/60), Cryptosporidium muris (2/60) and Cryptosporidium tyzzeri (2/60). At the gp60 locus, novel gp60 families XVIIa and XVIIIa were identified in Cryptosporidium apodemus genotype I and II, respectively, subtype IIaA16G1R1b was identified in C. parvum, and subtypes IXaA8 and IXcA6 in C. tyzzeri. Only animals infected with C. ditrichi, C. apodemi, and Cryptosporidium apodemus genotypes shed oocysts that were detectable by microscopy, with the infection intensity ranging from 2000 to 52,000 oocysts per gram of faeces. None of the faecal samples was diarrheic in the time of the sampling.     

DNA repair genes polymorphism and lung cancer risk with the emphasis to sex differences

Polymorphisms in nucleotide and base excision repair genes are associated with the variability in the risk of developing lung cancer. In the present study, we investigated the polymorphisms of following selected DNA repair genes: XPC (Lys939Gln), XPD (Lys751Gln), hOGG1 (Ser326Cys) and XRCC1 (Arg399Gln), and the risks they present towards the development of lung cancer with the emphasis to gender differences within the Slovak population. We analyzed 761 individuals comprising 382 patients with diagnosed lung cancer and 379 healthy controls. Genotypes were determined by polymerase chain reaction/restriction fragment length polymorphism method. We found out statistically significant increased risk for lung cancer development between genders. Female carrying XPC Gln/Gln, XPC Lys/Gln+Gln/Gln and XRCC1 Arg/Gln, XRCC1 Arg/Gln+Gln/Gln genotypes had significantly increased risk of lung cancer corresponding to OR = 2.06; p = 0.04, OR = 1.66; p = 0.04 and OR = 1.62; p = 0.04, OR = 1.69; p = 0.02 respectively. In total, significantly increased risk of developing lung cancer was found in the following combinations of genotypes: XPD Lys/Gln+XPC Lys/Lys (OR = 1.62; p = 0.04), XRCC1 Gln/Gln+hOGG1 Ser/Ser (OR = 2.14; p = 0.02). After stratification for genders, the following combinations of genotype were found to be significant in male: XPD Lys/Gln+XPC Lys/Lys (OR = 1.87; p = 0.03), XRCC1 Arg/Gln+XPC Lys/Lys (OR = 4.52; p = 0.0007), XRCC1 Arg/Gln+XPC Lys/Gln (OR = 5.44; p < 0.0001). In female, different combinations of the following genotypes were found to be significant: XRCC1 Arg/Gln+hOGG1 Ser/Ser (OR = 1.98; p = 0.04), XRCC1 Gln/Gln+hOGG1 Ser/Ser (OR = 3.75; p = 0.02), XRCC1 Arg/Gln+XPC Lys/Gln (OR = 2.40; p = 0.04), XRCC1 Arg/Gln+XPC Gln/Gln (OR = 3.03; p = 0.04). We found out decreased cancer risk in genotype combinations between female patients and healthy controls: XPD Lys/Lys+XPC Lys/Gln (OR = 0.45; p = 0.02), XPD Lys/Gln+XPC Lys/Lys (OR = 0.32; p = 0.005), XPD Lys/Gln+XPC Lys/Gln (OR = 0.48; p = 0.02). Our results did not show any difference between pooled smokers and non-smokers in observed gene polymorphisms in the association to the lung cancer risk. However, gender stratification indicated the possible effect of heterozygous constitution of hOGG1 gene (Ser/Cys) on lung cancer risk in female non-smokers (OR = 0.20; p = 0.01) and heterozygous constitution of XPC gene (Lys/Gln) in male smokers (OR = 2.70; p = 0.01).     

GBF/Gea mutant with a single substitution sustains fungal growth in the absence of BIG/Sec7

Golgi Arf1-guanine nucleotide exchange factors (GEFs) belong to two subfamilies: GBF/Gea and BIG/Sec7. Both are conserved across eukaryotes, but the physiological role of each is not well understood. Aspergillus nidulans has a single member of the early Golgi GBF/Gea-subfamily, geaA, and the late Golgi BIG/Sec7-subfamily, hypB. Both geaA and hypB are essential. hypB5 conditionally blocks secretion. We sought extragenic hypB5 suppressors and obtained geaA1. geaA1 results in Tyr1022Cys within a conserved GBF/Gea-specific S(Y/W/F)(L/I) motif in GeaA. This mutation alters GeaA localization. Remarkably, geaA1 suppresses hypBΔ, indicating that a single mutant Golgi Arf1-GEF suffices for growth.     

Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger

In early 2013, a Bengal tiger (Panthera tigris) in a zoo died of respiratory distress. All specimens from the tiger were positive for HPAI H5N1, which were detected by real-time PCR, including nose swab, throat swab, tracheal swab, heart, liver, spleen, lung, kidney, aquae pericardii and cerebrospinal fluid. One stain of virus, A/Tiger/JS/1/2013, was isolated from the lung sample. Pathogenicity experiments showed that the isolate was able to replicate and cause death in mice. Phylogenetic analysis indicated that HA and NA of A/Tiger/JS/1/2013 clustered with A/duck/Vietnam/OIE-2202/2012 (H5N1), which belongs to clade 2.3.2.1. Interestingly, the gene segment PB2 shared 98% homology with A/wild duck/Korea/CSM-28/20/2010 (H4N6), which suggested that A/Tiger/JS/1/2013 is a novel reassortant H5N1 subtype virus. Immunohistochemical analysis also confirmed that the tiger was infected by this new reassortant HPAI H5N1 virus. Overall, our results showed that this Bengal tiger was infected by a novel reassortant H5N1, suggesting that the H5N1 virus can successfully cross species barriers from avian to mammal through reassortment.     

Characteristics of TP53 germline variants and their correlations with Li-Fraumeni syndrome or Li-Fraumeni-like syndrome in Chinese tumor patients

Li-Fraumeni syndrome (LFS), a rare autosomal-dominant inheritance condition, is associated with a family cancer history as well as pathogenic/likely-pathogenic TP53 germline variants (P/LP TP53 GV). The current clinical methods for detecting LFS are limited. Here, we retrospectively investigate P/LP TP53 GV among Chinese cancer patients by next-generation sequencing and evaluate its relationship with a family cancer history. A total of 270 out of 19,226 cancer patients have TP53 GV, including 53 patients with P/LP TP53 GV. Patients with P/LP TP53 GV are mainly found in male with glioma, lung cancer or sarcoma. The median age of diagnosis for P/LP TP53 GV patients is significantly lower than that of non-P/LP TP53 GV patients (31-years vs. 53-years; P < 0.01). One LFS patient and 3 Li-Fraumeni-like syndrome (LFL) patients are among the 26 followed-up P/LP TP53 GV patients. Among 25 types of P/LP TP53 GV, the highest variant frequencies occurred at codon 175 and 248. p.M237I, p.R158H, p.C238Y and p.C275R, are firstly identified among the Chinese LFS/LFL patients. This study reports the (P/LP) TP53 GV characteristics of Chinese pan-cancer patients. These findings suggest analyzing the P/LP TP53 GV in cancer patients is an effective strategy for identifying cancer predisposition syndrome.     

Isolation and Functional Characterization of Calcitonin-Like Diuretic Hormone Receptors in Rhodnius prolixus

Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixus CT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogaster CT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC₅₀ = 200-300nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC₅₀ = 15nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.     

Cortical anchorages and cell type segregations of maternal postplasmic/PEM RNAs in ascidians

Ascidian postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs which include developmental determinants (macho-1 and pem-1). We have analyzed the localization, cortical anchorage and cell type segregation of postplasmic/PEM RNAs in Ciona intestinalis and Phallusia mammillata using very high-resolution fluorescent in situ hybridization. We also compared RNAs extracted from whole oocytes and from isolated cortices using microarrays and localized RNAs possessing clusters of xCACx motifs in their 3′UTRs.Based on these combined approaches we conclude that: (1) the vast majority of the 39 postplasmic/PEM RNAs (including vasa) are localized in the egg cortex. (2) Many postplasmic/PEM RNAs 3′UTR are enriched in xCACx motifs, allowing us to identify 2 novel postplasmic/PEM RNAs (PSD and MnK). (3) Postplasmic/PEM RNAs anchored to cortical Endoplasmic Reticulum (cER) and those associated with granules have different cell destinations.We propose that there are 2 distinct categories of postplasmic/PEM RNAs on the basis of their cortical anchorages and cell destinations: (1) macho-1-like postplasmic/PEM RNAs anchored to cER which segregate into somatic B8.11 cells. (2) vasa-like postplasmic/PEM RNAs associated with granules which in addition to B8.11 cells segregate into B8.12 germ cells.