Eggplant latent viroid,the candidate type species for a new genus within the family Avsunviroidae (hammerhead viroids)

Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name ELAVIROID:     

Male age and strain affect ejaculate quality in the Mexican fruit fly

Aging in all organisms is inevitable. Male age can have profound effects on mating success and female reproduction, yet relatively little is known on the effects of male age on different components of the ejaculate. Furthermore, in mass‐reared insects used for the Sterile Insect Technique, there are often behavioral differences between mass‐reared and wild males, while differences in the ejaculate have been less studied. The ejaculate in insects is composed mainly of sperm and accessory gland proteins. Here, we studied how male age and strain affected (i) protein quantity of testes and accessory glands, (ii) the biological activity of accessory gland products injected into females, (iii) sperm viability, and (iv) sperm quantity stored by females in wild and mass‐reared Anastrepha ludens (Diptera: Tephritidae). We found lower protein content in testes of old wild males and lower sperm viability in females mated with old wild males. Females stored more sperm when mated to young wild males than with young mass‐reared males. Accessory gland injections of old or young males did not inhibit female remating. Knowledge of how male age affects different ejaculate components will aid our understanding on investment of the ejaculate and possible postcopulatory consequences on female behavior.     

Circular and linear DNA molecules in the Entamoeba histolytica complex molecular karyotype

Entamoeba histolytica genome was analysed by pulsed field gel electrophoresis under conditions to separate linear chromosomes in the 170–1400 kb range. We identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three different methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric field and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at different pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and difficulties in the interpretation of the molecular karyotype of E. histolytica. Received: 19 January 1999 / Revised version: 3 November 1999 / Accepted: 22 November 1999     

Performance in working memory and attentional control is associated with the rs2180619 SNP in the CNR1 gene

Individual differences in cognitive performance are partly dependent, on genetic polymporhisms. One of the single‐nucleotide polymorphisms (SNP) of the CNR1 gene, which codes for cannabinoid receptor 1 (CB1R), is the rs2180619, located in a regulatory region of this gene (6q14–q15). The alleles of the rs2180619 are A > G; the G allele has been associated with addiction and high levels of anxiety (when the G allele interacts with the SS genotype of the 5‐HTTLPR gene). However, GG genotype is observed also in healthy subjects. Considering G allele as risk for ‘psychopathological conditions’, it is possible that GG healthy subjects do not be addicted or anxious, but would have reduced performance, compared to AA subjects, in attentional control and working memory processing. One hundred and sixty‐four healthy young Mexican‐Mestizo subjects (100 women and 64, men; mean age: 22.86 years, SD=2.72) participated in this study, solving a task where attentional control and working memory were required. GG subjects, compared to AA subjects showed: (1) a general lower performance in the task (P = 0.02); (2) lower performance only when a high load of information was held in working memory (P = 0.02); and (3) a higher vulnerability to distractors (P = 0.03). Our results suggest that, although the performance of GG subjects was at normal levels, a lower efficiency of the endocannabinoid system, probably due to a lowered expression of CB1R, produced a reduction in the performance of these subjects when attentional control and working memory processing is challenged .     

Molecular cloning and characterization of two novel autophagy-related genes belonging to the ATG8 family from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Rhipicephalus (Boophilus) microplus is an obligate haematophagous arthropod and the major problem for cattle industry due to economic losses it causes. The parasite shows a remarkable adaptability to changing environmental conditions as well as an exceptional ability to survive long-term starvation. This ability has been related to a process of intracellular protein degradation called autophagy. This process in ticks is still poorly understood and only few autophagy-related (ATG) genes have been characterized. The aim of the present study was to examine the ESTs database, BmiGI, of R. microplus searching for ATG homologues. We predicted five putative ATG genes, ATG3, ATG4, ATG6 and two ATG8s. Further characterization led to the identification of RmATG8a and RmATG8b, homologues of GABARAP and MAP1LC3, respectively, and both of them belonging to the ATG8 family. PCR analyses showed that the expression level of RmATG8a and RmATG8b was higher in egg and larval stages when compared to ovary and midgut from adult ticks. This up-regulation coincides with the period in which ticks are in a starvation state, suggesting that autophagy is active in R. microplus.     

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

Purification of HA-tagged P2Y₂ receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y₂ receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y₂ nucleotide receptors from metabolically [³²P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [³²P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y₂ receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y₂ receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y₂ receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y₂ nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005)     

Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus

Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.     

The Chlamydia pneumoniae Inclusion Membrane Protein Cpn1027 Interacts with Host Cell Wnt Signaling Pathway Regulator Cytoplasmic Activation/Proliferation-Associated Protein 2 (Caprin2)

We previously identified hypothetical protein Cpn1027 as a novel inclusion membrane protein that is unique to Chlamydia pneumoniae. In the current study, using a yeast-two hybrid screen assay, we identified host cell cytoplasmic activation/proliferation-associated protein 2 (Caprin2) as an interacting partner of Cpn1027. The interaction was confirmed and mapped to the C-termini of both Cpn1027 and Caprin2 using co-immunoprecipitation and GST pull-down assays. A RFP-Caprin2 fusion protein was recruited to the chlamydial inclusion and so was the endogenous GSK3β, a critical component of the β-catenin destruction complex in the Wnt signaling pathway. Cpn1027 also co-precipitated GSK3β. Caprin2 is a key regulator of the Wnt signaling pathway by promoting the recruitment of the β-catenin destruction complex to the cytoplasmic membrane in the presence of Wnt signaling while GSK3β is required for priming β-catenin for degradation in the absence of Wnt signaling. The Cpn1027 interactions with Caprin2 and GSK3β may allow C. pneumoniae to actively sequester the β-catenin destruction complex so that β-catenin is maintained even in the absence of extracellular Wnt activation signals. The maintained β-catenin can trans-activate Wnt target genes including Bcl-2, which may contribute to the chlamydial antiapoptotic activity. We found that the C. pneumoniae-infected cells were more resistant to apoptosis induction and the anti-apoptotic activity was dependent on β-catenin. Thus, the current study suggests that the chlamydial inclusion protein Cpn1027 may be able to manipulate host Wnt signaling pathway for enhancing the chlamydial anti-apoptotic activity.     

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.     

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.