Analysis of ascorbate peroxidase genes expressed in resistant and susceptible wheat lines infected by the cereal cyst nematode, Heterodera avenae

Changes in ascorbate peroxidase (APX) enzyme activity in response to nematode (Heterodera avenae) attack were studied in roots of three hexaploid wheat lines carrying Cre2, Cre5, or Cre7 nematode resistance genes and the susceptible Triticum aestivum cv. Anza. A spectrophotometric analysis was carried out with root extracts of infected plants 4, 7, 11, and 14 days after nematode inoculation using uninfected plant as control. APX induction in infected resistant genotypes was similar and higher than in the susceptible control. The introgression wheat/Aegilops ventricosa H-93-8 line, carrying the Cre2 gene, and its parental line H-10-15 as susceptible control were used to analyze whether this increase of activity was correlated with the induction of APX gene expression. Genes encoding cytosolic forms of APX were induced in roots of both lines in response to nematode infection. This induction took place both earlier and with greater intensity in the resistant line than in the susceptible one, and it was also higher in the root area at the site of nematode attachment.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as α-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies

Novel α-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group = 4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.     

Accounting for mating pair formation in plasmid population dynamics

Plasmids are important vehicles for horizontal gene transfer and rapid adaptation in bacteria, including the spread of antibiotic resistance genes. Conjugative transfer of a plasmid from a plasmid-bearing to a plasmid-free bacterial cell requires contact and attachment of the cells followed by plasmid DNA transfer prior to detachment. We introduce a system of differential equations for plasmid transfer in well-mixed populations that accounts for attachment, DNA transfer, and detachment dynamics. These equations offer advantages over classical mass-action models that combine these three processes into a single “bulk” conjugation rate. By decomposing the process of plasmid transfer into its constituent parts, this new model provides a framework that facilitates meaningful comparisons of plasmid transfer rates in surface and liquid environments. The model also allows one to account for experimental and environmental effects such as mixing intensity. To test the adequacy of the model and further explore the effects of mixing on plasmid transfer, we performed batch culture experiments using three different plasmids and a range of different mixing intensities. The results show that plasmid transfer is optimized at low to moderate shaking speeds and that vigorous shaking negatively affects plasmid transfer. Using reasonable assumptions on attachment and detachment rates, the mathematical model predicts the same behavior.     

Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.Dengue virus (DENV), a member of the Flaviviridae family, is a human pathogen causing dengue fever, the most common mosquito-borne viral disease in humans. The virus has become a major international public health concern, with 3 billion people at risk for infection and an estimated 50 million dengue cases worldwide every year (28). Neither specific antiviral therapies nor licensed vaccines are available, and the biology of the virus is poorly understood.DENV is a small enveloped virus containing a positive-stranded RNA genome with a length of approximately 10.7 kb. The virus encodes one large polyprotein that is co- and posttranslationally cleaved into 10 viral proteins. The structural proteins C, prM/M, and E are located in the N terminus, followed by the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (6, 10). NS5, the largest of the viral proteins, functions as an RNA-dependent RNA polymerase (RdRP) (29). The coding region is flanked at both ends by untranslated regions (UTR). The 5′ end has a type I cap structure (m⁷GpppAmp) mediating cap-dependent translation, but the virus can switch to a noncanonical translation mechanism under conditions in which translation factors are limiting (13). Cellular mRNAs are known to circularize via a protein-protein bridge between eIF4G and eIF4E (the cap binding complex) at the 5′ end and the poly(A) binding protein (PABP) at the 3′ end, enhancing translation efficiency. Despite the fact that the DENV 3′ UTR lacks a poly(A) tail, recent findings demonstrated binding of PABP to the 3′ UTR and an effect on RNA translation, suggesting a similar mechanism (12, 26).In addition to a presumed protein-mediated genome circularization regulating viral translation, an RNA-RNA-based 5′ and 3′ (5′-3′) end interaction, which can occur in the absence of proteins, leads to circularization of the viral genome (1, 3, 4, 18, 20, 30, 33, 34). This cyclization of the genome is necessary for viral RNA replication, and thus far, two complementary sequences at the 5′ and 3′ ends have been identified (3). The first are the cyclization sequences (CS) present in the capsid-coding region at the 5′ end (5′ CS) and upstream of the 3′ stem-loop (3′ SL) in the 3′ UTR (3′ CS) (2, 4, 18, 20, 30). A second sequence, known as the 5′ upstream AUG region (5′ UAR) element in the 5′ UTR, base pairs with its complementary 3′ UAR counterpart, which is located at the bottom part of 3′ SL (1, 4, 30). Recently, the structure of the 5′ end of the DENV genome hybridized to the 3′ end was determined in solution (25), confirming previous computer-predicted structures for genome cyclization (4, 20, 30). Besides the base pairing between 5′-3′ UAR and 5′-3′ CS sequences, a third stretch of nucleotides was identified to form a double-stranded (ds) region between the 5′ and 3′ ends.In addition to RNA sequences involved in 5′-3′-end interactions that are necessary for cyclization, the 5′ end of the viral genome harbors at least two more functional RNA elements, the stem-loop A (SLA) and capsid-coding region hairpin (cHP). The SLA consists of the first 70 nucleotides (nt) of the genome, forming a stable stem-loop structure. This structure has been confirmed in several studies and identified as a promoter element for RNA synthesis that recruits the viral RdRp NS5 (16, 22). Once NS5 is bound to the SLA at the 5′ end, it is believed to be delivered to the initiation site of minus-strand RNA synthesis at the 3′ end via 5′-3′ RNA-RNA circularization. In addition, a short poly(U) tract located immediately downstream of SLA has been shown to be necessary for RNA synthesis, although it is not involved in genome circularization (22). Finally, the cHP element resides within the capsid-coding region; it directs start codon selection and is essential for RNA replication (8, 9). The cHP structure is more important than its primary sequence. For start codon selection, it is believed that the cHP stalls the scanning initiation complex over the first AUG, favoring its recognition (9). In the case of RNA replication, the cHP likely stabilizes the overall 5′-3′ panhandle structure or participates in recruitment of factors associated with the replicase machinery (8).In this study, we demonstrate that in addition to the 5′ CS and 5′ UAR sequences, a third stretch of nucleotides in the 5′ end is required for RNA replication and appears to be involved in genome circularization. This new motif is located downstream of the AUG and was therefore designated the downstream AUG region (5′ DAR) element, with the predicted counterpart in the 3′ end designated the 3′ DAR. Our results indicate that the 5′ DAR modulates RNA-RNA interaction and RNA replication, and restoring complementarity between the 5′ DAR and 3′ DAR rescues detrimental effects caused by mutations in the 5′ DAR on genome circularization and RNA replication. Although the role of the predicted 3′ DAR counterpart is less conclusive, it may serve to make other structures and sequences in the 3′ end available for 5′-3′ RNA-RNA interaction to facilitate the replication-competent conformation of the DENV genome.Furthermore, we analyzed the functional interplay of RNA elements in the viral 5′ end, showing that two separate units are formed during replication. The first consists of the SLA, and it must be located at the very 5′ end of the genome. The second unit includes UAR, DAR, cHP, and CS elements, and the positional requirements are more flexible within the DENV RNA 5′ terminus. However, all four elements in the second unit must act in concert, forming a functional tertiary RNA structure of the circularized viral genome.     

Human Enterovirus 109: a Novel Interspecies Recombinant Enterovirus Isolated from a Case of Acute Pediatric Respiratory Illness in Nicaragua

Enteroviruses (Picornaviridae family) are a common cause of human illness worldwide and are associated with diverse clinical syndromes, including asymptomatic infection, respiratory illness, gastroenteritis, and meningitis. In this study, we report the identification and complete genome sequence of a novel enterovirus isolated from a case of acute respiratory illness in a Nicaraguan child. Unbiased deep sequencing of nucleic acids from a nose and throat swab sample enabled rapid recovery of the full-genome sequence. Phylogenetic analysis revealed that human enterovirus 109 (EV109) is most closely related to serotypes of human enterovirus species C (HEV-C) in all genomic regions except the 5′ untranslated region (5′ UTR). Bootstrap analysis indicates that the 5′ UTR of EV109 is likely the product of an interspecies recombination event between ancestral members of the HEV-A and HEV-C groups. Overall, the EV109 coding region shares 67 to 72% nucleotide sequence identity with its nearest relatives. EV109 isolates were detected in 5/310 (1.6%) of nose and throat swab samples collected from children in a pediatric cohort study of influenza-like illness in Managua, Nicaragua, between June 2007 and June 2008. Further experimentation is required to more fully characterize the pathogenic role, disease associations, and global distribution of EV109.The genus Enterovirus (EV) in the family Picornaviridae is a group of related viruses that are associated with a spectrum of disease, ranging from subclinical infections to acute respiratory and gastrointestinal illness to more severe manifestations, such as aseptic meningitis, encephalitis, and acute flaccid paralysis (16, 32). Enteroviruses are small, nonenveloped viruses that share a genomic organization. The RNA genome is a ∼7.5 kb single-stranded, positive-sense, polyadenylated molecule, with a single, long open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 5′ UTR is ∼700 nucleotides in length and contains highly structured secondary elements with internal ribosomal entry site (IRES) function. The ∼2,200-amino-acid (aa) polyprotein is cotranslationally processed by viral proteases to yield structural (VP4, VP2, VP3, and VP1) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (32). Current enterovirus classification is based on the high sequence divergence within the VP1 capsid region, which has been shown to correspond with serotype neutralization (27, 28). Human enterovirus (HEV) types are currently classified into four species, human enterovirus A (HEV-A), HEV-B, HEV-C (including poliovirus), and HEV-D, based on the four phylogenetic clusters observed in comparisons of the coding region sequences. An enterovirus is considered a new type within a species if it possesses <75% nucleotide identity and <85% amino acid identity with known members across the VP1 sequence (27, 30). Molecular identification methods play a crucial role in rapid, sensitive enterovirus diagnostics and have led to the recent discovery of several novel enteroviruses (29, 31, 40, 42, 44). Most approaches target a limited number of conserved regions in the 5′ UTR and VP4-VP2 junction or seek to ascertain serotype information by probing antigenic regions, such as VP1 (5).Picornavirus RNA-dependent RNA polymerases are highly error prone and lack proofreading ability, resulting in a misincorporation frequency of 1 per 10³ to 10⁴ nucleotides (48). The relative infidelity of these polymerases is believed to enable rapid adaptability under selective pressure. Large-impact evolutionary events, such as recombination within and between enterovirus serotypes, also contribute to their evolution and genetic diversity (3, 8, 26, 39) and may lead to changes in disease associations with human enterovirus infections. Human enteroviruses are classified into four species based on coding region sequence phylogeny, and intraspecies recombination events between enteroviruses that are closely related in the coding region are well documented (26, 38, 39). All known enterovirus 5′ UTR sequences, however, cluster into two groups containing either HEV-A and -B sequences or HEV-C and -D sequences. Recent findings have described enterovirus genomes with a coding region that clusters with one species and a 5′ UTR that clusters with a different species, suggesting possible interspecies recombination events (41, 44). Understanding the recombination-driven evolution of HEV-C viruses is of particular public health concern due to the viruses'' ability to recombine with vaccine poliovirus, resulting in circulating, highly neurovirulent vaccine-derived polioviruses (17, 21, 34). It is unclear whether recombination events between poliovirus and HEV-C viruses allow for the rapid acquisition of traits that increase pathogenic and circulation potential.The enterovirus pathogenicity spectrum is related to tissue tropism and is largely determined by cellular receptor usage. Most picornaviruses use receptors from the immunoglobulin superfamily of proteins, such as intracellular adhesion molecule-1 (ICAM-1) or coxsackievirus-adenovirus receptor (CAR) (36). A distinct subgroup of HEV-C viruses, which includes coxsackievirus (CAV) A1, A19, and A22 and enterovirus 104, has not yet been grown successfully in cell culture, and the receptor molecule for this subgroup is unknown (6). HEV-C viruses are believed to be the ancestral source of poliovirus, which resulted from a capsid mutation that caused a cellular receptor switch from ICAM-1 to CD155 (poliovirus receptor [PVR]) (17).In this study, we report the discovery and characterization of a novel human enterovirus type within species HEV-C, for which we propose the designation human enterovirus 109 (EV109). Sequence analysis reveals considerable nucleotide divergence in the 5′ UTR between EV109 and other HEV-C types, and scanning bootstrap analysis supports the hypothesis that EV109 is the product of an interspecies recombination event with an ancestral member of the HEV-A group. Viral capsid amino acid alignments and homology modeling reveal the predicted three-dimensional arrangement of divergent and conserved residues of EV109 compared with other related enteroviruses. We also report highly similar EV109 isolates within multiple cases of acute pediatric respiratory illness in Managua, Nicaragua.     

Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x_L, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.