Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex

During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus.     

Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component

Background

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.

Methodology/Principal Findings

NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.

Significance

The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00392015","term_id":"NCT00392015"}}NCT00392015     

Hydrogen peroxide alters splicing of soluble guanylyl cyclase and selectively modulates expression of splicing regulators in human cancer cells

Background

Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER).

Methodology/Principal Findings

Here we report that splicing of C-α1 sGC can be modulated by H₂O₂ treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H₂O₂ treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H₂O₂ occurs at the protein level with variable regulation observed in different breast cancer cells.

Conclusions/Significance

Our data demonstrate that H₂O₂ regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H₂O₂ treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress.     

Alpha1 soluble guanylyl cyclase (sGC) splice forms as potential regulators of human sGC activity

Soluble guanylyl cyclase (sGC), a key protein in the NO/cGMP signaling pathway, is an obligatory heterodimeric protein composed of one alpha- and one beta-subunit. The alpha(1)/beta(1) sGC heterodimer is the predominant form expressed in various tissues and is regarded as the major isoform mediating NO-dependent effects such as vasodilation. We have identified three new alpha(1) sGC protein variants generated by alternative splicing. The 363 residue N1-alpha(1) sGC splice variant contains the regulatory domain, but lacks the catalytic domain. The shorter N2-alpha(1) sGC maintains 126 N-terminal residues and gains an additional 17 unique residues. The C-alpha(1) sGC variant lacks 240 N-terminal amino acids, but maintains a part of the regulatory domain and the entire catalytic domain. Q-PCR of N1-alpha(1), N2-alpha(1) sGC mRNA levels together with RT-PCR analysis for C-alpha(1) sGC demonstrated that the expression of the alpha(1) sGC splice forms vary in different human tissues indicative of tissue-specific regulation. Functional analysis of the N1-alpha(1) sGC demonstrated that this protein has a dominant-negative effect on the activity of sGC when coexpressed with the alpha(1)/beta(1) heterodimer. The C-alpha(1) sGC variant heterodimerizes with the beta(1) subunit and produces a fully functional NO- and BAY41-2272-sensitive enzyme. We also found that despite identical susceptibility to inhibition by ODQ, intracellular levels of the 54-kDa C-alpha(1) band did not change in response to ODQ treatments, while the level of 83 kDa alpha(1) band was significantly affected by ODQ. These studies suggest that modulation of the level and diversity of splice forms may represent novel mechanisms modulating the function of sGC in different human tissues.     

Genetic Divergence of Mussels (Mollusca,Mytilidae) Based on the <Emphasis Type="Italic">28S</Emphasis> rRNA, <Emphasis Type="Italic">18S</Emphasis> rRNA,and <Emphasis Type="Italic">H3</Emphasis> Nuclear Gene Sequences

On the basis of nucleotide sequences of three nuclear genes and using molecular phylogenetic and evolutionary genetic approaches, the phylogeny of the main representatives of one of the largest taxa of bivalve mollusks, the family Mytilidae, was studied, and its system and taxonomy were refined. A phylogenetic system for the family Mytilidae and closely relative taxa of the order Mytilida, which currently has no consensus among specialists on the basis of traditional characters, is presented. Using nucleotide sequences of the 28S rRNA, 18S pRNA, and histone H3 genes, this consensus was established by the study of Mytilidae. Some concerns of mussel systematics were resolved; in particular, the monophyly of the family Mytilidae Rafinesque, 1815 was established with the strongest support for the subfamily Mytilinae Rafinesque, 1815. The data obtained disprove Distel’s conclusion on polyphyly of the subfamily Mytilinae Rafinesque, 1815. Isolation of the taxa in the rank of the Modiolinae G. Termier & H. Termier, 1950 and Bathymodiolinae Kenk & Wilson, 1985 subfamilies in the family Mytilidae and also the family Septiferidae Scarlato et Starobogatov, 1979 was confirmed, although the rank of the later taxon is not universally recognized and it remains to be clarified in an additional study.     

Sperm ultrastructure in representatives of six bivalve families from Peter the Great Bay, Sea of Japan

The ultrastructure of sperm in seven species of bivalves, the representatives of six families, Arcidae (Anadara broughtonii, Arca boucardi), Anomiidae (Pododesmus macrochisma), Tellinidae (Macoma tokyoensis), Ostreidae (Crassostrea gigas), Myidae (Mya japonica) and Trapezidae (Trapezium liratum) is described. All the studied sperm were typical tail sperm, adapted to external insemination, which, however, had a specific structure. Differences were revealed in the form of head, acrosome structure and number of mitochondria. The studied species of the above families had their specific morphology, the Arcidae species had a bullet- or barrel-shaped head with four or five mitochondria in the middle part; the Anomiidae had conic head, the acrosome with periacrosome material and four mitochondria (a basic feature of sperm is the axial core entering periacrosome material and consisting of bundle of actin filaments); the Myidae had a curved conic head and four mitochondria; in the Tellinidae the head was bullet-shaped, the periacrosome material contained a fibril component and four mitochondria; the Trapezidae had sperm of a conic form with spherical acrosome. The spherical sperm of C. gigas were similar to sperm of Saccostrea commercialis and Crassostrea virginica, but with some distinctions in the acrosome substructure. The morphology of sperm testified to the correct attribution of the Crassostreidae family as a synonym to the Ostreidae family.     

A population genetic study of the hybrid zone of Mytilus trossulus Gould, 1850 and an introduced species,M. galloprovincialis Lamarck, 1819, (Bivalvia: Mytilidae) in peter the great bay in the Sea of Japan

This paper examines the genetic variability of the Pacific mussel Mytilus trossulus and an introduced Atlantic species, M. galloprovincialis, in the northwestern Sea of Japan (Peter the Great Bay and Kievka Bay). The genotyping of individuals from eight populations was carried out using eight polymorphic enzyme loci and two nuclear DNA markers (Me-5 and ITS-1,2); the occurrence frequency of parent species and their hybrids was determined. The enzyme and nuclear markers demonstrated concordant genetic variation. The genotypes of the native species M. trossulus were predominant in the samples studied. The frequency of the introduced species M. galloprovincialis in the total material was relatively low; however, it reached 42 ± 2% in samples that were collected in Possjet Bay near the town of Zarubino in a zone of active international navigation. In this area the greatest number of hybrids was found as well. It is concluded that the invasion of M. galloprovincialis in the northwestern Sea of Japan is continuing; permanent populations of this mussel appeared in Possjet Bay that were not recorded here previously.     

Different messenger RNA expression for the antimicrobial peptides β-defensins between Meishan and crossbred pigs

β-defensins are cysteine-rich endogenously produced antimicrobial peptides that play an important role in innate immunity. In this study, the expressions of genes porcine β-defensins-1(pBD-1), pBD-2 and pBD-3 were determined using real-time PCR for Chinese Meishan pigs and Crossbred (Duroc × Yorkshire × Landrace) pigs of 7 days old in various tissues. The results showed that expressions of pBD-1, 2 and 3 of Meishan pigs in most tissues were higher than those of crossbred pigs and main expression sites for pBD-1 and pBD-3 were tongue and oral mucosa in two varieties of pigs, whereas pBD-2 of crossbred pig was mainly expressed in kidney and liver, and pBD-2 of Meishan pigs mainly in tongue and oral mucosa. The higher expression of pBDs might be the reason of Meishan pigs has higher immunity and disease resistance. The mechanisms of this need a further research.     

Zn2+ and l-isoleucine induce the expressions of porcine β-defensins in IPEC-J2 cells

The β-defensins, expressed in epithelial cells of multiple tissues including intestine, play a critical role in the mammalian innate immunity. However, it is little known about the role of functional nutrients in the regulation of porcine β-defensins’ expressions in intestinal epithelial cells. The present study was conducted to determine the hypothesis that zinc and l-isoleucine regulate the expressions of porcine β-defensins in IPEC-J2 cells. Cells were cultured in DMEM/F12 medium containing supplemental 0–500 μg/mL l-isoleucine or 0–500 μmol/mL zinc sulfate that was used to increase the concentration of Zn²⁺ in the medium. At 12 h after the treatment by the appropriate concentrations of l-isoleucine or Zn²⁺, the mRNA and protein expressions of porcine β-defensin 1, 2 and 3 were increased (P < 0.05), and reached their maximum after treatment with 25 or 100 μmol/mL zinc sulfate and 25 or 50 μg/mL isoleucine (P < 0.05). These results suggested that both Zn²⁺ and l-isoleucine could induce β-defensins’ expressions in porcine intestinal epithelial cells.