Chloroplast ultrastructure in leaves of tobacco plants with the introduced gene for the acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Synechococcus vulcanus</Emphasis> at normal and low temperature

Ultrastructural changes in chloroplasts of tobacco plants (Nicotiana tabacum L.) with the introduced desC gene for the acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were investigated during plant acclimation to cold. Control plants were transformed with an empty pGA482 binary vector. At optimum growth temperature, a decreased number of grana and thylakoids and an increased number of plastoglobules and their larger area were observed in transgenic plants when compared to control ones. In control plants, acclimation to cold (6 days at 10°C) resulted in the larger areas of chloroplasts and grana. These changes indicated starting cold-induced injuries manifested in swelling of the stroma and a slight decrease in the total number of thylakoids in the chloroplast. In contrast, transgenic plants responded to cold by reducing the chloroplast, granal, and plastoglobule areas. Meantime, the number of thylakoids per granum increased noticeably. The total number of thylakoids in the chloroplast increased from 123 to 203. It was concluded that expression of the acyl-lipid Δ9-desaturase gene in tobacco plants provided for the formation of the cell ultrastructure similar to one characteristic of cold-tolerant plants.     

Nyamanini and Midway Viruses Define a Novel Taxon of RNA Viruses in the Order Mononegavirales

Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.Nyamanini virus (NYMV) was first isolated in 1957 from a cattle egret (Bubulcus ibis) in South Africa (24). It has subsequently been isolated in Nigeria, Egypt, India, and Thailand from cattle egrets and Argas walkerae ticks (14, 16, 24). Although there has been no recognized human infection or disease associated with NYMV, suckling mice succumbed to NYMV infection 7 or 8 days after intracerebral inoculation (14). NYMV has not been definitively characterized or classified to date (10).Midway virus (MIDWV) was first isolated in 1966 from seabird ticks of two species [Ornithodoros (Alectorobius) spp.] collected on the Midway, Kure, and Manana islands in the Central Pacific and from northern Honshu, Japan. In addition, on Aomatsushima Island, nestling seabirds of two seabird species, Larus crassirostris and Nycticorax nycticorax, were found to have antibody to MIDWV. This virus is pathogenic for newborn Swiss mice but not 4-week-old Swiss mice injected intracranially. Also, the virus is cytopathic in BHK-21 cells and produces plaques in Vero cells. Efforts to classify MIDWV have revealed only that MIDWV is antigenically related to NYMV in cross-box complement fixation (CF) assays (22).To date, conventional approaches, such as serological analysis and electron microscopy (EM), have not yielded definitive characterization of MIDWV or NYMV. Heretofore, it was not known whether MIDWV and NYMV are DNA viruses or RNA viruses or to which virus family they belong. In this paper, we describe the application of unbiased high-throughput sequencing to define the genome sequences of NYMV and MIDWV. By analysis of their genomes, NYMV and MIDWV were determined to be negative-stranded RNA viruses highly divergent from all known viruses but most closely related to viruses in the order Mononegavirales. On the basis of the analysis presented herein, we propose that NYMV and MIDWV define a novel taxon within the order Mononegavirales.     

Heme interacts with c1q and inhibits the classical complement pathway

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.     

The Decreased Cold-Resistance of Chilling-Sensitive Plants Is Related to Suppressed CO2 Assimilation in Leaves and Sugar Accumulation in Roots

Tomato (Lycopersicon esculentum L., cv. Sibirskii skorospelyi) and cucumber (Cucumis sativus L., cv. Konkurent) plants were grown in a soil culture in a greenhouse at an average daily temperature of 20°C and ambient illumination until the development of five and eight true leaves, respectively. During the subsequent three days, some plants were kept in a climatic chamber at 6°C in the light, whereas other plants remained in a greenhouse (control). The cold-resistance of cucumber leaves and roots, as assayed from the electrolyte leakage, was reduced after cold exposure stronger than cold-resistance of tomato organs. The ratio photosynthesis/dark respiration was lower in cucumber than in tomato leaves at all measurement temperatures. The concentrations of sugars (sucrose + glucose + fructose) increased in chilled tomato roots but decreased in cucumber roots. Cold exposure changed the activities of various invertase forms (soluble and insoluble acidic and alkaline invertases). The total invertase activity and the ratio of mono- to disaccharides increased. The lower cucumber cold-resistance is related to the higher sensitivity of its photosynthetic apparatus to chilling and, as a consequence, insufficient root supply with sugars.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

Results of testing the comparatory method: The curvature of the shell valve frontal section is inappropriate as a systematic character for the freshwater pearl mussel of the genus Margaritifera

This paper continues a discussion on the number of pearl mussel species of the genus Margaritifera in northern Europe. A biometric study of 1711 pearl mussel Margaritifera margaritifera shells from 15 rivers in Russia and Latvia (basins of the White and Baltic seas) has been conducted. All the examined samples fall into two groups: the northern group (with the shells more flattened on average, f. margaritifera) and the southern one (with more convex shells, f. elongata); the boundary between these groups is at 63° N. Analysis of intrapopulation variation has shown that the samples contain individuals that correspond to f. margaritifera, f. elongata, and f. borealis. However, any hiatus between these forms is absent in all the samples, and individuals belonging to two intermediate forms are rather frequent. The hypothesis on the species specificity of the shell valve frontal section has not been confirmed based on examination of large shell samples. The pearl mussels inhabiting rivers of Northern Europe belong to a single species, M. margaritifera.     

Lipophylic compounds from Euphorbia peplis L.--a halophytic plant from the Bulgarian Black Sea coast

The chemical composition of the lipophylic fraction from the halophytic plant Euphorbia peplis L. was investigated. Compared to other terrestrial higher plants an increase of triacylglycerols and especially of glycolipids was observed. The main phospholipid was phosphatidyl choline, followed by almost equal concentrations of phosphatidyl ethanolamine and phosphatidyl glycerol. A relatively high concentration of phosphatidic acids (6.5% of the total phospholipids) was found. The main sterol appeared to be sitosterol and significant amounts of tetracyclic triterpene alcohols were found. The composition of the volatile compounds is relatively simple and only one chlorinated compound, identified as 2,2-diethoxy-1-chloroethane, was found. There was a strong toxicity of the total lipophylic extract towards Artemia salina.     

GC/MS analysis of some bioactive constituents from Carthamus lanatus L

Sterols, triterpenes, volatiles, polar and other constituents in aerial parts of Carthamus lanatus were analyzed by gas chromatography-mass spectrometry. Over 90 compounds were identified most of them new for the species. Sitosterol and stigmasterol were the most abundant of 10 sterols identified in the sterol fraction. Taraxasterol, alpha- and beta-amyrine prevailed in the triterpene fraction. Volatiles, sterols and a fraction of the dichloromethane extract showed strong cytotoxicity (Artemia salina assay).     

Somatic genome variations in vascular tissues and peripheral blood leukocytes in patients with atherosclerosis

The first data on the existence of multiple genomic rearrangements, such as copy number variation (CNV) and copy neutral loss of heterozygosity, in vascular tissues and peripheral blood leukocytes from patients with atherosclerosis, are presented. Compared to internal mammary arteries and peripheral blood leukocytes, right coronary arteries in the atherosclerotic plaque area presented with a higher CNV length and number of genes located in their vicinity. In each of the patients, 6–16% of CNVs were common to the three types of tissues examined. Therefore, most of the copy number variations in the tissues affected by atherosclerosis (from 68 to 91% in each of the patients) were of somatic origin. The gains in 3p21.31 (CACNA2D2), 7q32.1 (FLNC), 19p13.3 (C19orf29, PIP5K1C), and 21q22.3 (COL6A1) were detected in vascular tissues but not in peripheral blood leukocytes. Moreover, the gain in 7p15.2 (SKAP2), detected in the patients with atherosclerosis, did not overlap with any CNV regions currently reported in The Database of Genomic Variants. The loss of heterozygosity in 12 out of 13 chromosomal regions was copy neutral and covered tumor suppressor genes (SFRP1, CEBPD, RB1CC1, DIRAS3, TUSC3, and ZDHHC2).