Towards the production of high levels of eicosapentaenoic acid in transgenic plants: the effects of different host species,genes and promoters

Eicosapentaenoic acid (EPA, 20:5n-3) plays an important role in many aspects of human health. In our efforts towards producing high levels of EPA in plants, we investigated the effects of different host species, genes and promoters on EPA biosynthesis. Zero-erucic acid Brassica carinata appeared to be an outstanding host species for EPA production, with EPA levels in transgenic seed of this line reaching up to 25%. Two novel genes, an 18-carbon ω3 desaturase (CpDesX) from Claviceps purpurea and a 20-carbon ω3 desaturase (Pir-ω3) from Pythium irregulare, proved to be very effective in increasing EPA levels in high-erucic acid B. carinata. The conlinin1 promoter from flax functioned reasonably well in B. carinata, and can serve as an alternative to the napin promoter from B. napus. In summary, the judicious selection of host species and promoters, together with the inclusion of genes that enhance the basic very long chain polyunsaturated fatty acid biosynthetic pathway, can greatly influence the production of EPA in plants.     

HIV-1 Transmission by Dendritic Cell-specific ICAM-3-grabbing Nonintegrin (DC-SIGN) Is Regulated by Determinants in the Carbohydrate Recognition Domain That Are Absent in Liver/Lymph Node-SIGN (L-SIGN)

In this study, we identify determinants in dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) necessary for human immunodeficiency virus, type 1 (HIV-1), transmission. Although human B cell lines expressing DC-SIGN efficiently capture and transmit HIV-1 to susceptible target cells, cells expressing the related molecule liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) do not. To understand the differences between DC-SIGN and L-SIGN that affect HIV-1 interactions, we developed Raji B cell lines expressing different DC-SIGN/L-SIGN chimeras. Testing of the chimeras demonstrated that replacement of the DC-SIGN carbohydrate-recognition domain (CRD) with that of L-SIGN was sufficient to impair virus binding and prevent transmission. Conversely, the ability to bind and transmit HIV-1 was conferred to L-SIGN chimeras containing the DC-SIGN CRD. We identified Trp-258 in the DC-SIGN CRD to be essential for HIV-1 transmission. Although introduction of a K270W mutation at the same position in L-SIGN was insufficient for HIV-1 binding, an L-SIGN mutant molecule with K270W and a C-terminal DC-SIGN CRD subdomain transmitted HIV-1. These data suggest that DC-SIGN structural elements distinct from the oligosaccharide-binding site are required for HIV-1 glycoprotein selectivity.     

Identical TCR beta-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris

Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.     

Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux.     

Culture-independent characterization of the digestive-tract microbiota of the medicinal leech reveals a tripartite symbiosis

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the alpha-, gamma-, and delta-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.     

PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

Regulation of cardiac hypertrophy by intracellular signalling pathways

The mammalian heart is a dynamic organ that can grow and change to accommodate alterations in its workload. During development and in response to physiological stimuli or pathological insults, the heart undergoes hypertrophic enlargement, which is characterized by an increase in the size of individual cardiac myocytes. Recent findings in genetically modified animal models implicate important intermediate signal-transduction pathways in the coordination of heart growth following physiological and pathological stimulation.