Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.     

Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies

We investigated the suitability of transformed rice cell lines as a system for the production of therapeutic recombinant antibodies. Expression constructs encoding a single-chain Fv fragment (scFvT84.66, specific for CEA, the carcinoembryonic antigen present on many human tumours) were introduced into rice tissue by particle bombardment. We compared antibody production levels when antibodies were either secreted to the apoplast or retained in the endoplasmic reticulum (ER) using a KDEL retention signal. Production levels were up to 14 times higher when antibodies were retained in the ER. Additionally, we compared constructs encoding different leader peptides (plant codon optimised murine immunoglobulin heavy and light chain leader peptides) and carrying alternative 5 untranslated regions (the petunia chalcone synthase gene 5 UTR and the tobacco mosaic virus omega sequence). We observed no significant differences in antibody production levels among cell lines transformed with these constructs. The highest level of antibody production we measured was 3.8gg^–1 callus (fresh weight). Immunological analysis of transgenic rice callus confirmed the presence of functional scFvT84.66. We discuss the potential merits of cell culture for the production of recombinant antibodies and other valuable macromolecules.     

Isolation, characterization, and ecology of sulfur-respiring crenarchaea inhabiting acid-sulfate-chloride-containing geothermal springs in Yellowstone National Park

Elemental sulfur (S(0)) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S(0)-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72 degrees C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81 degrees C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S(0), and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S(0) flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.     

Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park

Elemental sulfur (S⁰) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S⁰-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S⁰, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S⁰ flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.     

Gene expression,protein profiling,and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.     

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.     

Conservation genetics of the short-beaked common dolphin (<Emphasis Type="Italic">Delphinus delphis</Emphasis>) in the Mediterranean Sea and in the eastern North Atlantic Ocean

Mediterranean Sea common dolphins have recently been listed as ‘endangered’ in the IUCN Red list, due to their reported decline since the middle of the 20th century. However, little is know about the number or distribution of populations in this region. We analysed 118 samples from the Black Sea, Mediterranean Sea and eastern North Atlantic at nine microsatellite nuclear loci and for 428 bps of the mtDNA control region. We found small but significant population differentiation across the basin between the eastern and the western Mediterranean populations at both nuclear and mtDNA markers (microsatellite F _ST = 0.052, mtDNA F _ST = 0.107, P values ≤ 0.001). This matched the differential distribution and habitat use patterns exhibited by this species in the eastern and the western parts of the Mediterranean Sea. The assignment test of a small number of samples from the central Mediterranean could not exclude further population structure in the central area of the basin. No significant genetic differentiation at either marker was observed among the eastern north Atlantic populations, though the Alboran population (inhabiting the Mediterranean waters immediately adjacent the Atlantic ocean) showed significant mtDNA genetic differentiation compared to the Atlantic populations. Directional estimates of gene flow suggested movement of females out of the Mediterranean, which may be relevant to the population decline. Phylogenetic analysis suggested that the observed population structure evolved recently.     

Specific interference between two unrelated internal ribosome entry site elements impairs translation efficiency

Internal ribosome entry site (IRES) elements allow simultaneous synthesis of multiple proteins in eukaryotic cells. Here, two unrelated IRESs that perform efficiently in bicistronic constructs, the picornavirus foot-and-mouth disease virus (FMDV) and the cellular immunoglobulin heavy chain binding protein (BiP) IRES, were used to generate a tricistronic vector. Functional analysis of the tricistronic RNA evidenced that the efficiency of protein synthesis under the control of BiP IRES was lower than that of the FMDV IRES, relative to the efficiency measured in bicistronic vectors. A specific competition between these elements was verified using two separate mono- or bicistronic constructs in vivo and in vitro. In contrast, no interference was detected with the hepatitis C virus (HCV) IRES. The interference effect of FMDV IRES was observed in cis and trans, in support of competition for common transacting factors different than those used in cap- and HCV-dependent initiation.