A Direct Role for the CD1b Endogenous Spacer in the Recognition of a Mycobacterium tuberculosis Antigen by T-Cell Receptors.

Lipids, glycolipids and lipopeptides derived from Mycobacterium tuberculosis (Mtb) are presented to T cells by monomorphic molecules known as CD1. This is the case of the Mtb-specific sulfoglycolipid Ac2SGL, which is presented by CD1b molecules and is recognized by T cells found in tuberculosis (TB) patients and in individuals with latent infections. Our group, using filamentous phage display technology, obtained two specific ligands against the CD1b-Ac2SGL complex: (i) a single chain T cell receptor (scTCR) from a human T cell clone recognizing the CD1b-AcSGL complex; and (ii) a light chain domain antibody (dAbκ11). Both ligands showed lower reactivity to a synthetic analog of Ac2SGL (SGL12), having a shorter acyl chain as compared to the natural antigen. Here we put forward the hypothesis that the CD1b endogenous spacer lipid (EnSpacer) plays an important role in the recognition of the CD1b-Ac2SGL complex by specific T cells. To support this hypothesis we combined: (a) molecular binding assays for both the scTCR and the dAbκ11 antibody domain against a small panel of synthetic Ac2SGL analogs having different acyl chains, (b) molecular modeling of the CD1b-Ac2SGL/EnSpacer complex, and (c) modeling of the interactions of this complex with the scTCR. Our results contribute to understand the mechanisms of lipid presentation by CD1b molecules and their interactions with T-cell receptors and other specific ligands, which may help to develop specific tools targeting Mtb infected cells for therapeutic and diagnostic applications.

Structural models of the different trimers present in the core of phycobilisomes from Gracilaria chilensis based on crystal structures and sequences.

Phycobilisomes (PBS) are accessory light harvesting protein complexes that directionally transfer energy towards photosystems. Phycobilisomes are organized in a central core and rods radiating from it. Components of phycobilisomes in Gracilaria chilensis (Gch) are Phycobiliproteins (PBPs), Phycoerythrin (PE), and Phycocyanin (PC) in the rods, while Allophycocyanin (APC) is found in the core, and linker proteins (L). The function of such complexes depends on the structure of each component and their interaction. The core of PBS from cyanobacteria is mainly composed by cylinders of trimers of α and ß subunits forming heterodimers of Allophycocyanin, and other components of the core including subunits αII and ß18. As for the linkers, Linker core (LC) and Linker core membrane (LCM) are essential for the final emission towards photoreaction centers. Since we have previously focused our studies on the rods of the PBS, in the present article we investigated the components of the core in the phycobilisome from the eukaryotic algae, Gracilaria chilensis and their organization into trimers. Transmission electron microscopy provided the information for a three cylinders core, while the three dimensional structure of Allophycocyanin purified from Gch was determined by X-ray diffraction method and the biological unit was determined as a trimer by size exclusion chromatography. The protein sequences of all the components of the core were obtained by sequencing the corresponding genes and their expression confirmed by transcriptomic analysis. These subunits have seldom been reported in red algae, but not in Gracilaria chilensis. The subunits not present in the crystallographic structure were modeled to build the different composition of trimers. This article proposes structural models for the different types of trimers present in the core of phycobilisomes of Gch as a first step towards the final model for energy transfer in this system.

Crecimiento y composición bioquímica de Limnothrix sp a diferentes salinidades y concentraciones de nitrato / Growth and biochemical composition of Limnothrix sp at different salinities and concentrations of nitrate

Se evalúo el efecto de la salinidad (15, 25 y 35 UPS) y concentración de nitrato (4, 8 y 16 mmoles L-1) sobre el crecimiento y composición bioquímica de la cianobacteria Limnothrix sp. (LAEP- 52) con miras a su explotación para fines biotecnológicos. La cianobacteria se cultivó durante 20 días a 25°C, 98 µmol m-2s-1, fotoperiodo 12:12 y aireación continua (200 mL min-1). El crecimiento fue evaluado cada 48 horas a través de la medición de la densidad óptica a 730 nm. Se evidenció que la salinidad y la concentración de nitrato modulan el crecimiento y la composición bioquímica de Limnothrix sp. El mayor crecimiento (6.3 ± 0.38 mg mL-1), contenidos de proteínas (57 ± 4.56 %), ficocianina (170.3 ± 13.6 µg mL-1) y clorofila a (16 ± 1.28 µg mL-1) se obtuvieron a la menor salinidad (15 UPS) y mayor concentración de nitrato (16 mmoles L-1). Por el contrario, las mayores concentraciones de lípidos (21.3± 1.19 %), carbohidratos (14.47 ± 1.15 %) y carotenoides (6 ± 0.48 µg mL-1) se lograron en la mayor salinidad (35 UPS) y menor concentración de nitrato (4 mmoles L-1). La producción de exopolisacáridos sólo fue influenciada por la salinidad, llegando a alcanzar sus mayores valores a 35 UPS (1600 ± 112.25 mg L-1). Los contenidos de proteínas, lípidos, carbohidratos y pigmentos obtenidos en esta cianobacteria permiten catalogarla como un organismo que puede ser usado en las industrias biotecnológicas, ya sea como alimento para organismos cultivados o como fuente de metabolitos de interés industrial.

In this research we evaluate the effect of salinity (15, 25 and 35 UPS) and nitrate concentration (4, 8 and 16 mmoles L-1) on growth and biochemical composition of the cyanobacterium Limnothrix sp. (LAEP-52) with a view to exploitation for biotechnological purposes. The cyanobacterium was grown in volumes of 1 L for 20 days. The culture conditions included 25 °C, irradiance of 98 µmol m-2 s-1, photoperiod 12:12 and continuous aeration (200 mL min-1). Growth was evaluated every 48 hours through the measurement of optical density at 730 nm. It showed that salinity and concentration of nitrate modulate the growth and biochemical composition of Limnothrix sp. The highest values of growth (6.3 ± 0.38 mg mL-1), protein content (57 ± 4.56%), phycocyanin (170.3 ± 13.6 mg mL-1) and chlorophyll a (16 ± 1.28 mg mL-1) were obtained at the lowest salinity (15 UPS) and highest levels of nitrate (16 mmolesL-1). By contrast, higher concentrations of lipids (21.3 ± 1.19%), carbohydrate (14.47 ± 1.15%) and carotenoids (6 ± 0.48 mg mL-1) were achieved in the highest salinity (35 UPS) and the lowest concentrations of nitrate (4 mmoles L-1). The production of exopolysaccharides was only influenced by salinity, reaching its highest values at 35 UPS (1600 ± 112.25 mg L-1). The content of proteins, lipids, carbohydrates and pigments obtained in this cyanobacterium allow cataloged as an organism which can be used in biotechnology industries, either as feed for farmed organisms or as a source of metabolites of industrial interest.

Carotenogénesis de cinco cepas del alga Dunaliella sp. (Chlorophyceae) aisladas de lagunas hipersalinas de Venezuela / Carotenogenesis of five strains of the algae Dunaliella sp. (Chlorophyceae) isolated from Venezuelan hypersaline lagoons

Se realizaron cultivos discontinuos (medio Algal con 0.5 mM de NaNO3 y 27% de NaCl) de cinco cepas de Dunaliella sp., aisladas de diferentes lagunas hipersalinas de Venezuela (Araya, Coche, Peonía, Cumaraguas y Boca Chica) y una cepa de referencia (Dunaliella salina LB1644). Los bioensayos se mantuvieron a 25 ± 1 °C con aireación constante, fotoperiodo 12:12 y dos intensidades luminosas (195 y 390 µE.m-2.s-1) durante 30 días. El crecimiento celular se determinó diariamente mediante conteo celular en cámara de Neubaüer. La clorofila a y los carotenoides totales se analizaron al final del ensayo. Las mayores densidades celulares correspondieron a los ensayos de menor intensidad luminosa. La cepa que alcanzó la mayor densidad celular fue la aislada de Boca Chica (8 x106 y 2.5 x106 cel.ml-1 a 195 y 390 µE.m-2.s-1, respectivamente). El incremento de la intensidad luminosa en los cultivos produjo una disminución significativa de las tasas de crecimiento en todas las cepas. Los carotenoides totales por volumen fueron mayores a 390 µE.m-2 .s-1; siendo las cepas de referencia LB1644, Coche y Araya las que produjeron mayor cantidad (38.4; 32.8 y 21.0 µg.ml-1, respectivamente). El contenido de carotenoides totales por célula en los dos tratamientos fue significativamente diferente, obteniéndose la mayor concentración a 390 µE.m-2.s-1. Las cepas LB1644 y Coche fueron las que produjeron los valores más altos de carotenos (137.14 y 106.06 pg.cel-1, respectivamente). La cepa LB1644 presentó la mayor relación carotenoides totales:clorofila a (20:1) a 195 µE.m-2.s-1, mientras que en la cepa Coche no se evidenciaron diferencias significativas entre las dos intensidades (15:1). El resto de las cepas mostraron relaciones inferiores a uno. Nuestros resultados sugieren que las cepas Coche y Araya pueden ser potencialmente utilizadas en la biotecnología de producción de carotenoides

We evaluated discontinuous cultures (Algal medium at 0.5 mM of NaNO3 , and 27% NaCl) of five strains of Dunaliella sp. isolated from Venezuelan hypersaline lagoons (Araya, Coche, Peonía, Cumaraguas, and Boca Chica) and one strain from a reference collection (Dunaliella salina, LB1644). Cultures were maintained to 25±1 °C, with constant aeration, photoperiod 12:12, and two light intensities (195 and 390 µE.m-2 .s-1) during 30 days. Cell count was recorded on a daily basis using a Neubaüer camera. Totals of chlorophyll a and carotenoids were measured at the end of the experiment. The largest cellular densities were measured during the smallest light intensities. The strain with the largest cellular density was isolated from Boca Chica (8 x106 and 2.5 x106 cel.ml-1 a 390 and 195µE.m-2 .s-1, respectively). The increment of light intensity produced a significant reduction of growth rates in all strains. Totals of carotenoids by volume were as large as 390 µE.m-2 .s-1. Strains LB1644, from Coche and Araya were those that produced the largest amount of carotenoids (38.4; 32.8 and 21.0 µg.ml-1 , respectively). Differences total carotenoids by cell between treatments were significant. The largest concentration was 390 µE.m-2 .s-1 . The strains LB1644 and Coche produced the highest values of carotenes (137.14 and 106.06 pg.cel-1, respectively). Differences in the relation carotenoid:chlorophyll a between the strains at various light intensities was significant. Strains LB1644 presented the largest value of the relation carotenoids:chlorophyll a (20:1) at 195 µE.m-2 .s-1. No significant differences were detected in the strain Coche (15:1). All the other strains showed relations lower than one. Our results suggest that the strains of Coche and Araya show potential to be used in the biotechnology of carotenoids production

[Carotenogenesis of five strains of the algae Dunaliella sp. (Chlorophyceae) isolated from Venezuelan hypersaline lagoons]. / Carotenogénesis de cinco cepas del alga Dunaliella sp. (chlorophyceae) aisladas de lagunas hipersalinas de Venezuela.

We evaluated discontinuous cultures (Algal medium at 0.5 mM of NaNO3, and 27% NaCI) of five strains of Dunaliella sp. isolated from Venezuelan hypersaline lagoons (Araya, Coche, Peonia, Cumaraguas. and Boca Chica) and one strain from a reference collection (Dunaliella salina, LB1644). Cultures were maintained to 25+/-1 degrees C, with constant aeration, photoperiod 12:12, and two light intensities (195 and 390 microE.m(-2).s(-1)) during 30 days. Cell count was recorded on a daily basis using a Neubaüer camera. Totals of chlorophyll a and carotenoids were measured at the end of the experiment. The largest cellular densities were measured during the smallest light intensities. The strain with the largest cellular density was isolated from Boca Chica (8 xl0(6) and 2.5 xl0(6) cel.ml(-1) a 390 and 195microE.m(-2).s(-1), respectively). The increment of light intensity produced a significant reduction of growth rates in all strains. Totals of carotenoids by volume were as large as 390 microE.m(-2).s(-1). Strains LB 1644, from Coche and Araya were those that produced the largest amount of carotenoids (38.4; 32.8 and 21.0 microg.ml(-1), respectively). Differences total carotenoids by cell between treatments were significant. The largest concentration was 390 microE.m(-2).s(-1). The strains LB 1644 and Coche produced the highest values of carotenes (137.14 and 106.06 pg.cel(-1), respectively). Differences in the relation carotenoid:chlorophyll a between the strains at various light intensities was significant. Strains LB1644 presented the largest value of the relation carotenoids:chlorophyll a (20:1) at 195 microE.m(-2).s(-1). No significant differences were detected in the strain Coche (15:1). All the other strains showed relations lower than one. Our results suggest that the strains of Coche and Araya show potential to be used in the biotechnology of carotenoids production.