Alternative splicing creates a pseudo-strictosidine ß-d-glucosidase modulating alkaloid synthesis in Catharanthus roseus.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by ß-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine ß-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks ß-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of ß-glucosidase multimerization, an organization common to many defensive GH1 members.

Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus).

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

Vacuole-Targeted Proteins: Ins and Outs of Subcellular Localization Studies.

Accurate and efficient demonstrations of protein localizations to the vacuole or tonoplast remain strict prerequisites to decipher the role of vacuoles in the whole plant cell biology and notably in defence processes. In this chapter, we describe a reliable procedure of protein subcellular localization study through transient transformations of Catharanthus roseus or onion cells and expression of fusions with fluorescent proteins allowing minimizing artefacts of targeting.

Contribución al conocimiento de la vegetación de las sabanas de casanare (colombia) / Contribution to knowledge of savannas vegetation of casanare (colombia)

RESUMEN Se estudió la diversidad vegetal en parcelas de bosques de galería, bosques de vega, morichales, matas de monte, bancos de sabana, bajos de sabana y esteros, así como de inventarios ad libitum. En total, se registraron 318 especies y 206 géneros de 80 familias, siendo la más diversa la familia Poaceae, con 29 especies, seguida de Euphorbiaceae, con 17 especies. Los géneros más diversos fueron Phyllantus y Panicum, con siete especies. Los tipos de vegetación con mayor riqueza fueron los bosques de galería, los bajos de sabana y los morichales. La composición, a nivel de familias en los bosques, es similar a la reportada en otros estudios, pero las especies son diferentes, sugiriendo dominancia local, debida a la heterogeneidad espacial. Con este estudio, se incrementó el número de especies registradas para el departamento de Casanare, de 1.479 a 1.562.

SUMMARY We studied plant diversity in forest plots of bosques de galería, bosques de vega, morichales, matas de monte, bancos de sabana, bajos de sabana y esteros, as well as ad libitum inventories. A total of 318 species and 80 genera of 206 families were found, being Poaceae the most diverse family with 29 species, followed by Euphorbiaceae with 17 species. The most diverse genera were Phyllantus and Panicum with seven species. The types of vegetation with the greatest diversity were bosques de galería, bajos de sabana and morichales. The composition of families in forests is similar to that reported in other studies, but species composition is different suggesting local dominance due to spatial heterogeneity. With this study, there was an increase in the number of species recorded for the department of Casanare from 1479 to 1562.

A BAHD acyltransferase catalyzing 19-O-acetylation of tabersonine derivatives in roots of Catharanthus roseus enables combinatorial synthesis of monoterpene indole alkaloids.

While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to tabersonine metabolism have been identified to date, namely tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxytabersonine derivatives from roots, such as minovincinine and hörhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of tabersonine in tailor-made MIAs bearing both leaf and root-type decorations.

Autoantibodies and Gastrointestinal Symptoms in Colombian Children with Juvenile Idiopathic Arthritis.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is the most common inflammatory joint disease in children. JIA and autoimmune inflammatory Gastrointestinal (GI) diseases share common etiologic mechanisms, including genetic predisposition and environmental influences. OBJECTIVE: To Investigate association between gastrointestinal, rheumatologic clinical variables and the presence of autoantibodies in patients with JIA in treatment. METHODOLOGY: In a cross-sectional study of patients with JIA according to diagnostic criteria and the ILAR classification. GI symptoms and autoantibody expression were evaluated with respect to their association with JIA clinical variables. Anti-Saccharomyces Cerevisiae IgG/IgA (ASCA), 6 antigen associated with anti polymorphonuclear neutrophil (ANCA), anti Transglutaminase (tTG) IgG/IgA, anti deaminated gliadin peptide (DGP) IgG/IgA autoantibodies, ANAS and IgA were measured in all patients. The association between clinical variables and auto-antibodies were evaluated using the Fisher test with significant value of p <0.05. The study was approved by the ethics committee of the all institutions. RESULTS: Samples were collected from ninety-seven patients, 63% of whom were female. The average age was 14 years. The JIA subtype associated with the most common GI symptoms was enthesitis- related arthritis. Of these patients, 44.3% and 14% reported abdominal pain and diarrhea, respectively. Anti-DPG and anti-tTG antibodies were found in 9.28% and 7.22%, respectively and 11.34% were positive for p-ANCA, and 2% were positive for ASCA. CONCLUSION: GI symptoms and autoantibodies associated with inflammation of the GI mucosa were detected in JIA patients but were not associated with autoantibodies or clinical variables. However, it is the monitoring of these patients diagnosis is important.

Tiroidopatías autoimmunes juveniles y poliautoinmunidad / Tiroidopatias juvenile autoimmune and poliautoinmunidad

ntroducción y objetivo: Las tiroidopatias autoimmunes juveniles (TAIJ) son las enfermeda-des órgano específicas más comunes y con frecuencia hacen parte de la enfermedad autoin-mune múltiple (EAIM). El objetivo del estudio es determinar el tipo y frecuencia de TAIJ en pacientes con poliautoinmunidad. Métodos: En un registro de pacientes juveniles que du-rante el seguimiento en 13 centros de reumatología pediátrica, han asociado enfermedades autoinmunes (EAI), se identificaron pacientes con TAIJ.

¿Existen diferencias en la expresión clínica del síndrome antifosfolipídico juvenil en función del género? / Are there differences in the clinical expression of the gender youth antiphospholipid syndrome?

Introduccion: El síndrome antifosfolipídico (SAF) condiciona hipercoagulabilidad y pue-de provocar manifestaciones no trombóticas. Se asocia o no a otra enfermedad autoinmune (EAI). En el presente estudio se exploraron diferencias entre géneros en cohortes de pacientes juveniles con SAF. Métodos: Estudio retrospectivo descriptivo. Se revisaron historias clínicas de pacientes con SAF seguidos en 13 centros de reumatología pediátrica en Colombia y en Hospital San Joan (Barcelona, España). Se determinaron variables demográficas y frecuencia de manifestaciones clínicas y resultados serológicos.

Uveítis crónica en la artritis idiopática juvenil. Impacto pronóstico / Chronic Uveitis in juvenile idiopathic arthritis. Impact forecasting

Introducción y objetivo: La uveitis en la artritis idiopática juvenil (AIJ) es causa de importan-te morbilidad. La cronicidad de inflamación intraocular determina severidad de complicacio-nes y secuelas. El objetivo del estudio es evaluar la extensión, tratamiento y complicaciones de la uveítis crónica en pacientes con AIJ. Métodos: Estudio descriptivo, retrospectivo. Se in-cluyeron pacientes con seguimiento mínimo de 24 meses por oftalmología y reumatología. Se analizaron variables dermográficas, clínicas, complicaciones y tratamiento. La uveitis se eva-luó según clasificación SUN (standardization of uveítis nomenclature).

Porphyromonas Gingivalis and IgG1 and IgG2 Subclass Antibodies in Patients with Juvenile Idiopathic Arthritis.

PURPOSE: The purpose of this study was to evaluate the periodontal status and the presence and concentration of Porphyromonas gingivalis (P. gingivalis) and immunoglobulin G (IgG) subclass against P. gingivalis in juvenile idiopathic arthritis (JIA) and its association with rheumatic clinical activity parameters. METHODS: Rheumatologic conditions and periodontal status were clinically assessed in 51 patients with JIA. P. gingivalis, IgG1 and IgG2, high-sensitivity C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), and human leukocyte antigen B27 were also evaluated. RESULTS: Periodontitis was observed in 21.5 percent of the patients, 23.5 percent of whom were positive for P. gingivalis, which was associated with enthesitis-related arthritis (P<0.035). IgG1 against P gingivalis was associated with RF autoantibodies (P=0.05), and all patients positive for ACPAs had higher anti-P gingivalis IgG1 levels. A significant correlation was found between the presence of limited joint mobility and the plaque index in polyarticular JIA (r=0.55, P=0.028). CONCLUSIONS: An association between IgG and rheumatic disease activity markers in JIA was evident. It is important to investigate the familiar periodontal status and clinical course in JIA, especially in enthesitis-related arthritis.

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris / Caracterización de beta-hexosaminidasas lisosomales humanas recombinantes producidas en la levadura metilotrófica Pichia pastoris / Caracterização de beta-hexosaminidases lisossomais humanas recombinantes produzidas na levedura metilotrófica Pichia pastoris

Abstract β-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded by HEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant α-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14,606 U .mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at 4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant β-hexosaminidases produced within the same host.

Resumen Las β-hexosaminidasas (Hex) son enzimas diméricas involucradas en la degradación lisosomal de glicolípidos y glicanos. Estas enzimas están formadas por las subunidades α- y/o β-codificadas por los genes HEXA and HEXB respectivamente. Las mutaciones de estos genes conducen a las enfermedades de Tay Sachs o Sandhoff, que son desórdenes neurodegenerativos causados por la acumulación de glicolípidos no degradados. Aunque las Hex derivadas de tejido han sido ampliamente caracterizadas, la información disponible sobre las p-hexosaminidasas recombinantes es limitada. En este estudio se produjeron Hex recombinantes lisosomales (rhHex-A, rhHex-B y rhHex-S) en la levadura metilotrófica Pichia pastoris GS115. Las actividades específicas más altas de las enzimas fueron 13.124, 12.779, 14.606 U .mg-1 para rhHex-A, rhHex-B y rhHex-S, respectivamente. Estos resultados fueron 25 a 50 veces más altos que los obtenidos de leucocitos humanos normales. Las proteínas se purificaron y se caracterizaron a diferentes condiciones de pH y temperatura. Todas las proteínas fueron estables a pH ácido y a 4°C y 37°C. A 45°C la rhHex-S se inactivó completamente, mientras que rhHex-A y rhHex-B mostraron alta estabilidad. Este estudio demuestra el potencial de P. pastoris GS115 para la producción de enzimas lisosomales poliméricas y presenta la caracterización de distintas β-hexosaminidasas recombinantes producidas en un único hospedero.

Resumen As β-hexosaminidases (Hex) são enzimas diméricas envolvidas na degradação lisossomal de glicolipídeos e glicanos. Essas enzimas são formadas por subunidades a- e/ou p-codificadas pelos genes HEXA e HEXB, respectivamente. As mutações nesses genes causam a doença de Sandhoff ou Tay Sachs, que são desordens neurodegenerativas causadas pela acumulação de glicolipídeos não degradados. Embora Hex derivadas de tecido hajam sido caracterizadas extensivamente, as informações disponíveis sobre as p-hexosaminidases recombinantes são limitadas. Esse estudo produziu Hex recombinantes lisossomais (rhHex-A, rhHex-B e rhHex-S) na levedura metilotrófica Pichia pastoris GS115. As atividades específicas mais altas das enzimas foram 13.124, 12.779, 14.606 U .mg-1 para rhHex-A, rhHex-B y rhHex-S, respectivamente. Esses resultados foram 25 a 50 vezes mais altos do que os obtidos a partir de leucócitos humanos normais. As proteínas foram purificadas e caracterizadas em diferentes condições de pH e temperatura. Todas as proteínas foram estáveis a pH ácido e a 4°C e 37°C. A 45°C a rhHex-S foi completamente inativada, enquanto rhHex rhHex-A e B se mostraram altamente estáveis. Esse estudo demonstra o potencial de P. pastoris GS115 para a produção de enzimas lisossomais poliméricas e apresenta a caracterização de diferentes p-hexosaminidases recombinantes produzidas em único hospedeiro.

Human recombinant lysosomal enzymes produced in microorganisms.

Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD.

Effect of culture conditions and signal peptide on production of human recombinant N-acetylgalactosamine-6-sulfate sulfatase in Escherichia coli BL21.

The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.