High-fat diet alters N-glycosylation of PTPRJ in murine liver.

Protein tyrosine phosphatases (PTPs) regulate multiple signaling pathways. Disruption of tyrosine phosphorylation through imbalanced action between protein tyrosine kinases (RTKs) and PTPs is a hallmark of metabolic disorders, including insulin resistance. A representative member of the receptor-type PTP family, PTPRJ (DEP-1), was previously identified as a negative regulator of insulin signaling and possesses post-translational glycosylation sites. In this regard, it seems of great importance to decipher the structure of PTPRJ's glycosylation, particularly in the context of metabolic disturbances, but this has not been done in detail. Thus, here we aimed at characterizing the glycosylation pattern of PTPRJ in liver. We show that N-glycosylation accounts for up to half of PTPRJ's molecular weight. Applying mass spectrometry, we detected increased levels of high-mannose structures in PTPRJ in liver tissue of obese mice compared to lean littermates. In addition, complex neutral structures without fucose were also elevated in PTPRJ of high-fat diet (HFD) mice. Conversely, complex fucosylated N-glycans as well as sialylated bi- and triantennary N-glycans, were significantly reduced in PTPRJ of HFD-derived liver tissue compared to LFD by ∼two fold (P≤.01, P≤.0001 and P≤.001, respectively). In congruence with these findings, the mannosidase MAN2A1, responsible for the conversion of high-mannose to complex N-glycans, was significantly downregulated under HFD conditions. Here we present for the first time that HFD-induced obesity impacts on the glycosylation pattern of the insulin signaling component PTPRJ in liver. These findings may inspire new research on the glycosylation of PTPs in metabolic diseases and may open up new therapeutic approaches.

Cannabis sativa L. from Seized Drug Material: In Vitro Germination and Establishment.

Background: With the expansion of the cannabis-derived product market, there is a growing need for seedling development to produce raw material for pharmaceutical applications and medicinal research. However, cannabis cultivation is illegal in many countries, and legal producers do not sell cannabis seeds in these countries. In Brazil, cannabis is still illegal, and the only way to obtain access to cannabis plants for research or as medicine is through importation, which is costly and requires authorization from the National Health Surveillance Agency (ANVISA), or from material seized by the police from drug trafficking. Methods: Therefore, since cannabis seeds obtained from drug trafficking have never been tested regarding their viability and use in in vitro cultivation, the aim of this study was to analyze the in vitro establishment of cannabis from seeds derived from Brazilian drug trafficking seizures that were provided by the police to investigate seed disinfestation procedures and further multiplication of nodal segments, with the purpose of obtaining material for medicinal research in the country. Seeds were subjected to four disinfestation treatments. Results: The best disinfestation treatment consisted in submerging the seeds in a 2 g·L-1 Captan® solution for 30 min before following the standard procedure with 70% ethanol for 30 sec and then 20 min in 2.5% sodium hypochlorite. The in vitro establishment of cannabis from seeds originating from Brazilian drug trafficking seizures was successful. The germination rate ranged from 10% to 90% according to the sample material. Non-brick weed, which consisted of dry leaves, stalks, and flowers, was more suitable for seed extraction and germination. Clones originating from BW4b showed the best development results compared with others. Conclusions: This is the first report of in vitro cannabis use in Brazil and opens great prospects for future work on its cultivation and research for medicinal applications in the country without relying on seed importation.

GPx3 dysregulation impacts adipose tissue insulin receptor expression and sensitivity.

Insulin receptor signaling is crucial for white adipose tissue (WAT) function. Consequently, lack of insulin receptor (IR) in WAT results in a diabetes-like phenotype. Yet, causes for IR downregulation in WAT of patients with diabetes are not well understood. By using multiple mouse models of obesity and insulin resistance, we identify a common downregulation of IR with a reduction of mRNA expression of selenoproteins Txnrd3, Sephs2, and Gpx3 in gonadal adipose tissue. Consistently, GPX3 is also decreased in adipose tissue of insulin-resistant and obese patients. Inducing Gpx3 expression via selenite treatment enhances IR expression via activation of the transcription factor Sp1 in 3T3-L1 preadipocytes and improves adipocyte differentiation and function. Feeding mice a selenium-enriched high-fat diet alleviates diet-induced insulin resistance with increased insulin sensitivity, decreased tissue inflammation, and elevated IR expression in WAT. Again, IR expression correlated positively with Gpx3 expression, a phenotype that is also conserved in humans. Consequently, decreasing GPx3 using siRNA technique reduced IR expression and insulin sensitivity in 3T3-L1 preadipocytes. Overall, our data identify GPx3 as a potentially novel regulator of IR expression and insulin sensitivity in adipose tissue.

Assessment of allelopathic, cytotoxic, genotoxic and antigenotoxic potential of Smilax brasiliensis Sprengel leaves.

Smilax brasiliensis (Smilacaceae) is a native Brazilian plant found in the Cerrado biome and commonly used in folk medicine. The aim of this study was to evaluate the allelopathic, cytotoxic, genotoxic, and antigenotoxic potential of extract and fractions of Smilax brasiliensis leaves. Quercetin and rutin isomers were observed in the subfractions. The dichloromethane fraction (1000 µg/mL) decreased lettuce (Lactuca sativa) seed vigor, while and ethyl acetate and hydromethanol fractions (1000 µg/mL) affected the germination, and quercetin and rutin affected the vigor and germination of onion seeds. The extract, fractions, quercetin, and rutin inhibited or promoted lettuce hypocotyl and radicle growth. The extract and fractions inhibited onion hypocotyl growth at all concentrations. With regards to radicle growth, the results were diversified: growth was either inhibited or promoted. Rutin and quercetin inhibited onion hypocotyl and radicle growth at all concentrations. The extract and fractions of Smilax brasiliensis, rutin, and quercetin did not cause cytotoxic effect evaluated by mitotic index. The extract and fractions showed genotoxic effects. Quercetin and rutin did not cause genotoxic effects. On the other hand, the extract and fractions showed antigenotoxic effects at all tested concentrations, where they were able to revert chromosomal abnormalities caused by glyphosate. However, additional studies are required to evaluate the possible use of the S. brasiliensis leaf methanol extract and fractions as natural sources of bioherbicides.

Embryogenic potential of the callus of gabirobeira, Campomanesia adamantium(Cambess) O. Berg

Campomanesia adamantiumis a native plant species of Brazilian Cerrado with diverse economic potential and great medicinal importance. Its sexual propagation is impaired by the recalcitrance of its seeds, which prevents effective and profitable propagation. With the purpose of establishing commercial crops and minimizing the extractive use of vegetal resources, the aim of the present study was to induce embryogenic calli in nodal segments of gabirobeira, and to determine and characterize their embryogenic phase through the establishment of a growth curve based on cellular characteristics. Calli were induced using nodal segments inoculated in WPM culture medium without the addition of hormones (control) and with different concentrations of 2,4-D, IAA, IBA, NAA or picloram. Cytochemical and SEM analyses revealed cellular characteristics of the formation of meristematic centers that indicated 4.14 μM of picloram to be the best treatment for induction of embryogenic calli, and demonstrating their embryogenic potential. The treatment was used to establish a callus growth curve, from which it was inferred that calli should be transferred to new culture media on the 28thday to maintain cell viability.

Morpho and Cytological Differentiation of Calli of Eucalyptus grandis x Eucalyptus urophylla During Somatic Embryogenesis

ABSTRACT The aim of this study was to induce and analyze embryogenic calli from two types of explants (leaves and meristems) of the hybrid Eucalyptus grandis x Eucalyptus urophylla. Leaves and meristems of plants kept in a nursery were disinfected and inoculated in Petri dishes containing MS culture medium supplemented with different concentrations of the growth regulator dicamba (1.13, 4.52, and 9.04 µM) and without it. At 60 days of culturing, the calli were analyzed by scanning electron microscopy and at 90 days were evaluated by light microscopy in regard to the embryogenic characteristics of the cells. Different type of calli were induced in leaf explants, designated as Type I with light yellow coloring, Type II with dark yellow coloring, and Type III of brown coloring; however, only Type I had embryogenic characteristics. In the meristematic explants, only one type of callus was induced, and it had embryogenic characteristics. At 90 days of culturing, the formation of somatic embryos in the different embryogenic stages was observed and the formation of procambium, protoderm, and ground meristem tissues. At 150 days of culturing, the concentration of 1.13 µM of dicamba was prominent in the formation of somatic embryos in the different embryogenic stages.

Non-linear impact of glutathione depletion on C. elegans life span and stress resistance.

The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH) representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM), an α,ß-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1mM. In contrast, low and moderate doses of DEM (10-100µM) increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100µM DEM, but not 1mM DEM, whereas only 1mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1) expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly hatched and developing worms.

Influence of hemodialysis on the plasma concentration of adenosine deaminase in patients with chronic kidney disease / Influência da hemodiálise na concentração plasmática da adenosina deaminase em pacientes com doença renal crônica

ABSTRACT Introduction: Over the past years there has been a significant increase in hospitalizations and treatments due to kidney complications that eventually resulted in the increased number of patients on dialysis. The adenosine deaminase (ADA) enzyme mediates the formation of some defense cells of the organism and is therefore a marker of inflammation. Objective: The objective of this study was to evaluate biomarkers of renal function and serum ADA of hemodialysis patients. Materials and methods: Blood samples were collected from 80 patients – 40 women and 40 men – between 19 and 60 years old, before and after the completion of hemodialysis. Results: There was a significant difference in levels of creatinine, urea and ADA in pre- and post-hemodialysis periods (p < 0.0001). There was a significant increase in post-dialysis ADA regardless of sex; however there was a significantly greater increase in men. Conclusion: The results showed a reduction in urea and creatinine parameters, evidencing the main purpose of hemodialysis. This study suggests that the determination of ADA activity could be used to monitor inflammation in hemodialysis patients, however wider and more specific studies are needed to show the effectiveness of serum ADA activity as an inflammatory marker in patients with chronic kidney disease. .

RESUMO Introdução: No decorrer dos últimos anos, houve aumento significativo nas internações e nos tratamentos decorrentes de complicações renais que resultaram, consequentemente, no aumento de pacientes sujeitos a diálise. A adenosina deaminase (ADA) atua como enzima mediadora na formação de algumas células de defesa do organismo, sendo, portanto, marcadora de processos inflamatórios. Objetivos: O objetivo deste trabalho foi avaliar biomarcadores da função renal e da ADA sérica de pacientes em hemodiálise Materiais e métodos: Amostras de sangue foram coletadas de 80 pacientes – 40 mulheres e 40 homens – entre 19 e 60 anos, antes e após a realização da hemodiálise. Resultados: Houve diferença significativa nas dosagens de creatinina, ureia e ADA no pré e pós-hemodiálise (p < 0,0001). Observou-se aumento significativo da ADA no pós-hemodiálise independentemente do sexo, no entanto houve aumento considerável nos homens. Conclusão: Os resultados mostraram redução nos parâmetros de ureia e creatinina, evidenciando o propósito principal da hemodiálise. Por meio deste estudo, sugere-se que a determinação da atividade da ADA pode ser utilizada para monitorar o processo inflamatório de pacientes em hemodiálise, contudo estudos mais amplos e específicos são necessários para mostrar a eficiência da dosagem de ADA sérica como marcador inflamatório para pacientes com doença renal crônica. .

Induction and Morpho-Ultrastructural Analysis of Organogenic Calli of a Wild Passionfruit

This work studied a new protocol for organogenic calli induction and characterization of the morphology and ultrastructure of callogenesis in leaf explants of Passiflora gibertii N. E. Brown, a native passion fruit species from Brazil. Calli induction was performed in different growth conditions (light and dark), different MS medium salt concentrations (MS and MS half strength) and the presence or absence of coconut water. The leaf explants maintained in the dark were more responsive to bud formation. In order to reduce spending on in vitro culture, the most suitable induction medium for P. gibertii organogenesis could, therefore be the MS half strength salt concentration medium maintained in the dark. The addition of coconut water to the culture medium was essential for both calli induction and bud formation. The morphological and ultrastructural features of the organogenic calli were isodiametric cells, characterized by an organized cellular system, nucleus with prominent nucleoli, presence of starch grains and dense cytoplasm rich in endoplasmic reticulum. The scanning electron microscopy demonstrated that buds were present on these calli.

Induction and characterization of oil palm (Elaeis guineensis Jacq.) pro-embryogenic masses.

Oil palm is one of the most economically valuable oil seed plants, but the expansion of plantations has been limited by availability of seedlings, as the conventional propagation is through seeds, which have low germination rates. One possible solution for the large-scale production is the use of somatic embryogenesis. The aim of this study was evaluate the effects auxins 2,4-D and picloram on the induction of pro-embryogenic masses in E.guineenesis hybrid leaf explants and characterize, regarding embryogenic characteristics, with cytochemical and ultrastructural analysis. Specifically, in vitro plantlets leaves fragments were inoculated in Y3 culture medium supplemented by 2.4-D or picloram at different concentrations (0.0, 1.0, 3.0, 6.0 and 9.0 mg l⁻¹). After 90 days the presence/ absence of cell masses were evaluated. Both growth regulators efficiently induced cellular masses regardless of the concentrations applied. As the cell masses were not homogeneously formed, they were classified according to color and shape into four types: TYPE 1--elongated and translucent, TYPE 2--uneven and translucent, TYPE 3--globular and beige, TYPE 4--globular and white. Based on the anatomical and ultrastructural features, TYPE 2, 3 and 4 cell masses were considered to have the highest embryogenic potential and therefore may be most suited to large-scale vegetative propagation of oil palm.

Growth curve and development of the internal calli structure of Eucalyptus camaldulensis Dehn

The objective of this work was to elucidate the growth curve of Eucalyptus camaldulensis Dehn. calli analyzing their anatomical modifications. A sigmoid aspect of the growth curve of the calli fresh matter was observed, with five different phases (lag, exponential, linear, deceleration and decline). In the lag phase, the highest growth percentage 87%, was observed, which reduced during the evaluation period to 17% in the linear phase. As for the anatomical analyses, cellular multiplications was observed during the lag and exponential phases and increase in cell size during the linear phase, promoting the calli volume growth and the establishment of the globular conformation.

Efeito de meios de cultura, concentrações de GA3 e pH sobre a germinação in vitro de mangabeira (Hancornia speciosa Gomes) / Effect of culture media, GA3 concentrations and pH on in vitro germination of Hancornia speciosa Gomes

A mangabeira (Hancornia speciosa Gomes) destaca-se por possuir um grande potencial como planta frutífera e produtora de borracha. As dificuldades encontradas no seu processo de propagação por meio de sementes, devido, principalmente, à baixa taxa de germinação e à recalcitrância, valorizam a busca por soluções alternativas para a produção de mudas dessa espécie, de maneira rápida e eficiente. Objetivou-se, neste trabalho, realizar o estudo da germinação de sementes de mangabeira em condições in vitro, tendo como precedente a obtenção de explantes, para posterior utilização no cultivo in vitro. Neste estudo foram avaliados os efeitos de diferentes meios de cultura, concentrações de sacarose e GA3 e de três níveis de pH na germinação da mangabeira. Frutos maduros foram coletados, passaram por processo de beneficiamento e tiveram suas sementes retiradas e utilizadas como explantes. Maior porcentagem de germinação de sementes de mangabeira in vitro foi obtida com a utilização dos meios de cultura WPM e MS/2, suplementados com 15,0 g L-1 de sacarose, 0,2 mg L-1 de GA3 e com pH corrigido para 5,8.

The Hancornia speciosa Gomes species presents potential for fruit and rubber production. Propagation is difficult primarily due, to a reduced seed germination and occurrence of recalcitrant seeds that stimulate the search of rapid and efficient propagation alternatives. In this context, the aim of this work was to study in vitro seed germination conditions in order to produce explants to be used on in vitro culture. The effect of different culture media, sucrose and GA3 concentrations and three pH levels were evaluated. Seeds were extracted from mature fruits after being harvested and processed. Higher in vitro germination was obtained using WPM and MS/2 media supplemented with 15.0 g L-1, 0.2 mg L-1 GA3 and pH adjusted to 5.8.

Germinação in vitro e ex vitro de Inga vera Willd. subsp. affinis (DC.) T.D. Penn. / Germination in vitro and ex vitro of Inga vera Willd. subsp. affinis (DC.) T.D. Penn

O Inga vera Will subsp. affinis (DC). T.D. Penn. é uma espécie frutífera nativa do Cerrado, importante na recuperação de matas ciliares degradadas. Entretanto, apresenta sua propagação dificultada pelo fato de suas sementes serem recalcitrantes, ou seja, não tolerarem a perda de água. O objetivo deste trabalho foi estudar aspectos da germinação ex vitro e in vitro de ingazeiro. Para tanto, foram avaliados os efeitos de diferentes substratos: areia, Plantmax® e Areia+ Plantmax®; diferentes concentrações de sais: WPM, WPM/2, MS e MS/2, e diferentes concentrações de GA3 (0, 5, 10, 17 e 20 µM) no meio de cultura. Observou-se que, na germinação ex vitro, o substrato Plantmax® proporcionou maior porcentagem de germinação (82 por cento). Com relação à germinação in vitro, a maior percentagem de germinação foi obtida utilizando-se meio de cultura WPM/2 (96 por cento). A adição de GA3 no meio de cultura não foi estatisticamente significativa, no entanto, a concentração de 20 µM de GA3 proporcionou um aumento na germinabilidade de sementes de ingazeiro.

The Inga vera Will subsp. affinis (DC). T.D. Penn. is fruit native specie from cerrado, commonly for recovering devastated areas. However, its propagation is complicated due to the fact that the seeds are recalcitrant and does not support water loss. The objective of this work was to study Inga vera Willd. subsp. affinis (DC.) T.D. Penn. ex vitro and in vitro germination aspects. For this purpose, different substrates: sand, Plantmax® and sand+ Plantmax®; different salt concentrations: WPM, WPM/2, MS and MS/2, and different GA3 concentrations (0, 5, 10, 17 and 20 µM) were evaluated. The results showed that, in the ex vitro germination, the use of Plantmax® provided the highest germination percentage (82 percent). Regarding the in vitro germination, highest percentage was observed using WPM/2 (96 percent). The addition of GA3 was not statistically significant although the concentration of 20 µM promoted an increase in the germination of Inga vera seeds.