Studies of Mucosal Irritation and Cellular Uptake Mechanisms of Xingnaojing Nanoemulsion

Abstract Xingnaojing (XNJ) injection was used to treat pneumonia and stroke in clinic in China, but with poor patient compliance. Xingnaojing nanoemulsion for intranasal delivery was developed to improve it. This article tried to evaluate the mucosal irritation of Xingnaojing nanoemulsion and investigate cellular uptake mechanism of its encapsulated lipophilic drugs. The toad palate model and rat nasal mucosa model were used to study the nasal ciliotoxicity and nasal mucosal irritation of nanoemulsion to evaluate its safety intranasally. The cellular uptake mechanism was studied by Calu-3 cell model. Coumarin 6 was encapsulated in nanoemulsion and the endocytic pathways were studied by cellular uptake experiments after being treated with different inhibitors. In toad palate model, the cilia movement of Xingnaojing nanoemulsion group last for 467.40 ± 39.02 min, which was obviously longer than deoxycholate group (90.60 ± 15.40 min). Studies on rats showed that the damage caused by nanemulsion is capable of being recovered. Nanoemulsion uptake was reduced obviously when cells were treated with wortmannin, and it also decreased about 13% when the temperature reduced from 37ºC to 4ºC. Mucosal irritation caused by nanoemulsion is low and the damage is recoverable. The cellular uptake of Xingnaojing nanoemulsion is energy-dependent, and macropinocytosis was the most important pathway for cellular uptake.

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells

BACKGROUND The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

El síndrome urémico hemolítico y manejo renal de proteínas / Hemolytic uremic syndrome and renal handling of proteins

El síndrome urémico hemolítico (SUH) está definido por la tríada de anemia hemolítica microangiopática, trombocitopenia e insuficiencia renal aguda. En Argentina constituye la primera causa de insuficiencia renal aguda en pediatría. Aproximadamente, del 2% al 4% de los pacientes mueren durante la fase aguda de la enfermedad, y solo un tercio del 96% restante que sobrevive lo hace con secuelas renales, como la persistencia de la proteinuria. Un individuo adulto sano filtra alrededor de 5000 mg/día de proteínas, si bien la excreción en orina es escasa (150 mg/día). La escasa cantidad de proteínas excretadas indica la presencia de un mecanismo de reabsorción a nivel del túbulo proximal. Por lo tanto, la reabsorción tubular renal desempeña un papel muy importante ya que, ante una función glomerular normal, es el principal mecanismo encargado de evitar la depleción proteica corporal. Desde hace aproximadamente 30 años se sabe que la albúmina es reabsorbida en el túbulo proximal. La reabsorción proteica se produce por un mecanismo de endocitosis mediada por el receptor dependiente de clatrina y por endocitosis de fase líquida. Clásicamente se ha descrito que el mecanismo básico del daño renal en el SUH típico y en el atípico es una microangiopatía trombótica, pero de diferentes causas. Sin embargo, debe tenerse en cuenta que la fisiopatología de esta enfermedad es más compleja de lo que se creía, ya que la alteración tubular que surge va a evolucionar en fallas en el mecanismo de endocitosis de proteínas que se suman a las eliminadas por las alteraciones a nivel de la barrera de filtración glomerular.

Hemolytic uremic syndrome (HUS) is defined by the triad of hemolytic anemia microangiopathic, thrombocytopenia and acute renal failure. In Argentina it constitutes the first cause of acute renal failure in Pediatrics. Approximately 2-4% of patients die during the acute phase of the disease, and only a third of the remaining 96% survive with renal sequelae, such as the persistence of proteinuria. A healthy adult filters around 5000 mg/day of proteins, with an excretion in urine of 150 mg/day. The little quantity of proteins excreted indicates the presence of a reabsorption mechanism at the level of the proximal tubule. Therefore, the tubular reabsorption plays a very important role since it is the main mechanism responsible for preventing the depletion of protein. For approximately 30 years, it has been known that albumin is reabsorbed in the proximal tubule. Protein reabsorption occurs by a clathrin-dependent receptor mediated endocytosis mechanism and by fluid phase endocytosis. The basic mechanism of renal damage in typical and atypical HUS has been described as a thrombotic microangiopathy, but of different causes. However, the pathophysiology of this disease is more complex than what was believed since the emerging tubular alteration will ewvolve into failures of the protein endocytosis mechanism that are added to the alterations at the level of the glomerular filtration barrier.

Enhanced apoptosis and inhibition of gastric cancer cell invasion following treatment with LDH@Au loaded Doxorubicin

Background: The suppression of cancer cell growth and invasion has become a challenging clinical issue. In this study, we used nanotechnology to create a new drug delivery system to enhance the efficacy of existing drugs. We developed layered double hydroxide by combing Au nanosol (LDH@Au) and characterized the compound to prove its function as a drug delivery agent. The anti-cancer drug Doxorubicin was loaded into the new drug carrier to assess its quality. We used a combination of apoptosis assays, cell cycle assays, tissue distribution studies, cell endocytosis, transwell invasion assays, and immunoblotting to evaluate the characteristics of LDH@Au as a drug delivery system. Results: Our results show that the LDH@Au-Dox treatment significantly increased cancer cell apoptosis and inhibited cell invasion compared to the control Dox group. Additionally, our data indicate that LDH@Au-Dox has a better target efficiency at the tumor site and improved the following: cellular uptake, anti-angiogenesis action, changes in the cell cycle, and increased caspase pathway activation. Conclusions: Our findings suggest the nano drug is a promising anti-cancer agent and has potential clinical applications.

Uptake of DNA by cancer cells without a transfection reagent

BACKGROUND: Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. METHODS: A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). RESULTS: The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. CONCLUSIONS: In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting reagent.

The Study of Otoacoustic Emissions and the Suppression of Otoacoustic Emissions in Subjects with Tinnitus and Normal Hearing: An Insight to Tinnitus Etiology

Introduction Analysis of the suppression effect is a simple method to evaluate cochlear status and central auditory mechanisms and, more specifically, the medial olivocochlear system. This structure may be involved in the generation of mechanisms that cause tinnitus and in the pathophysiology of tinnitus in patients with tinnitus and normal hearing. Objective To review the literature of the etiology of tinnitus on the lights of otoacoustic emissions in patients with normal hearing. Data Synthesis Individuals with tinnitus and normal hearing have a higher prevalence of alterations in transient-evoked otoacoustic emissions and distortion-product otoacoustic emissions than normal subjects. This fact suggests that dysfunctions of the outer hair cells (OHCs) might be important in the generation of the tinnitus; however, this feature is not always present in those who have the symptoms of tinnitus. Final Comments These findings suggest that OHC dysfunction is not necessary for tinnitus development—that is, there might be mechanisms other than OHC damage in the tinnitus development. On the other hand, OHC dysfunction alone is not sufficient to cause the symptom, because a great many individuals with OHC dysfunction did not complain about tinnitus. .

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Estudo do efeito da autofagia sobre a endocitose e a adesão celular em macrófago murino in vitro / Study of autophagy the effect on endocytosis and cell adhesion in a murine macrophage in vitro

INTRODUÇÃO: A influência da autofagia em processos celulares que participam da homeostase celular, como a endocitose e a adesão celular, até o momento, foi pouco estudada. A endocitose consiste na internalização de material extracelular,quando as vesículas endocíticas são menores que 500nm é chamada de endocitose em microescala e quando as vesículas formadas são maiores que essa medida trata-se de endocitose em macroescala. Foi demonstrado que a conexão da via endocítica com a via autofágica é fundamental para a degradação de material citosólico e, subsequente, produção de energia e disponibilização de substrato para o metabolismo celular. Estudos controversos da literatura mostraram que a autofagia pode favorecer ou não interferir com a endocitose em macroescala. Além disso, alguns trabalhos demonstraram que o processo autofágico foi capaz de reduzir a reciclagem de integrinas para a membrana plasmática por alterar a endocitose em microescala envolvida na internalização desse tipo de proteína, reduzindo a capacidade de adesão e, consequentemente, a migração celular. Assim, em conjunto, esses achados evidenciam que a autofagia pode interagir e interferir com eventos celulares dependentes da participação da membrana plasmática como a endocitose e a adesão celular.OBJETIVO: No presente estudo, hipotetizamos que a prévia indução de autofagia em macrófagos é capaz de reduzir a endocitose em micro e macroescala, além de reduzir a capacidade de adesão celular. Desta forma, o objetivo desse estudo foi determinar o efeito da indução de autofagia, in vitro, sobre a endocitose e a adesão de macrófagos murino. MATERIAL E MÉTODOS: Macrófagos foram induzidos à autofagia por privação de nutrientes (starvation) ou pelo tratamento com um indutor farmacológico, a rapamicina, seguida da exposição a macromoléculas ou grandes partículas de diferentes naturezas. Além disso, após indução de autofagia, macrófagos em suspensão foram incubados em superfícies como o vidro ou uma matriz de colágeno e fibronectina para avaliação da capacidade de adesão.Os percentuais de endocitose em microescala, em macroescala e de adesão foram estimados. RESULTADOS: Mostramos que a indução de autofagia promoveu redução da capacidade fagocítica em cerca de 60% no percentual de macrófagos que internalizam grandes partículas, como levedo, sendo um mecanismo precoce e reversível. Ao passo que a indução de autofagia por privação de aminoácidos ou farmacológica não interferiu na endocitose em microescala. A indução de autofagia não alterou a endocitose de transferrina (endocitose mediada por receptores) e endocitose de BSA (endocitose de fase fluida). Em contraste, a indução de autofagia promoveu redução em aproximadamente 70% da quantidade de macrófagos que aderem a matriz de colágeno e fibronectina. Uma possível explicação para a redução da endocitose em macroescala pode estar relacionada à autofagia diminuir a disponibilidade de grandes extensões de membrana necessárias à internalização de partículas ma iores que 500nm. Alternativamente, a indução de autofagia pode estar levando a célula a uma indisponibilidade de receptores na membrana plasmática que justificaria a redução da capacidade fagocítica e de adesão do 11 macrófago murino. CONCLUSÕES: A indução de autofagia diminui a capacidade fagocítica e a capacidade de adesão do macrófago murino.

INTRODUCTION: The influence of autophagy on cellular processes that participate in cellular homeostasis, such as endocytosis and cell adhesion has been poorly evaluated. Endocytosis consists in the internalization of extracellular material and includes microscale endocytosis, when endocytic vesicles are smaller than 500nm, and macroscale endocytosis, when the formed vesicles are larger than this measure. It has been shown that the connection between the endocytic and the autophagic pathways is essential for degradation of cytosolic material and, subsequently, power generation and provision of substrate for cellular metabolism. Controversial studies showed that autophagy can improve or do not interfere with macroscale endocytosis. Furthermore, some studies demonstrated that the autophagic process reduced integrin recycling to the plasma membrane through the modulation of microscale endocytosis involved in the internalization of this protein, reducing cell adhesion and migration. Taken together, these findings show that autophagy can interact and interfere with cellular events that depend on plasma membrane participation, such as endocytosis and cell adhesion. OBJECTIVES: In the present study, we hypothesized that prior autophagy induction in macrophage reduces micro and macroscale endocytosis, as well as cell adhesion. Thus, the aim of this study was to determine the effect of autophagy induction, in vitro, on endocytosis and adhesion of murine macrophages. MATERIAL AND METHODS: Autophagy by nutrient deprivation (starvation) or by treatment with an inducer drug, rapamycin, was induced in macrophages, followed by exposure to macromolecules or large particles of different natures. Furthermore, after autophagic induction, macrophages were plated on different surfaces like glass or collagen-fibronectin matrix to evaluate cell adhesiveness. After that, the percentage of endocytosis in micro and macroscale and adhesion were determined...

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae

BACKGROUND: A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae. RESULTS: A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked. CONCLUSIONS: Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

Interferon-γ inhibits group B Streptococcus survival within human endothelial cells

Endothelial dysfunction is a major component of the pathophysiology of septicaemic group B Streptococcus (GBS) infections. Although cytokines have been shown to activate human umbilical vein endothelial cells (HUVECs), the capacity of interferon (IFN)-γ to enhance the microbicidal activity of HUVECs against GBS has not been studied. We report that the viability of intracellular bacteria was reduced in HUVECs activated by IFN-γ. Enhanced fusion of lysosomes with bacteria-containing vacuoles was observed by acid phosphatase and the colocalisation of Rab-5, Rab-7 and lysosomal-associated membrane protein-1 with GBS in IFN-γ-activated HUVECs. IFN-γ resulted in an enhancement of the phagosome maturation process in HUVECs, improving the capacity to control the intracellular survival of GBS.

Adhesive and invasive capacities of Edwarsiella tarda isolated from South American sea lion

Edwarsiella tarda is a zoonotic bacterium that can be isolated from humans, animals and the environment. Although E. tarda is primarily considered a fish pathogen, it is the only species of its genus considered to be pathogenic for humans as well. A survey of zoonotic intestinal bacteria in fresh feces from South American sea lions (SASL) Otaria flavescens, reported E. tarda as the most frequently isolated species. In this study, we used HEp-2 cells to establish in vitro the adherence and invasive ability of 17 E. tarda strains isolated from SASL fecal material. All the strains were able to adhere and invade HEp-2 cells with adhesion and invasion percentages ranging from 56 to 100% and 21 to 74%, respectively. Despite the expression of these pathogenic factors, further investigation is needed to determine whether this bacterium could play a role as primary pathogen for this and other species of pinnipeds.

The balance between GMD and OFUT1 regulates Notch signaling pathway activity by modulating Notch stability

The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.

Noncrystalline uric acid inhibits proteoglycan and glycosaminoglycan synthesis in distal tubular epithelial cells (MDCK)

Hyperuricemia is associated with renal stones, not only consisting of uric acid (UrAc) but also of calcium oxalate (CaOx). Glycosaminoglycans (GAGs) are well-known inhibitors of growth and aggregation of CaOx crystals. We analyzed the effect of noncrystalline UrAc on GAG synthesis in tubular distal cells. MDCK (Madin-Darby canine kidney) cells were exposed to noncrystalline UrAc (80 µg/mL) for 24 h. GAGs were labeled metabolically and characterized by agarose gel electrophoresis. The expression of proteoglycans and cyclooxygenase 2 (COX-2) was assessed by real-time PCR. Necrosis, apoptosis and prostaglandin E2 (PGE2) were determined by acridine orange, HOESCHT 33346, and ELISA, respectively. CaOx crystal endocytosis was evaluated by flow cytometry. Noncrystalline UrAc significantly decreased the synthesis and secretion of heparan sulfate into the culture medium (UrAc: 2127 ± 377; control: 4447 ± 730 cpm) and decreased the expression of perlecan core protein (UrAc: 0.61 ± 0.13; control: 1.07 ± 0.16 arbitrary units), but not versican. Noncrystalline UrAc did not induce necrosis or apoptosis, but significantly increased COX-2 and PGE2 production. The effects of noncrystalline UrAc on GAG synthesis could not be attributed to inflammatory actions because lipopolysaccharide, as the positive control, did not have the same effect. CaOx was significantly endocytosed by MDCK cells, but this endocytosis was inhibited by exposure to noncrystalline UrAc (control: 674.6 ± 4.6, CaOx: 724.2 ± 4.2, and UrAc + CaOx: 688.6 ± 5.4 geometric mean), perhaps allowing interaction with CaOx crystals. Our results indicate that UrAc decreases GAG synthesis in MDCK cells and this effect could be related to the formation of UrAc and CaOx stones.

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.

Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56 percent lipids and 9 and 7 percent carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Trypanosoma cruzi: contribuição ao estudo da endocitose dependente e independente de clatrina em formas epimastigotas / Trypanosoma cruzi: contribution to the study of the dependent and independent endocytosis of clatrina in epimastigote forms

Endocitose em células eucarióticas é o processo de incorporação de macromoléculas por diferentes vias, com diversas proteínas associadas. Este processo ocorre através do brotamento de vesículas na membrana plasmática e endereçamento destas vesículas a compartimentos endossomais no citoplasma. Os tripanossomatídeos são protozoários flagelados patogênicos de grande importância médica e veterinária que apresentam diferentes formas adaptativas aolongo de seu ciclo de vida. Estes parasitas estão estruturalmente organizados dentro de um arcabouço de microtúbulos subpeliculares, o que dificulta invaginações de membrana na maior parte do corpo celular. Entretanto, este arcabouço é descontinuado na região da bolsa flagelar, de onde o flagelo emerge do corpo. Formas epimastigotas do Trypanosoma cruzi apresentam além da bolsa flagelar, uma segunda invaginação de membrana, o citóstoma/citofaringe. Esta estrutura é sustentada por microtúbulos especializados que participam ativamente no processo de endocitose. O objetivo desta tese foi investigar as vias endocíticas presentes nos dois sítios competentes para a captação de nutrientes (bolsa flagelar e citóstoma) de epimastigotas de T. cruzi. Após incubação dos parasitas a 28(graus) C com albumina, transferrina e LDL conjugadas a ouro coloidal e posterior processamento para microscopia eletrônica de transmissão (MET), vesículas endocíticas revestidas contendo albumina foram observadas brotando da bolsa flagelar. Através da análise do conteúdo protéico dos protozoários foi detectada a expressão de clatrina, confirmada por citometria de fluxo e análise in silico da base de dados genômicos do T. cruzi. Por microscopia confocal localizou-se a clatrina na região compreendida entre o núcleo e a bolsa flagelar dos parasitas. Como transferrina foi visualizada em vesículas sem revestimento localizadas majoritariamente no citóstoma, frações de membrana detergente-resistentes foram purificadas por fracionamento celular e o...

Toxoplasma gondii: aspectos estruturais e funcionais da parede de cistos teciduais e cistogênese em células musculares esqueléticas, in vitro / Toxoplasma gondii: structural and functional aspects of the wall of tissue cysts and cystogenesis in skeletal muscle cells, in vitro

Toxoplasmose é uma importante doença parasitária de distribuição mundial, causada pelo Toxoplasma gondii. A necessidade de novos agentes terapêuticos contra a toxoplasmose tem despertado o interesse: (i) pela melhor caracterização da superfície celular das diferentes formas evolutivas do parasito, e (ii) por um melhor entendimento sobre os mecanismos de aquisição de nutrientes por esse patógeno assim como (iii) dos eventos relacionados à sua conversão para bradizoítos e formação do cisto tecidual. Assim, nosso primeiro objetivo foi investigar a carga elétrica de superfície dos cistos de T. gondii, através de marcadores policatiônicos, tais como o vermelho de rutênio (VR) e a ferritina cationizada (FC). Através do uso do VR foi possível identificar a localização de glicosaminoglicanos por toda a superfície da parede cística, como uma camada homogênea granular eletrondensa. A incubação de cistos vivos por 20 min a 4°C com FC confirmou que a sua superfície é carregada negativamente, havendo a subseqüente incorporação destes sítios aniônicos após a elevação da temperatura para 37°C. Nestes ensaios, inicialmente, os sítios aniônicos estavam presentes em vesículas e túbulos, e posteriormente localizados na matriz cística, próximos elou em contato direto com a membrana de bradizoítos presentes no interior dos cistos. A seguir, como o intercâmbio molecular entre os parasitos encistados e o citoplasma da célula hospedeira infectada é ainda pouco conhecido, nosso segundo objetivo foi avaliar a incorporação de traçadores endocíticos de fase fluída pelos cistos de T. gondii.

A microscopia de varredura confocal a laser (MVCL) utilizando reconstrução 3D de cistos isolados e incubados com albumina bovina (BSA) conjugada a FITC mostrou a associação do ligante à parede cística, assim como sua localização na matriz dos cistos. A atividade endocítica foi adicionalmente avaliada por estudos ultraestruturais utilizando-se diferentes traçadores (ferritina nativa, peroxidase e BSA) conjugados a partículas de ouro coloidal. A análise por microscopia eletrônica de transmissão (MET) confirmou a presença dos ligantes na superfície do cisto tecidual, dentro de vesículas e em túbulos dispersos na sua matriz, bem como associados à membrana dos bradizoítos intracísticos. Esses resultados sugerem que, pelo menos uma das vias relacionadas à aquisição de nutrientes pelos parasitos intracísticos , envolva a incorporação de moléculas disponíveis no citoplasma da célula hospedeira. Na última etapa da presente tese nosso objetivo foi acompanhar a cistogênese do T. gondii in vitro, durante a interação de bradizoítos com culturas primárias de células musculares esqueléticas (CME). A conversão de bradizoíto para taquizoíto foi monitorada entre 12-168 h de interação através de ensaios de imunofluorescência, utilizando-se anticorpos monoclonais estágio-específicos, anti-BAG1 e anti-SAG1, respectivamente.

Nossos resultados revelaram que até 24 h de interação, não houve conversão bradizoíto-taquizoíto, e que somente a partir de 48 h, vacúolos parasitóforos (VPs) positivos para SAG1 ou BAG1 puderam ser identificados numa mesma célula, sendo que os primeiros sempre continham um maior número de parasitas. Adicionalmente, a análise da interação bradizoítos-CME por microscopia eletrônica de varredura revelou a associação do parasito à célula hospedeira pela região anterior do parasito assim como, pela sua porção posterior e lateral havendo inclusive, expansão da membrana das CME. Estes dados sugerem que a invasão deste tipo celular por bradizoítos ocorra tanto por penetração ativa como por endocitose. Por fim, a análise por MET mostrou a participação de elementos do retículo endoplasmático rugoso das CME na formação do VP e permitiu evidenciar a completa cistogênese do T. gondii a partir de 96 h de infecção. Os presentes resultados fornecem importantes informações relacionadas à natureza da parede cística e sua capacidade endocítica, além de apresentar dados inéditos sobre o processo de cistogênese do T. gondii em CME. Este conjunto de informações pode efetivamente contribuir para a formulação de novas abordagens terapêuticas para toxoplasmose crônica além de revelar que o cultivo primário de CME representa uma excelente ferramenta para o estudo dos fatores envolvidos na interconversão do T. gondii.

Expressão do receptor para manose em células de Schwann e Schwannoma ST88-14 / Expression of the receiver for mannose in cells of Schwann and Schwannoma ST88-14

O receptor para manose receptor (RM) é uma glicoproteína transmembrana que é expressa em vários tipos celulares, mas, pouca ou nenhuma informação sobre RM existe em células de Schwann (CS). Mostramos que CS de rato em culturas primárias de células dissociadas ou em explantes de nervo bem como, numa linhagem de Schwannoma humano (ST88-14) liga uma neoglicoproteína manosil/albumina de soro bovino - isotiocianato de fluoresceína (man/BSA-FITC) de uma maneira altamente específica. Após incubação com man/BSA-FITC, a análise por citometria de fluxo demonstra 62por cento de células ST88-14 e 90por cento de CS positivas, uma ligação dose-dependente de ligantes marcads e inibição quase total pela competição com 250 mM D-manose ou com a proteína (altamente manosilada) peroxidase de raiz forte (HRP) - 1.1(miu)M. O tratamento de CS cultivadas com dexametasona (0.1 (miu)g/ml) ou interferon gama (IFN-gama - 100 U/ml) seguido pela marcação com man/BSA-FITC e análise por citometria de fluxo mostra um acréscimo e um decréscimo, respectivamente, da captação do ligante. Explantes de nervo ciático cultivados na presence de IFN-gama mostram colocalização de man/BSA-FITC e imunoreatividade para MHC classe II. A análise ultra-estrutural de células ST88-14 após incubação a 4grausC por 40min com HRP-Au coloidal com ou sem subseqüente elevação da temperatura para 37grausC mostra uma localização inicial na superfície, seguida da internalização do traçador temperatura e tempo-dependente. Células ST88-14 e CS dissociadas bem como nervos recentemente esgarçados mostram reatividade a um anticorpo policlonal contra o domínio C-terminal do RM de macrófagos murinos (anti-cMR). No caso de nervos esgarçados, a imunorreatividade é particularmente abundante em regiões topograficamente homólogas às alças para-nodais. Além disso, a imuno-histoquímica ultra-estrutural de nervo ciático incluído em Lowicryl mostra reatividade ao anti-cMR, com máxima deposição do anticorpo secundário-Au...periférico.

Envolvimento do proteoglicano de heparam sulfato no transplante de proteínas para o sistema endossomal/lisossomal / Envolvement of the Proteoglicano de Heparam Sulphate in the Protein Transport for the Endossomal/Lisossomal System

A membrana plasmática é a fronteira que delimita a célula e representa uma barreira para os componentes da matriz extracelular. Os avanços obtidos em biologia molecular resultaram no desenvolvimento de novas estratégias terapêuticas (ex: terapia gênica e entrega de proteínas), que na maioria das vezes, baseia-se na capacidade que macromoléculas carregadas possuem de atravessar a membrana plasmática e atingir o compartimento celular. O presente trabalho mostra o papel do proteoglicano de heparam sulfato no processo de internalização da catepsina X e da crotamina, um peptídeo carregado positivamente, e também o complexo crotamina-ácido nucléico. Esse trabalho mostra a importância do proteoglicano de heparam sulfato como uma molécula de interface entre componentes da matriz extracelular e o meio intracelular.