Biogenesis of EVs in Trypanosomatids.

Trypanosomes are protozoan parasites responsible for human diseases such as Chagas disease, African trypanosomiasis, and leishmaniasis. These organisms' growth in various environments and exhibit multiple morphological stages, while adapting their surface components. They acquire and release materials extensively to get nutrients and manage interactions with the extracellular environment. They acquire and utilize proteins, lipids, and carbohydrates for growth via using membrane transport and endocytosis. Endocytosis takes place through distinct membrane areas known as the flagellar pocket and cytostome, depending on the parasite species and its developmental stage. Some forms establish a complex endocytic system to either store or break down the absorbed materials. In contrast, membrane transport facilitates the uptake of small molecules like amino acids, carbohydrates, and iron via particular receptors on the plasma membrane. Concurrently, these parasites secrete various molecules such as proteins, enzymes, nucleic acids, and glycoconjugates either in soluble form or enclosed in extracellular vesicles, which significantly contribute to their parasitic behavior. These activities require exocytosis through a secretory pathway in certain membrane domains such as the flagellum, flagellar pocket, and plasma membrane, which are controlled at various developmental stages. The main features of the endocytic and exocytic mechanisms, as well as the organelles involved, are discussed in this chapter along with their connection to the formation of exosomes and extracellular vesicles in the Tritryp species.

The interaction of galectin-8 C-terminal domain with cell surface glycoconjugates modulates membrane elasticity to stimulate antigen uptake and presentation to CD4 T cells.

Galectins constitute a family of soluble lectins with unique capacity to induce macroscale rearrangements upon interacting with cell membrane glycoconjugates. Galectin-8 (Gal-8) is acknowledged for its role in facilitating antigen uptake and processing upon engaging with cell surface glycoconjugates on antigen-presenting cells (APCs). Gal-8 consists of two covalently fused N- and C-terminal carbohydrate recognition domains (N- and C-CRD), each exhibiting distinct glycan specificity. In this study, we utilized single N- and C-CRD recombinant proteins to dissect the nature of Gal-8-glycan interactions during antigen internalization enhancement. Single C-CRD was able to replicate the effect of full-length Gal-8 (FLGal-8) on antigen internalization in BMDCs. Antigen uptake enhancement was diminished in the presence of lactose or when N-glycosylation-deficient macrophages served as APCs, underscoring the significance of glycan recognition. Measurement of the elastic modulus using Atomic Force Microscopy unveiled that FLGal-8- and C-CRD-stimulated macrophages exhibited heightened membrane stiffness compared to untreated cells, providing a plausible mechanism for their involvement in endocytosis. C-CRD proved to be as efficient as FLGal-8 in promoting antigen degradation, suggesting its implication in antigen-processing induction. Lastly, C-CRD was able to replicate FLGal-8-induced antigen presentation in the MHC-II context both in vitro and in vivo. Our findings support the notion that Gal-8 binds through its C-CRD to cell surface N-glycans, thereby altering membrane mechanical forces conducive to soluble antigen endocytosis, processing, and presentation to cognate CD4 T-cells. These findings contribute to a deeper comprehension of Gal-8 and its mechanisms of action, paving the way for the development of more efficacious immunotherapies.

Lysosomal Activation Mediated by Endocytosis in J774 Cell Culture Treated with N-Trimethyl Chitosan Nanoparticles.

Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 µg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.

Receptor-mediated endocytosis in kidney cells during physiological and pathological conditions.

Mammalian cell membranes are very dynamic where they respond to several environmental stimuli by rearranging the membrane composition by basic biological processes, including endocytosis. In this context, receptor-mediated endocytosis, either clathrin-dependent or caveolae-dependent, is involved in different physiological and pathological conditions. In the last years, an important amount of evidence has been reported that kidney function involves the modulation of different types of endocytosis, including renal protein handling. In addition, the dysfunction of the endocytic machinery is involved with the development of proteinuria as well as glomerular and tubular injuries observed in kidney diseases associated with hypertension, diabetes, and others. In this present review, we will discuss the mechanisms underlying the receptor-mediated endocytosis in different glomerular cells and proximal tubule epithelial cells as well as their modulation by different factors during physiological and pathological conditions. These findings could help to expand the current understanding regarding renal protein handling as well as identify possible new therapeutic targets to halt the progression of kidney disease.

The angiotensin II/type 1 angiotensin II receptor pathway is implicated in the dysfunction of albumin endocytosis in renal proximal tubule epithelial cells induced by high glucose levels.

It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10-7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.

Expression and function of the vitellogenin receptor in the hypopharyngeal glands of the honey bee Apis mellifera (Hymenoptera: Apidae) workers.

The vitellogenin receptor (VgR) is essential for the uptake and transport of the yolk precursor, vitellogenin (Vg). Vg is synthesized in the fat body, released in the hemolymph, and absorbed in the ovaries, via receptor-mediated endocytosis. Besides its important role in the reproductive pathway, Vg occurs in nonreproductive worker honey bee, suggesting its participation in other pathways. The objective was to verify if the VgR occurs in the hypopharyngeal glands of Apis mellifera workers and how Vg is internalized by these cells. VgR occurrence in the hypopharyngeal glands was evaluated by qPCR analyses of VgR and immunohistochemistry in workers with different tasks. The VgR gene is expressed in the hypopharyngeal glands of workers with higher transcript levels in nurse honey bees. VgR is more expressed in 11-day-old workers from queenright colonies, compared to orphan ones. Nurse workers with developed hypopharyngeal glands present higher VgR transcripts than those with poorly developed glands. The immunohistochemistry results showed the co-localization of Vg, VgR and clathrin (protein that plays a major role in the formation of coated vesicles in endocytosis) in the hypopharyngeal glands, suggesting receptor-mediated endocytosis. The results demonstrate that VgR performs the transport of Vg to the hypopharyngeal glands, supporting the Ovary Ground Plan Hypothesis and contributing to the understanding of the role of this gland in the social context of honey bees.

PLC-γ-Ca2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling.

Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca2+ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca2+ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca2+ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.

Toll like receptor 4 mediates the inhibitory effect of SARS-CoV-2 spike protein on proximal tubule albumin endocytosis.

Tubular proteinuria is a common feature in COVID-19 patients, even in the absence of established acute kidney injury. SARS-CoV-2 spike protein (S protein) was shown to inhibit megalin-mediated albumin endocytosis in proximal tubule epithelial cells (PTECs). Angiotensin-converting enzyme type 2 (ACE2) was not directly involved. Since Toll-like receptor 4 (TLR4) mediates S protein effects in various cell types, we hypothesized that TLR4 could be participating in the inhibition of PTECs albumin endocytosis elicited by S protein. Two different models of PTECs were used: porcine proximal tubule cells (LLC-PK1) and human embryonic kidney cells (HEK-293). S protein reduced Akt activity by specifically inhibiting of threonine 308 (Thr308) phosphorylation, a process mediated by phosphoinositide-dependent kinase 1 (PDK1). GSK2334470, a PDK1 inhibitor, decreased albumin endocytosis and megalin expression mimicking S protein effect. S protein did not change total TLR4 expression but decreased its surface expression. LPS-RS, a TLR4 antagonist, also counteracted the effects of the S protein on Akt phosphorylation at Thr308, albumin endocytosis, and megalin expression. Conversely, these effects of the S protein were replicated by LPS, an agonist of TLR4. Incubation of PTECs with a pseudovirus containing S protein inhibited albumin endocytosis. Null or VSV-G pseudovirus, used as control, had no effect. LPS-RS prevented the inhibitory impact of pseudovirus containing the S protein on albumin endocytosis but had no influence on virus internalization. Our findings demonstrate that the inhibitory effect of the S protein on albumin endocytosis in PTECs is mediated through TLR4, resulting from a reduction in megalin expression.

MSC-derived exosomes protect auditory hair cells from neomycin-induced damage via autophagy regulation.

BACKGROUND: Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear. RESULTS: Exosomes derived from MSCs reduced hearing and hair cell loss caused by neomycin-induced damage in models in vivo and in vitro. In addition, MSC-derived exosomes modulated autophagy in hair cells to exert a protective effect. Mechanistically, exogenously administered exosomes were internalized by hair cells and subsequently upregulated endocytic gene expression and endosome formation, ultimately leading to autophagy activation. This increased autophagic activity promoted cell survival, decreased the mitochondrial oxidative stress level and the apoptosis rate in hair cells, and ameliorated neomycin-induced ototoxicity. CONCLUSIONS: In summary, our findings reveal the otoprotective capacity of exogenous exosome-mediated autophagy activation in hair cells in an endocytosis-dependent manner, suggesting possibilities for deafness treatment.

Multiple Labeling of Compartmentalized Cortical Neurons in Microfluidic Chambers.

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells. Key features • Preparation and attachment of PDMS microfluidic chambers to glass coverslips. • Primary culture of cortical neurons and plating cortical neurons in microfluidic chamber. • Confirmation of compartmentalization using the retrograde transport of the fluorescently labeled form of cholera toxin subunit B (f-Ctb). • Immunofluorescence and multilabeling of compartmentalized cortical neurons. • Retrograde transport of fluorescently labeled BDNF.

Caveolae-mediated endocytosis of extracellular QSOX1b modulates the migration of fibroblasts.

Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.

MSC-derived exosomes protect auditory hair cells from neomycin-induced damage via autophagy regulation

BACKGROUND: Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear. RESULTS: Exosomes derived from MSCs reduced hearing and hair cell loss caused by neomycin-induced damage in models in vivo and in vitro. In addition, MSC-derived exosomes modulated autophagy in hair cells to exert a protective effect. Mechanistically, exogenously administered exosomes were internalized by hair cells and subsequently upregulated endocytic gene expression and endosome formation, ultimately leading to autophagy activation. This increased autophagic activity promoted cell survival, decreased the mitochondrial oxidative stress level and the apoptosis rate in hair cells, and ameliorated neomycin-induced ototoxicity. CONCLUSIONS: In summary, our findings reveal the otoprotective capacity of exogenous exosome-mediated autophagy activation in hair cells in an endocytosis-dependent manner, suggesting possibilities for deafness treatment.

Endocytosis in anaerobic parasitic protists

The incorporation of different molecules by eukaryotic cells occurs through endocytosis, which is critical to the cell's survival and ability to reproduce. Although this process has been studied in greater detail in mammalian and yeast cells, several groups working with pathogenic protists have made relevant contributions. This review analysed the most relevant data on the endocytic process in anaerobic protists (Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, and Tritrichomonas foetus). Many protozoa can exert endocytic activity across their entire surface and do so with great intensity, as with E. histolytica. The available data on the endocytic pathway and the participation of PI-3 kinase, Rab, and Rho molecular complexes is reviewed from a historical perspective.

Human Papilloma Virus-Infected Cells.

Human papillomavirus (HPV) is associated with infection of different tissues, such as the cervix, anus, vagina, penis, vulva, oropharynx, throat, tonsils, back of the tongue, skin, the lungs, among other tissues. HPV infection may or may not be associated with the development of cancer, where HPVs not related to cancer are defined as low-risk HPVs and are associated with papillomatosis disease. In contrast, high-risk HPVs (HR-HPVs) are associated with developing cancers in areas that HR-HPV infects, such as the cervix. In general, infection of HPV target cells is regulated by specific molecules and receptors that induce various conformational changes of HPV capsid proteins, allowing activation of HPV endocytosis mechanisms and the arrival of the HPV genome to the human cell nucleus. After the transcription of the HPV genome, the HPV genome duplicates exponentially to lodge in a new HPV capsid, inducing the process of exocytosis of HPV virions and thus releasing a new HPV viral particle with a high potential of infection. This infection process allows the HPV viral life cycle to conclude and enables the growth of HPV virions. Understanding the entire infection process has been a topic that researchers have studied and developed for decades; however, there are many things to still understand about HPV infection. A thorough understanding of these HPV infection processes will allow new potential treatments for HPV-associated cancer and papillomatosis. This chapter focuses on HPV infection, the process that will enable HPV to complete its HPV life cycle, emphasizing the critical role of different molecules in allowing this infection and its completion during the HPV viral life cycle.

Respiratory syncytial virus entry mechanism in host cells: A general overview.

Respiratory syncytial virus (RSV) is a virus that causes acute respiratory infections in neonates and older adults. To infect host cells, the attachment glycoprotein (G) interacts with a cell surface receptor. This interaction determines the specific cell types that are susceptible to infection. RSV possesses a type I fusion protein F. Type I fusion proteins are metastable when rearrangement of the prefusion F occurs; the fusion peptide is exposed transforming the protein into postfusion form. The transition between the prefusion form and its postfusion form facilitates the viral envelope and the host cell membrane to fuse, enabling the virus to enter the host cell. Understanding the entry mechanism employed by RSV is crucial for developing effective antiviral therapies. In this review, we will discuss the various types of viral fusion proteins and explore the potential entry mechanisms utilized by RSV. A deeper understanding of these mechanisms will provide valuable insights for the development of novel approaches to treat RSV infections.

A SNARE-like protein from Solanum lycopersicum increases salt tolerance by modulating vesicular trafficking in tomato.

Intracellular vesicular trafficking ensures the exchange of lipids and proteins between endomembrane compartments. This is relevant under high salinity conditions, since both the removal of transporters and ion channels from the plasma membrane and the compartmentalization of toxic ions require the formation of vesicles, which can be maintained as multivesicular bodies or be fused to the central vacuole. SNARE proteins (Soluble N-ethylmaleimide-sensitive factor attachment receptor) participate in the vesicle fusion process and give specificity to their destination. Plant genome studies have revealed a superfamily of genes that encode for proteins called SNARE-like. These proteins appear to be participating in vesicular trafficking with similar functions to those of SNARE proteins. A SNARE-like, named SlSLSP6, in Solanum lycopersicum plants has been shown to be induced under high salinity conditions. A phylogenetic relationship of SlSLSP6 with SNARE-like proteins of salinity-tolerant plants, including Salicornia brachiata, Zostera marina and Solanum pennelli, was determined. Considering its amino acid sequence, a putative clathrin adapter complex domain and palmitoylation site was predicted. Subcellular localization analysis evidenced that SlSLSP6 is mostly localized in the plasma membrane. Using transgenic tomato plants, we identified that overexpression of SlSLSP6 increased tolerance to salt stress. This tolerance was evident when we quantified an improvement in physiological and biochemical parameters, such as higher chlorophyll content, performance index, efficiency of photosystem II and relative water content, and lower malondialdehyde content, compared to control plants. At the subcellular level, the overexpression of SlSLSP6 reduced the presence of H2O2 in roots and increased the compartmentalization of sodium in vacuoles during salt stress. These effects appear to be associated with the higher endocytic rate of FM4-64, determined in the plant root cells. Taken together, these results indicate that SlSLSP6 increases tolerance to salt stress by modulating vesicular trafficking through over-induction of the endocytic pathway. This work contributes to understanding the role of this type of SNARE-like protein during salt stress and could be a potential candidate in breeding programs for tolerance to salt stress in tomato plants.

More Than Pigments: The Potential of Astaxanthin and Bacterioruberin-Based Nanomedicines.

Carotenoids are natural products regulated by the food sector, currently used as feed dyes and as antioxidants in dietary supplements and composing functional foods for human consumption. Of the nearly one thousand carotenoids described to date, only retinoids, derived from beta carotene, have the status of a drug and are regulated by the pharmaceutical sector. In this review, we address a novel field: the transformation of xanthophylls, particularly the highly marketed astaxanthin and the practically unknown bacterioruberin, in therapeutic agents by altering their pharmacokinetics, biodistribution, and pharmacodynamics through their formulation as nanomedicines. The antioxidant activity of xanthophylls is mediated by routes different from those of the classical oral anti-inflammatory drugs such as corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs): remarkably, xanthophylls lack therapeutic activity but also lack toxicity. Formulated as nanomedicines, xanthophylls gain therapeutic activity by mechanisms other than increased bioavailability. Loaded into ad hoc tailored nanoparticles to protect their structure throughout storage and during gastrointestinal transit or skin penetration, xanthophylls can be targeted and delivered to selected inflamed cell groups, achieving a massive intracellular concentration after endocytosis of small doses of formulation. Most first reports showing the activities of oral and topical anti-inflammatory xanthophyll-based nanomedicines against chronic diseases such as inflammatory bowel disease, psoriasis, atopic dermatitis, and dry eye disease emerged between 2020 and 2023. Here we discuss in detail their preclinical performance, mostly targeted vesicular and polymeric nanoparticles, on cellular models and in vivo. The results, although preliminary, are auspicious enough to speculate upon their potential use for oral or topical administration in the treatment of chronic inflammatory diseases.

Two-Step Preparation of Protein-Decorated Biohybrid Quantum Dot Nanoparticles for Cellular Uptake.

Decoration of nanoparticles with specific molecules such as antibodies, peptides, and proteins that preserve their biological properties is essential for the recognition and internalization of their specific target cells. Inefficient preparation of such decorated nanoparticles leads to nonspecific interactions diverting them from their desired target. We report a simple two-step procedure for the preparation of biohybrid nanoparticles containing a core of hydrophobic quantum dots coated with a multilayer of human serum albumin. These nanoparticles were prepared by ultra-sonication, crosslinked using glutaraldehyde, and decorated with proteins such as human serum albumin or human transferrin in their native conformations. These nanoparticles were homogeneous in size (20-30 nm), retained the fluorescent properties of quantum dots, and did not show a "corona effect" in the presence of serum. The uptake of transferrin-decorated quantum dot nanoparticles was observed in A549 lung cancer and SH-SY5Y neuroblastoma cells but not in non-cancerous 16HB14o- or retinoic acid dopaminergic neurons differentiated SH-SY5Y cells. Furthermore, digitoxin-loaded transferrin-decorated nanoparticles decreased the number of A549 cells without effect on 16HB14o-. Finally, we analyzed the in vivo uptake of these biohybrids by murine retinal cells, demonstrating their capacity to selectively target and deliver into specific cell types with excellent traceability.

Direct antitumoral effects of sulfated fucans isolated from echinoderms: a possible role of neuropilin-1/ß1 integrin endocytosis and focal adhesion kinase degradation.

Hypercoagulability, a major complication of metastatic cancers, has usually been treated with heparins from natural sources, or with their synthetic derivatives, which are under intense investigation in clinical oncology. However, the use of heparin has been challenging for patients with risk of severe bleeding. While the systemic administration of heparins, in preclinical models, has shown primarily attenuating effects on metastasis, their direct effect on established solid tumors has generated contradictory outcomes. We investigated the direct antitumoral properties of two sulfated fucans isolated from marine echinoderms, FucSulf1 and FucSulf2, which exhibit anticoagulant activity with mild hemorrhagic potential. Unlike heparin, sulfated fucans significantly inhibited tumor cell proliferation (by ~30-50%), and inhibited tumor migration and invasion in vitro. We found that FucSulf1 and FucSulf2 interacted with fibronectin as efficiently as heparin, leading to loss of prostate cancer and melanoma cell spreading. The sulfated fucans increased the endocytosis of ß1 integrin and neuropilin-1 chains, two cell receptors implicated in fibronectin-dependent adhesion. The treatment of cancer cells with both sulfated fucans, but not with heparin, also triggered intracellular focal adhesion kinase (FAK) degradation, with a consequent overall decrease in activated focal adhesion kinase levels. Finally, only sulfated fucans inhibited the growth of B16-F10 melanoma cells implanted in the dermis of syngeneic C57/BL6 mice. FucSulf1 and FucSulf2 arise from this study as candidates for the design of possible alternatives to long-term treatments of cancer patients with heparins, with the advantage of also controlling local growth and invasion of malignant cells.

Evidence on differential role for alpha 1 and alpha 2 subtypes of AP-2 adaptin in the central nervous system.

Two subtypes of alpha (α)subunits, α1and α2, belonging to AP-2 complex have been described in the central nervous system (CNS). The specific role of each subtype is still unclear. In this study, we evaluated the expression and interaction with cell membranes of both subtypes in the postnatal developing cerebral cortex and cerebellum in two rat strains that display distinct developmental features. We observed that α2 displays higher variations than α1 during development, and at lesser extent in the rats with delayed rate of development. Additionally, by in vitro binding assays we evaluated the interaction of α subunits with bovine brain membranes. Both subtypes displayed clear differences in their performance, maximum binding of α1 was higher and α2 reached it faster than α1. In addition, both subtypes displayed different binding to membranes when bivalent cations or nucleotides were added. We conclude that both subtypes interact differently with membranes and that they may play different roles in clathrin-mediated endocytosis in the CNS.