Leaf colleters in Tabernaemontana catharinensis (Apocynaceae, Rauvolfioideae): structure, ontogenesis, and cellular secretion

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

End-to-end Distance Distribution in Fluorescent Derivatives of Bradykinin in Interaction with Lipid Vesicles

Cellular membranes have relevant roles in processes related to proteases like human kallikreins and cathepsins. As enzyme and substrate may interact with cell membranes and associated co-factors, it is important to take into account the behavior of peptide substrates in the lipid environment. In this paper we report an study based on energy transfer in two bradykinin derived peptides labeled with the donor-acceptor pair Abz/Eddnp (ortho-aminobenzoic acid/N-[2,4-dinitrophenyl]-ethylenediamine). Time-resolved fluorescence experiments were performed in phosphate buffer and in the presence of large unilamelar vesicles of phospholipids, and of micelles of sodium dodecyl sulphate (SDS). The decay kinetics were analyzed using the program CONTIN to obtain end-to-end distance distribution functions f(r). Despite of the large difference in the number of residues the end-to-end distance of the longer peptide (9 amino acid residues) is only 20 % larger than the values obtained for the shorter peptide (5 amino acid residues). The proline residue, in position 4 of the bradykinin sequence promotes a turn in the longer peptide chain, shortening its end-to-end distance. The surfactant SDS has a strong disorganizing effect, substantially broadening the distance distributions, while temperature increase has mild effects in the flexibility of the chains, causing small increase in the distribution width. The interaction with phospholipid vesicles stabilizes more compact conformations, decreasing end-to-end distances in the peptides. Anisotropy experiments showed that rotational diffusion was not severely affected by the interaction with the vesicles, suggesting a location for the peptides in the surface region of the bilayer, a result consistent with small effect of lipid phase transition on the peptides conformations.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Differential cellular FGF-2 upregulation in the rat facial nucleus following axotomy, functional electrical stimulation and corticosterone: a possible therapeutic target to Bell's palsy

Abstract Background The etiology of Bell's palsy can vary but anterograde axonal degeneration may delay spontaneous functional recovery leading the necessity of therapeutic interventions. Corticotherapy and/or complementary rehabilitation interventions have been employed. Thus the natural history of the disease reports to a neurotrophic resistance of adult facial motoneurons leading a favorable evolution however the related molecular mechanisms that might be therapeutically addressed in the resistant cases are not known. Fibroblast growth factor-2 (FGF-2) pathway signaling is a potential candidate for therapeutic development because its role on wound repair and autocrine/paracrine trophic mechanisms in the lesioned nervous system. Methods Adult rats received unilateral facial nerve crush, transection with amputation of nerve branches, or sham operation. Other group of unlesioned rats received a daily functional electrical stimulation in the levator labii superioris muscle (1 mA, 30 Hz, square wave) or systemic corticosterone (10 mgkg-1). Animals were sacrificed seven days later. Results Crush and transection lesions promoted no changes in the number of neurons but increased the neurofilament in the neuronal neuropil of axotomized facial nuclei. Axotomy also elevated the number of GFAP astrocytes (143% after crush; 277% after transection) and nuclear FGF-2 (57% after transection) in astrocytes (confirmed by two-color immunoperoxidase) in the ipsilateral facial nucleus. Image analysis reveled that a seven days functional electrical stimulation or corticosterone led to elevations of FGF-2 in the cytoplasm of neurons and in the nucleus of reactive astrocytes, respectively, without astrocytic reaction. Conclusion FGF-2 may exert paracrine/autocrine trophic actions in the facial nucleus and may be relevant as a therapeutic target to Bell's palsy.

Dual effect of Lutzomyia longipalpis saliva on Leishmania braziliensis infection is mediated by distinct saliva-induced cellular recruitment into BALB/c mice ear

Abstract Background Leishmania parasites are transmitted to their vertebrate hosts by infected Phlebotomine sand flies during the blood meal of the flies. Sand fly saliva is known to enhance Leishmania spp. infection, while pre-exposure to saliva protects mice against parasitic infections. In this study, we investigated the initial inflammatory leucocyte composition induced by one or three inocula of salivary gland extract (SGE) from Lutzomyia longipalpis in the presence or absence of Leishmania braziliensis. Results We demonstrated that inoculating SGE once (SGE-1X) or three times (SGE-3X), which represented a co-inoculation or a pre-exposure to saliva, respectively, resulted in different cellular infiltrate profiles. Whereas SGE-1X led to the recruitment of all leucocytes subtypes including CD4+ T cells, CD4+CD25+ T cells, dendritic cells, macrophages and neutrophils, the immune cell profile in the SGE-3X group differed dramatically, as CD4+ T cells, CD4+CD25+ T cells, dendritic cells, macrophages and neutrophils were decreased and CD8+ T cells were increased. The SGE-1X group did not show differences in the ear lesion size; however, the SGE-1X group harbored a higher number of parasites. On the other hand, the SGE-3X group demonstrated a protective effect against parasitic disease, as the parasite burden was lower even in the earlier stages of the infection, a period in which the SGE-1X group presented with larger and more severe lesions. These effects were also reflected in the cytokine profiles of both groups. Whereas the SGE-1X group presented with a substantial increase in IL-10 production, the SGE-3X group showed an increase in IFN-γ production in the draining lymph nodes. Analysis of the inflammatory cell populations present within the ear lesions, the SGE-1X group showed an increase in CD4+FOXP3+ cells, whereas the CD4+FOXP3+ population was reduced in the SGE-3X group. Moreover, CD4+ T cells and CD8+ T cells producing IFN-γ were highly detected in the ears of the SGE-3X mice prior to infection. In addition, upon treatment of SGE-3X mice with anti-IFN-γ monoclonal antibody, we observed a decrease in the protective effect of SGE-3X against L. braziliensis infection. Conclusions These results indicate that different inocula of Lutzomyia longipalpis salivary gland extract can markedly modify the cellular immune response, which is reflected in the pattern of susceptibility or resistance to Leishmania braziliensis infection.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Quantification of cellular action fibers alignment from confocal microscopic images.

The cellular rheology has recently undergone a rapid development with particular attention to the cytoskeleton mechanical properties and its main components - actin filaments, intermediate filaments, microtubules and crosslinked proteins. However it is not clear what are the cellular structural changes that directly affect the cell mechanical properties. Thus, in this work, we aimed to quantify the structural rearrangement of these fibers that may emerge in changes in the cell mechanics. We created an image analysis platform to study smooth muscle cells from different arteries: aorta, mammary, renal, carotid and coronary and processed respectively 31, 29, 31, 30 and 35 cell image obtained by confocal microscopy. The platform was developed in Matlab (MathWorks) and it uses the Sobel operator to determine the actin fiber image orientation of the cell, labeled with phalloidin. The Sobel operator is used as a filter capable of calculating the pixel brightness gradient, point to point, in the image. The operator uses vertical and horizontal convolution kernels to calculate the magnitude and the angle of the pixel intensity gradient. The image analysis followed the sequence: (1) opens a given cells image set to be processed; (2) sets a fix threshold to eliminate noise, based on Otsu's method; (3) detect the fiber edges in the image using the Sobel operator; and (4) quantify the actin fiber orientation. Our first result is the probability distribution II(Δθ) to find a given fiber angle deviation (Δθ) from the main cell fiber orientation θ0. The II(Δθ) follows an exponential decay II(Δθ) = Aexp(-αΔθ) regarding to its θ0. We defined and determined a misalignment index α of the fibers of each artery kind: coronary αCo = (1.72 ‘+ or =’ 0.36)rad POT -1; renal αRe = (1.43 + or - 0.64)rad POT -1; aorta αAo = (1.42 + or - 0.43)rad POT -1; mammary αMa = (1.12 + or - 0.50)rad POT -1; and carotid αCa = (1.01 + or - 0.39)rad POT -1. The α of coronary and carotid are statistically different (p < 0.05) among all analyzed cells. We discussed our results correlating the misalignment index data with the experimental cell mechanical properties obtained by using Optical Magnetic Twisting Cytometry with the same group of cells.

Cellular and molecular effects of cross reacting material 197 (CRM197) on HT-29 colon cancer cell line

Cross Reacting Material 197(CRM197) is a Diphteria toxin non toxic mutant that had shown anti-tumor activity in mice and humans. CRM197 is utilized as a specific inhibitor of heparin-binding epidermal growth factor (HB-EGF), that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. We evaluated the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible effects on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment, or changes in cell cycle distribution, in EGFR localization or activation, they show a change in gene expression profile analyzed by microarray. This is the first study where the CRM197 treatment on HT-29 show the alteration of a specific and selected number of genes.

Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles

This study addresses the cellular uptake and intracellular trafficking of 15-nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air-liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG-coated than citrate-stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150-1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin- and clathrin-mediated endocytosis by methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG-coated than citrate-stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MbetaCD. With prolonged exposure times, both NPs are preferentially localized in larger-sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG-coated NPs in the cytosol.

Synthesis, binding and cellular uptake properties of oligodeoxynucleotides containing cationic bicyclo-thymidine residues

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6′)-β position is described. Compared to unmodified DNA these oligonucleotides show Tm-neutral behavior when paired against complementary DNA and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic distribution which was not observed in the case of an unmodified control oligonucleotide.

Long-term cellular and regional specificity of the photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina

This study investigated the anatomical consequences of a photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina. Retinae were examined 2 weeks, 1, 3, and 6 months after systemic IAA injection. The retinae were processed using standard histological methods to assess the gross morphology and topographical distribution of damage, and by immunohistochemistry to examine specific cell populations in the retina. Degeneration was restricted to the photoreceptors and was most common in the ventral retina and visual streak. In damaged regions, the outer nuclear layer was reduced in thickness or eliminated entirely, with a concomitant loss of immunoreactivity for rhodopsin. However, the magnitude of the effect varied between animals with the same IAA dose and survival time, suggesting individual differences in the bioavailability of the toxin. In all eyes, the inner retina remained intact, as judged by the thickness of the inner nuclear layer, and by the pattern of immunoreactivity for protein kinase C-alpha (rod bipolar cells) and calbindin D-28 (horizontal cells). Müller cell stalks became immunoreactive for glial fibrillary acidic protein (GFAP) even in IAA-treated retinae that had no signs of cell loss, indicating a response of the retina to the toxin. However, no marked hypertrophy or proliferation of Müller cells was observed with either GFAP or vimentin immunohistochemistry. Thus the selective, long lasting damage to the photoreceptors produced by this toxin did not lead to a reorganization of the surviving cells, at least with survival as long as 6 months, in contrast to the remodeling of the inner retina that is observed in inherited retinal degenerations such as retinitis pigmentosa and retinal injuries such as retinal detachment.

Osteopontin (OPN) and neoplastic disease: Circulating levels and tissue distribution

OPN is a secreted phosphate containing protein which is expressed by osteoblasts and a variety of other cells in vivo. Data from in vitro studies has accumulated which relates OPN to cellular transformation. We hypothesize that OPN expression is associated with neoplastic disease in humans as suggested by cell culture models. The overall objective of the current study was to determine the tissue distribution of OPN in human malignancy and to determine whether or not a correlation exists between OPN serum levels and malignancy. At the inception of this project, no study had been made demonstrating the relevance of OPN expression with naturally occurring neoplastic disease in humans. To date, few studies have reported OPN distribution in human neoplasia and are limited by either the number of specimens analyzed or the technique used in analysis. In this dissertation study, OPN was purified from human milk and $\alpha$-OPN antiserum developed and characterized. Following antibody development, the distribution and prevalence of OPN in human oral squamous cell carcinoma and human prostate carcinoma was evaluated using immunohistochemical localization. OPN immunolocalization was found in a high percentage of oral epithelial dysplasia and oral squamous cell carcinoma in humans. One oral squamous cell carcinoma cells line, UMSCC-1, was found to express OPN mRNA using Northern blotting. OPN localized to a high percentage of primary prostate adenocarcinomas. OPN localized to 52% of androgen dependent cases and 100% of androgen independent cases. Androgen dependent cell lines such as LNCap and NbE showed minimal OPN mRNA expression while the androgen independent lines C4-2 and PC3 produced ample OPN mRNA. An OPN sandwich assay was developed and used to determine the serum level of OPN in normal males, patients with BPH (benign prostate hypertrophy), and patients with prostate carcinoma. No statistically significant difference was found in OPN serum levels among the three groups. However, a trend of increasing OPN in the serum was noted in patients with BPH and prostate cancer. ^

Mechanical constraints imposed by 3D cellular geometry and arrangement modulate growth patterns in the Arabidopsis embryo

Morphogenesis occurs in 3D space over time and is guided by coordinated gene expression programs. Here we use postembryonic development in Arabidopsis plants to investigate the genetic control of growth. We demonstrate that gene expression driving the production of the growth-stimulating hormone gibberellic acid and downstream growth factors is first induced within the radicle tip of the embryo. The center of cell expansion is, however, spatially displaced from the center of gene expression. Because the rapidly growing cells have very different geometry from that of those at the tip, we hypothesized that mechanical factors may contribute to this growth displacement. To this end we developed 3D finite-element method models of growing custom-designed digital embryos at cellular resolution. We used this framework to conceptualize how cell size, shape, and topology influence tissue growth and to explore the interplay of geometrical and genetic inputs into growth distribution. Our simulations showed that mechanical constraints are sufficient to explain the disconnect between the experimentally observed spatiotemporal patterns of gene expression and early postembryonic growth. The center of cell expansion is the position where genetic and mechanical facilitators of growth converge. We have thus uncovered a mechanism whereby 3D cellular geometry helps direct where genetically specified growth takes place.

Interplay between the prostaglandin transporter OATP2A1 and prostaglandin E2-mediated cellular effects.

Prostaglandins such as prostaglandin E2 (PGE2) play a pivotal role in physiological and pathophysiological pathways in gastric mucosa. Little is known about the interrelation of the prostaglandin E (EP) receptors with the prostaglandin transporter OATP2A1 in the gastric mucosa and gastric carcinoma. Therefore, we first investigated the expression of OATP2A1 and EP4 in normal and carcinoma gastric mucosa. Different PGE2-mediated cellular pathways and mechanisms were investigated using human embryonic kidney cells (HEK293) and the human gastric carcinoma cell line AGS stably transfected with OATP2A1. Colocalization and expression of OATP2A1 and EP4 were detected in mucosa of normal gastric tissue and of gastric carcinomas. OATP2A1 reduced the PGE2-mediated cAMP production in HEK293 and AGS cells overexpressing EP4 and OATP2A1. The expression of OATP2A1 in AGS cells resulted in a reduction of [(3)H]-thymidine incorporation which was in line with a higher accumulation of AGS-OATP2A1 cells in S-phase of the cell cycle compared to control cells. In contrast, the expression of OATP2A1 in HEK293 cells had no influence on the distribution in the S-phase compared to control cells. OATP2A1 also diminished the PGE2-mediated expression of interleukin-8 mRNA (IL-8) and hypoxia-inducible-factor 1α (HIF1α) protein in AGS-OATP2A1 cells. The expression of OATP2A1 increased the sensitivity of AGS cells against irinotecan which led to reduced cell viability. Taken together, these data show that OATP2A1 influences PGE2-mediated cellular pathways. Therefore, OATP2A1 needs to be considered as a key determinant for the understanding of the physiology and pathophysiology of prostaglandins in healthy and tumorous gastric mucosa.

Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^