Paraformaldehyde Solution Research Articles

Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison

Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE(2)) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE(2) and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE(2) induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE(2) receptors EP2 and EP4 eliminated the PGE(2)-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE(2)-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE(2) response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE(2) stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE(2) plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter.

The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.

Visual and Both Non-visual Arrestins in Their “Inactive” Conformation Bind JNK3 and Mdm2 and Relocalize Them from the Nucleus to the Cytoplasm

Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a "nuclear exclusion assay" to study their interactions with two proteins involved in "life-and-death" decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants "frozen" in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely "preloaded" with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration.

A Phage Display Screen and Binding Studies with Acetylated Low Density Lipoprotein Provide Evidence for the Importance of the Scavenger Receptor Cysteine-rich (SRCR) Domain in the Ligand-binding Function of MARCO

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.

Serial Analysis of Gene Expression in Mouse Uterus at the Implantation Site

Although oligonucleotide chips, cDNA microarrays, differential display reverse transcription-PCR, and other approaches have been used to screen implantation-related molecules, the mechanism by which embryo implantation occurs is still unknown. The aim of this study was to profile the differential gene expression between interimplantation site and implantation site in mouse uterus on day 5 of pregnancy by serial analysis of gene expression (SAGE). In our two SAGE libraries of 11-bp tags, the total numbers of tags sequenced were 48,121 for the interimplantation site and 50,227 for the implantation site. There were 1,039 tags specifically expressed at interimplantation site, and 1,252 tags specifically expressed at the implantation site. Based on the p value, there were 195 tags significantly up-regulated at the interimplantation site and 261 tags significantly up-regulated at the implantation site, of which 100 genes were single matched at the interimplantation site and 127 genes were single matched at the implantation site, respectively. By reverse transcription-PCR, the tag ratio between the implantation site and interimplantation site was verified on 14 significantly changed genes. Using in situ hybridization, 1810014L12Rik, Psmb5, Cd63, Npm1, Fads3, and Tagln2 were shown to be highly expressed at the implantation site compared with the interimplantation site. Compared with the interimplantation site, Ddx39 was strongly expressed in the subluminal stromal cells at the implantation site on day 5 of pregnancy. Ddx39 expression at the implantation site was specifically induced by active blastocysts. Additionally, Ddx39 expression was significantly up-regulated by estrogen in the ovariectomized mice. In our SAGE data, many implantation-related genes were identified in mouse uterus. Our data could be a valuable source for future study on embryo implantation.

A Regulatory Role for 1-Acylglycerol-3-phosphate-O-acyltransferase 2 in Adipocyte Differentiation

Mutations in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene have been identified in individuals affected with congenital generalized lipodystrophy (CGL). AGPAT2 catalyzes acylation of lysophosphatidic acid to phosphatidic acid, a precursor for both triacylglycerol (TAG) and phospholipid synthesis. Recent studies suggest that reduced AGPAT2 enzymatic activity may underlie the CGL clinical phenotype. To gain insight into how altered AGPAT2 activity causes lipodystrophy, we examined the effect of knockdown of AGPAT2 expression in preadipocytes on TAG synthesis and storage, and on adipocyte differentiation. We show that AGPAT2 mRNA expression is induced 30-fold during adipocyte differentiation and that AGPAT2 enzymatic activity is required for TAG mass accumulation in mature adipocytes. We demonstrate that small interference RNA-mediated knockdown of AGPAT2 expression prevents appropriate early induction of C/EBPbeta and PPARgamma, key transcriptional activators of the adipogenic program, and delays expression of multiple adipocyte-related genes. The unexpected finding, that levels of several phospholipid species, including phosphatidic acid (PA), are elevated in TAG-depleted adipocytes with AGPAT2 knockdown, suggests that impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synthesis. These findings underscore the importance of an AGPAT2-mediated metabolic pathway in adipocyte differentiation.

Targeting and Trafficking of the Human Thiamine Transporter-2 in Epithelial Cells

Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.

274 LOCALIZATION OF MACROPHAGE COLONY STIMULATING FACTOR (M-CSF) GENE IN THE ENDOMETRIUM OF JAPANESE BLACK COW

Macrophage colony stimulating factor (M-CSF), a member of the group of hemopoietic cytokines, plays an important role in placental physiology of humans and mice. The objective of this study was to investigate the localization of M-CSF gene in bovine endometrial tissues during pregnancy using in situ hybridization (ISH). Twelve Japanese Black cows aged between 1.4 and 14.5 years, with normal estrous cycles, were used in this study. They were observed daily for estrous behavior, and the day of estrus was considered as Day 0. Cows were artificially inseminated, and pregnancy was confirmed by presence of conceptus and/or corpus luteum using ultrasonography. They were euthanized and their uteri were collected on Days 20 to 21 (n = 2), 36 to 42 (n = 3), 59 to 64 (n = 3), 127 to 137 (n = 2), and 225 to 226 (n = 2) of pregnancy. Pregnancy was confirmed finally by the presence of a conceptus in the uterus, and the uterus was isolated by dissection, avoiding damage of the uterine artery. The uterus was perfused with 4% paraformaldehyde (PFA) solution using catheters inserted into the uterine artery, and placental tissues were isolated by dissection. Each tissue was cut into small pieces (5 mm thick) and fixed in 4% PFA solution. After being fixed in 4% PFA solution for 20 to 24 h, tissue pieces were embedded in paraffin using routine procedures. Several tissue pieces were collected from each individual cow. Sections (6 mm thick) were cut and placed on MAS-coated glass slides (Matsunami Glass, Kishiwada, Japan). The sections were dried in an oven for 3 days at 40�C. Antisense and sense biotin-labeled oligonucleotide probes for bovine M-CSF were designed from the sequence information (GenBank accession number D87917). Sense probe was used as the negative control. ISH was carried out using the GenPoint System (DakoCytomation, Glostrup, Denmark) according to the manufacturer's instructions. The sections were observed under an Eclipse 800 microscope (Nikon, Tokyo, Japan), and the positive signal for M-CSF gene was detected. Density analysis was performed by using Scion Image (Scion Corporation, Frederick, MD, USA). Data were analyzed by one-way ANOVA and Tukey-Kramer's HSD, using 0.05 as a significant level. The M-CSF gene was expressed in the stromal and luminal epithelial cells of endometrium, stromal cells of the fetal cotyledonary villus and caruncular crypt, and mono- and multi-nuclear epithelium cells of the fetal cotyledonary villus and caruncular crypt. Intensities of M-CSF gene-positive signals in all positive cells at Days 225 to 226 of pregnancy were stronger than those at other periods (P < 0.05). These results suggest that M-CSF is produced in several cell types in the endometrium and placental tissues and may play important roles in bovine pregnancy.

Flow-Sorted chromosomes: a fine material for plant gene physical mapping

Fluorescence in situ hybridization (FISH) on flow-sorted plant chromosomes is described. The protocol involves flow cytometric sorting of metaphase chromosomes, then fixing them with 4% paraformaldehyde solution, and re-sorting these chromosomes directly onto a spot on poly-lysine coated slides after stained. FISH mapping ex- periments were performed on the sorted chromosomes from oat and barley using 45S and 5S ribosomal DNAs as probes. The chromosome morphology was very well preserved and there were nearly no chromosomes lost after pro- cedures of hybridization and fluorescent staining. Sorted chromosomes are advantageous over metaphase chromo- somes as targets for FISH mapping studies because a large number of target chromosomes with better chromosome morphology on a small area on the slide are easy to gain by flow-sorting, background is very clear and hybridization sensitivity is enhanced. The successful and reliable location of rDNA genes in these two plant genomes, combining with the data published before in maize proves that flow-sorted chromosomes are fine materials for gene physical mapping in plants.

Modification of injection mapping of vascular-capillary bed in cerebral nervous tissue

A simple original method of simultaneous staining of the cell elements on cerebral cortex and its vascular bed is developed. It is based on perfusion of polyvinyl acetate emulsion prepared on 2% paraformaldehyde solution with phosphate buffer at pH 7.4.

Expression and Function of C/EBP Homology Protein (GADD153) in Podocytes

Podocytes are crucial for the permeability of the glomerular filtration barrier. In glomerular disease, however, reactive oxygen species (ROS) may be involved in podocyte injury and subsequent proteinuria. Here, we describe ROS-dependent gene induction in differentiated podocytes stimulated with H(2)O(2) or xanthine/xanthine-oxidase. Superoxide anions and H(2)O(2) increased mRNA and protein expression of GAS5 (growth arrest-specific protein 5) and CHOP (C/EBP homology protein). Cultured podocytes overexpressing CHOP showed increased generation of superoxide anions compared to controls. In addition, the expression of alpha(3)/beta(1) integrins, crucial for cell-matrix interaction of podocytes, was down-regulated, leading to increased cell-matrix adhesion and cell displacement. The altered cell-matrix adhesion was antagonized by the ROS scavenger 1,3-dimethyl-2-thiourea, and the increase in cell displacement could be mimicked by stimulating untransfected podocytes with puromycin, an inductor of ROS. We next performed immunohistochemical staining of human kidney tissue (normal, membranous nephropathy, focal segmental glomerulosclerosis, and minimal change nephropathy) as well as sections from rats with puromycin nephrosis, a model of minimal change nephropathy. CHOP was weakly expressed in podocytes of control kidneys but up-regulated in most proteinuric human kidneys and in rat puromycin nephrosis. Our data suggest that CHOP-via increased ROS generation-regulates cell-matrix adhesion of podocytes in glomerular disease.

Influence of chronic administration of zearalenone on the processes of apoptosis in the porcine ovary

Zearalenone (ZEA), a micotoxin produced by Fusarium sp. is regarded as a phytoestrogen. Although cytotoxic and genotoxic activity of ZEA was detected, the majority of its toxic influence is related to the ability of binding to estrogen receptors and disrupting the endocrine regulation of the reproductory functions in females. It was previously found that ZEA inhibits proliferation of cells in porcine ovaries, as detected with immunostaining for proliferating cells nuclear antigen (PCNA). The number of PCNA-positive cells was inversely proportional to the dose of ZEA. We decided to answer the question of whether ZEA induces apoptosis in porcine ovaries. Experimental gilts (before first estrus) were fed ZEA in a dosage of 20 (group II) or 40 (group III) µg/kg of body weight/day for 63 days. Control animals (group I) were fed a placebo. After that period animals were sacrificed, ovaries were extirpated, fixed in paraformaldehyde solution, cut into sections with a cryostat and studied for apoptosis with TUNEL kit, and for the presence of apoptosis-promoting protein Bax with immunohistochemistry. It was found that apoptosis was detected with TUNEL only in medium-sized antral ovarian follicles in animals of groups I and II. No apoptosis signal was found in the ovaries of animals in group III. No differences in the distribution and intensity of staining for Bax were found between animals of all investigated groups. The results indicate that ZEA do not induce apoptosis in porcine ovaries, and the inhibition of proliferation must be associated with other mechanisms.

Morphometric aspects of the human hypoglossal nerve in adults and the elderly

Perform a morphometric analysis of the myelinic fibers of the right hypoglossal nerve, in two age groups; to verify quantitative changes as a result of the aging process. Anatomic. A 1 cm fragment of the right hypoglossal nerve was collected from 12 male corpses without any medical history of diseases such as: diabetes, alcoholism, and malignant neoplasia. The sample was divided in two groups: group with six corpses under sixty years old (adult), and another group with six corpses sixty years old or above (elderly). The material was fixed at 2.5% glutaldehyde and 2% paraformaldehyde solution; post-fixed at 2% osmium tetroxide; dehydrated with increasing ethanol concentrations, and included in epoxy resin. Semi-thin sections of 0.3 microm were obtaining, colored in 1% toluidine blue, and evaluated with light microscope combined with image analyzing system. The following morphometric data were quantified: intraperineural transversal section area, number, and diameter of the myelinic fibers. The intraperineural area of the hypoglossal nerve was similar in both age groups (p=0.8691). The average area in the adult group was 1.697 mm2 and in the elderly group it was 1.649 mm2. The total number of myelinic fibers of the hypoglossal nerve was similar in both age groups (p=0.9018). The adult group presented an average of 10,286 +/- 2,308 myelinic fibers, and the elderly group presented an average of 10,141 +/- 1,590 myelinic fibers. A bimodal distribution of the myelinic fibers was observed, with a significant peak on the 9 microm fibers, and another smaller peak on the 2 microm fibers. The intraperineural area and the total number of myelinic fibers of the right hypoglossal nerve are similar in both age groups.

Pathological alterations of strial capillaries in dominant white spotting W/ Wv mice

Dominant white spotting W/ W v and W v/ W v mice are well-known mutants that lack strial intermediate cells in their cochlea and manifest hereditary sensorineural hearing loss. We recently reported marked thickening of and IgG deposition on the basement membrane of strial capillaries in W/ W v mutant mice, similar to observations made in aged animals and in animals with autoimmune sensorineural hearing loss. The present study aimed to clarify the age-dependent changes in these pathological findings of strial capillaries in the W/ W v mice. Male WBB6F1 +/+ and dominant white spotting W/ W v mutant mice were sacrificed by transcardiac perfusion with paraformaldehyde solution. The cochlear ducts were isolated and subjected to light- and electron-microscopy, immunohistochemistry, immunoelectron microscopy. Alternatively, lanthanum chloride tracer examination in the isolated cochlear ducts was performed in order to compare the permeability of the strial capillaries between +/+ and W/ W v mice. In the W/ W v mice, thickening of and IgG deposition on the basement membrane of strial capillaries were observed as early as 1 week after birth and became more noticeable with age. Deposited IgG was preferentially localized to the thickened basement membrane and was also observed in partially the intercellular space between adjacent of endothelial cells. In addition, pinocytotic vesicles both in the apical and basal lesions of such cells also showed IgG deposition. Lanthanum chloride was retained along apical plasma membrane of the endothelial cells in the +/+ mice but penetrated through the endothelial layer in the W/ W v mice. These results indicate that active transport via pinocytotic vesicles as well as increased permeability of strial capillaries in the W/ W v mice occur in the early stage after birth, resulting in the morphological alterations in the strial capillaries of these mice.

An examination of non‐formalin‐based fixation methods for Xenopus embryos

Despite the growing availability of non-formalin-based fixatives, the vast majority of researchers in developmental biology continue to fix embryos and tissue in 4% paraformaldehyde. This fixation method has proven useful for both immunohistochemistry and in situ hybridization, yet working with paraformaldehyde has distinct disadvantages in its toxicity and the short shelf life of prepared solutions. In a search for viable alternative fixatives, we have evaluated two non-formalin-based commercial products, FineFIX (Milestone Microwave Laboratory System) and NOTOXhisto (Scientific Device Laboratory). These products were tested side-by-side with a commonly used 4% paraformaldehyde solution (MEMPFA) on Xenopus laevis embryos and assayed using whole mount immunohistochemistry and whole mount in situ hybridization. The results indicate that NOTOXhisto can be used as a substitute for MEMPFA in both tested Xenopus protocols with no loss of sensitivity or tissue morphology.

Modulation of the Interleukin-6 Receptor Subunit Glycoprotein 130 Complex and Its Signaling by LMO4 Interaction

The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with protein-tyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The effect of LMO4 in IL-6 signaling was subsequently examined. Overexpression of LMO4 enhanced the transcriptional activity and target gene expression of Stat 3 (signal transducers and activators of transcription 3). Consistent with this, silencing LMO4 expression in stable cell lines expressing the small interfering RNA of LMO4 decreased Stat3 activity. Furthermore, the half-life of gp130 was shortened, and the production of acute phase proteins induced by IL-6 was reduced. Together, our data reveal a positive regulatory role of LMO4 in IL-6 signaling, possibly by acting as a scaffold for stabilization of the gp130 complex. These studies may open up a link between the oncogenic effect of LMO proteins and their regulatory role in cytokine signaling in general.

The Pro-inflammatory Mediator Leukotriene D4 Induces Phosphatidylinositol 3-Kinase and Rac-dependent Migration of Intestinal Epithelial Cells

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.

Quantitative evaluation of the fibrin clot extension on different implant surfaces: An in vitro study

The aim of the present study was a quantitative evaluation of the in vitro fibrin clot extension on different implant surfaces. Forty-five disk-shaped commercially pure Grade 2 titanium samples with three different surface topographies (machined, DPS, and Plus) were used in the present study. For the quantitative evaluation of the fibrin clot, 30 specimens were used (10 per group); human whole blood was employed. Venous blood was drawn from three healthy adult volunteers, and 0.2 mL were immediately dropped onto the surface of each specimen. Contact time was 5 min at room temperature; then the samples were rinsed with saline solution and fixed in a buffered solution of glutaraldehyde and paraformaldehyde. Samples were washed again with buffer and dehydrated in an ascending alcohol series. Specimens belonging to all groups were observed under SEM at a magnification of 1000x. From each sample, 50 random micrographs were collected in .tif format with an N x M 1024 x 768 grid of pixels. Quantitative analysis of fibrin clot extension showed the following results: in machined samples fibrin clot extension was 345987.2 +/- 63747.7 pixels(2) (mean +/- SD), in DPS samples fibrin clot extension was 375930.9 +/- 54726.86 pixels(2) (mean +/- SD), and in Plus samples, fibrin clot extension was 612333.6 +/- 46268.42 pixels(2) (mean +/- SD). With ANOVA it was possible to find that there were significant differences among the groups. The Tukey test revealed that the extension of the fibrin clot of Plus samples was statistically higher compared to both machined and DPS samples. The results of this in vitro study indicate that there is a correlation between implant surface morphology and fibrin clot extension. Improvement in surface microtexture complexity seems to determine the formation of a more extensive and three dimensionally complex fibrin scaffold. Further investigations are necessary to explain in more detail the mechanisms that regulate the fibrin clot formation on different implant surfaces.

Induction of Corneal Lesion and Nerve Fiber Sprouting by Neonatal Capsaicin Application Depends on the Dose of the Drug and Survival Time after Treatment

The effects of capsaicin on the rat cornea and its NsAchE (non specific acetylcholinesterase)-positive nerve fibers were investigated after long and short survival periods following subcutaneous (s.c) injection of the drug. Sixteen rats were injected once s.c with capsaicin on postnatal day 2 at a dose of 50 mg/kg. Age-matched rats were injected only vehicle, and served as control (n = 16). After 4 (n = 6), 8 (n = 6) and 12 (n = 4) mos., both sides of the corneas were examined under a binocular microscope to look for corneal abnormalities. Immediately after the enucleation, bilateral corneas were excised with a thin scleral margin and their ciliary body and iris were removed in DPBS solution. Then, they were fixed in a 4% paraformaldehyde solution containing 8% sucrose for 1 h at 4 degrees C, and processed for staining by the NsAchE method. For comparison, 15 rats were injected 3 times with capsaicin (total dose: 150 mg/kg) at a dose of 50 mg/kg on days 1, 2 and 3 after birth. Age-matched rats (n = 16) were injected vehicle as controls. Five (n = 4), 17 (n = 4), 45 (n = 3) and 75 (n = 4) days later, their corneas were similarly handled as described above. Corneal lesions and sprouting of the NsAchE-positive subepithelial nerve fibers appeared 4 mos. after the treatment with capsaicin (50 mg/kg). In particular, all the treated corneas (8/8) at 12 mos. showed corneal abnormalities. Contrary to the results from the single injection of capsaicin, corneal changes had already appeared on day 17 after treatment with capsaicin at a high dose (150 mg/kg). These data reinforce the suggestion that yielding of corneal lesions is closely associated with the longer survival time at lower doses and with the short survival time at a high dose of the drug. Further, the relationship between sprouting of corneal nerve fibers and corneal wounds was discussed on the basis of the previous reports.

273 APOPTOSIS EVALUATION OF IN VITRO-PRODUCED PIG EMBRYOS (PARTIAL RESULT)

Apoptosis is a physiological event involved with death and tissue replication, fulfilling an important function of tissue organizations during embryogenesis. This mechanism occurs in in vivo as well as in vitro pre-implantation embryos, but most frequently in the latter. The transcriptional activation of pig embryos occurs at the four-cell stage, which is the longer stage during the pre-implantation period. This stage is characterized by embryonic developmental blockage that decreases the production rates (embryos loss). The aim of this study was to evaluate a correlation between apoptosis mechanism and developmental blockage of IVP porcine embryos. Immature oocytes after IVM/IVF were submitted to IVC in PZM-1 medium containing BSA 3 mg/mL at 38.5�C, 5% CO2 in air and high humidified atmosphere. The embryo development was analyzed at 96 h of cultute (Day 4) in order to verify cleavage rate and blockage (4 cells) and non-blockage (e8 cells) embryo rates. Out of 625 grade I, II, and III oocytes submitted to IVP, 70.3 � 5.2% (430/625) cleaved from which 27.1 � 10.3% (166/625) were blocked and 43 � 10.8% (264/625) were non-blocked. Blocked and non-blocked embryos were assessed to evaluate apoptosis rates. Qualitative assays of embryo cells were achieved with two different DNA stains: YOPRO-1 (Molecular Probe�; Invitrogen Brasil, Ltd., Sao Paulo, Brazil), permeable though plasma membrane in the early stage of apoptosis, and TUNEL (Roche�; Amersham Biosciences, Sao Paulo, Brazil), which detects DNA fragmentation in the last stages of apoptosis. The embryos were stained with 0.1 �M YOPRO-1/mL PBS, incubated 15 min at 38�C, 5% CO2 in air and high humidified atmosphere, and immediately observed by means of confocal microscopy. For the TUNEL assay, embryos were fixed in 4% paraformaldehyde solution (w/v) in PBS for 1 h at room temperature, and incubated in permeabilization solution [0.5% (v/v) Triton X-100, 0.1% (w/v) sodium citrate in PBS] for 2 h. For positive control, samples were treated with DNase-I at 37�C for 1 h. The negative control and experimental samples were incubated with buffer solution under the same conditions. The positive control and experimental samples were incubated with enzymatic and stain solution (FITC) at 37�C for 1 h; the negative control was incubated with only enzymatic solution. The embryos were stained with Hoechst 33342 (5 �/mL) and observed by means of fluorescence microscopy. Blocked embryos showed more apoptosis (66% and 40% to YOPRO-1 and TUNEL, respectively) than non-blocked embryos (25% and 0% to YOPRO-1 and TUNEL, respectively). In conclusion, the developmentally blocked embryos suffered more apoptosis, although morphologic apoptosis assays (light and electronic microscopic) must be performed to confirm this finding. This work was supported by FAPESP 04/01252-4.