Ethylene and polyamine interactions in morphogenesis of Passiflora cincinnata: effects of ethylene biosynthesis and action modulators, as well as ethylene scavengers

Ethylene is a plant hormone that is of fundamental importance to in vitro morphogenesis, but in many species, it has not been thoroughly studied. Its relationship with polyamines has been studied mainly because the two classes of hormones share a common biosynthetic precursor, S-adenosylmethionine (SAM). In order to clarify whether competition between polyamines and ethylene influences in vitro morphogenetic responses of Passiflora cincinnata Mast., a climacteric species, different compounds were used that act on ethylene biosynthesis and action, or as ethylene scavengers. Treatment with the ethylene inhibitor, aminoethoxyvinylglycine (AVG) caused a greater regeneration frequency in P. cincinnata, whereas treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC) lessened regeneration frequencies. The data suggested that levels of polyamines and ethylene are not correlated with morphogenic responses in P. cincinnata. It was ascertained that neither the absolute ethylene and polyamine levels, nor competition between the compounds, correlated to the obtained morphogenic responses. However, sensitivity to, and signaling by, ethylene appears to play an important role in differentiation. This study reinforces previous reports regarding the requirement of critical concentrations and temporal regulation of ethylene levels for morphogenic responses. Temporal regulation also appeared to be a key factor in competition between the two biosynthetic pathways, without having any effects on morphogenesis. Further studies investigating the silencing or overexpression of genes related to ethylene perception, under the influence of polyamines in cell differentiation are extremely important for the complete understanding of this process.

Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L. are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Speed 1 1 is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.

Effects of short chain fatty acids on effector mechanisms of neutrophils

Short chain fatty acids (SCFAs) are metabolic by products of anerobic bacteria fermentation. These fatty acids, despite being an important fuel for colonocytes, are also modulators of leukocyte function. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate, and butyrate) on function of neutrophils, and the possible mechanisms involved. Neutrophils obtained from rats by intraperitoneal lavage 4 h after injection of oyster glycogen solution (1%) were treated with non toxic concentrations of the fatty acids. After that, the following measurements were performed: phagocytosis and destruction of Candida albicans, production of ROS (O(2)(center dot-), H(2)O(2), and HOCl) and degranulation. Gene expression (p47(phox) and p22(phox)) and protein phosphorylation (p47(phox)) were analyzed by real time reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. Butyrate inhibited phagocytosis and killing of C. albicans. This SCFA also had an inhibitory effect on production of O(2)(center dot-), H(2)O(2), and HOCI by neutrophils stimulated with PMA or fMLP. This effect of butyrate was not caused by modulation of expression of NADPH oxidase subunits (p47(phox) and p22(phox)) but it was in part due to reduced levels of p47(phox) phosphorylation and an increase in the concentration of cyclic AMP. Acetate increased the production of O(2)(center dot-) and H(2)O(2), in the absence of stimuli but had no effect on phagocytosis and killing of C. albicans. Propionate had no effect on the parameters studied. These results suggest that butyrate can modulate neutrophil function, and thus could be important in inflammatory neutrophil-associated diseases. Copyright (C) 2008 John Wiley & Sons, Ltd.

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-2 (CINC-2 alpha beta)] by rat neutrophils. The involvement of nuclear factor kappa B (NF-kappa B) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-alpha, CINC-2 alpha beta and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-kappa B activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

CHANGES OF GENE EXPRESSION IN ELECTRICALLY STIMULATED AND CONTRALATERAL RAT SOLEUS MUSCLES

In this study we investigate the effect of a single session of high-intensity contractions on expression of pleiotropic genes and, in particular, those genes associated with metabolism in soleus muscle from electrically stimulated (ES) and contralateral (CL) limbs. The right limbs of male Wistar rats were submitted to contractions by 200-ms trains of electrical stimulation at 100-Hz frequency with pulses of 0.1 ms (voltage 24 3 V) delivered each second for 1 hour. Soleus muscles were isolated 1 hour after contraction, and gene expression was analyzed by a macroarray technique (Atlas Toxicology 1.2 Array; Clontech Laboratories). Electrical stimulation increased expression in 92 genes (16% of the genes present in the membrane). Sixty-six genes were upregulated in both ES and CL soleus muscles, and expression of 26 genes was upregulated in the ES muscle only. The most altered genes were those related to stress response and metabolism. Electrical stimulation also raised expression of transcription factors, translation and posttranslational modification of proteins, ribosomal proteins, and intracellular transducers/effectors/modulators. The results indicate that a single session of electrical stimulation upregulated expression of genes related to metabolism and oxidative stress in soleus muscle from both ES and CL limbs. These findings may indicate an association with tissue hypertrophy and metabolic adaptations induced by physical exercise training not only in the ES but also in the CL non-stimulated muscle, suggesting a cross-education phenomenon. Muscle Nerve 40: 838-846, 2009

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Three-dimensional Reconstruction of Ovaries of Leaf-cutting Ant (Atta sexdens rubropilosa) Queens (Hymenoptera: Formicidae)

In this study, the ovary morphology of newly emerged ant queens of Atta sexdens rubropilosa was studied in whole mount preparations by confocal microscopy. The ovaries are composed of approximately 40 ovarioles, showing non-synchronic oocyte maturation. The terminal filament with clusters of undifferentiated cells was found at the distal end of the ovarioles. Next to this region is the germarium, composed of several elongated cystocytes interconnected by cytoplasmic bridges. The nurse cells (23-28 cells) result from asymmetric mitosis. Cytoskeleton analysis showed F-actin concentrated at the muscle cells of the external tunica and in fusomes inside the ovarioles. Microtubules were concentrated around the nuclei of the nurse and follicular cells. In contrast, the oocytes and the external tunica showed faint staining for tubulin.

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Phosphoproteomics Profiling Suggests a Role for Nuclear beta IPKC in Transcription Processes of Undifferentiated Murine Embryonic Stem Cells

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal and differentiation However, the function of specific PKC Isoenzymes have yet to be determined Of the PKCs expressed in undifferentiated ESCs, beta IPKC was the only isoenzyme abundantly expressed in the nuclei To investigate the role of beta IPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one beta IPKC-specific inhibitor peptide We identified 13 nuclear proteins that are direct or indirect beta IPKC substrates in undifferentiated ESCs These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation Inhibiting beta IPKC had no effect on DNA synthesis in undifferentiated ESCs However, upon differentiation many cells seized to express beta IPKC and beta IPKC was frequently found in the cytoplasm Taken together, our results suggest that beta IPKC takes part in the processes that maintain ESCs in their undifferentiated state

Fragment-Based QSAR and Molecular Modeling Studies on a Series of Discodermolide Analogs as Microtubule-Stabilizing Anticancer Agents

Inhibition of microtubule function is an attractive rational approach to anticancer therapy. Although taxanes are the most prominent among the microtubule-stabilizers, their clinical toxicity, poor pharmacokinetic properties, and resistance have stimulated the search for new antitumor agents having the same mechanism of action. Discodermolide is an example of nontaxane natural product that has the same mechanism of action, demonstrating superior antitumor efficacy and therapeutic index. The extraordinary chemical and biological properties have qualified discodermolide as a lead structure for the design of novel anticancer agents with optimized therapeutic properties. In the present work, we have employed a specialized fragment-based method to develop robust quantitative structure - activity relationship models for a series of synthetic discodermolide analogs. The generated molecular recognition patterns were combined with three-dimensional molecular modeling studies as a fundamental step on the path to understanding the molecular basis of drug-receptor interactions within this important series of potent antitumoral agents.

Structure-Based Approach for the Study of Estrogen Receptor Binding Affinity and Subtype Selectivity

Estrogens exert important physiological effects through the modulation of two human estrogen receptor (hER) subtypes, alpa (hER alpha) and beta (hER beta). Because the levels and relative proportion of hER alpha and hER beta differ significantly in different target cells, selective hER ligands could target specific tissues or pathways regulated by one receptor subtype without affecting the other. To understand the structural and chemical basis by which small molecule modulators are able to discriminate between the two subtypes, we have applied three-dimensional target-based approaches employing a series of potent hER-ligands. Comparative molecular field analysis (CoMFA) studies were applied to a data set of 81 hER modulators, for which binding affinity values were collected for both hER alpha and hER beta. Significant statistical coefficients were obtained (hER alpha, q(2) = 0.76; hER beta, q(2) = 0.70), indicating the internal consistency of the models. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. Five hER crystal structures were used in GRID/PCA investigations to generate molecular interaction fields (MIF) maps. hER alpha and hER beta were separated using one factor. The resulting 3D information was integrated with the aim of revealing the most relevant structural features involved in hER subtype selectivity. The final QSAR and GRID/PCA models and the information gathered from 3D contour maps should be useful for the design or novel hER modulators with improved selectivity.

Morphometric analysis of the urethra of castrated female rats treated with tamoxifen

Objectives: The aim of this study was to evaluate the effects of tamoxifen on the weight and thickness of the urethral epithelium of castrated female rats. Methods: Forty castrated adult female Wistar-Hannover rats were randomly divided into two groups: Group I (n = 20) in which the animals received only the vehicle (propylene glycol) and Group 11 (n = 20) in which the rats received tamoxifen 250 mu g/day by gavage. After 30 days of treatment, all animals were sacrificed and the urethra was immediately removed for weighing. Next, the urethra was divided into the proximal and distal segments, which were fixed in 10% formaldehyde and submitted to routine histological techniques for morphometric study. The data were analyzed using the weighted minimum mean-square error method and Student`s t-test for two independent samples (p < 0.05). Results: There was a significant increase in the mean weight of the urethra in the rats of Group 11 compared to the control group, 32.0 +/- 2.0 mg and 22.0 +/- 1.6 mg, respectively (p < 0.001). The mean thickness of the distal urethral epithelium of the animals treated with tamoxifen was significantly greater than that of the control group, 42.8 +/- 2.0 mu m and 36.6 +/- 1.5 mu m, respectively (p < 0.001). There was no statistically significant difference between the two groups with respect to the epithelial thickness of the proximal urethra (p = 0.514). Conclusion: Treating castrated adult rats with 250 mu g/day of tamoxifen for 30 days may increase the weight of the urethra and the thickness of the distal urethral epithelium. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Effects of Raloxifene on Ki-67 and CD34 Antigen Expression in Breast Cancer

Background: The aim of this study was to evaluate the effect of raloxifene on CD34 and Ki-67 antigen expression in breast cancer specimens from postmenopausal women. Methods: Sixteen postmenopausal patients with operable, stage II (>= 3 cm), estrogen receptor-positive breast cancer, who took 60 mg of raloxifene daily for 28 days, participated in this study. Immunohistochemistry was carried out in tumor samples prior to and following raloxifene treatment to evaluate CD34 and Ki-67 protein expression. Angiogenesis was quantified in 10 randomly selected fields per slide, and Ki-67-stained nuclei were counted in 1,000 cells per slide using an image capture and analysis system with 400 ! magnification. Student`s t test for paired samples was used for the statistical analysis of data. Statistical significance was established at p < 0.05. Results: The mean number of microvessels was 44.44 +/- 3.54 prior to raloxifene therapy and 22.63 +/- 1.61 following therapy (p < 0.001), and the mean percentage of Ki-67-stained nuclei was 19.28 +/- 8 1.61 and 12.13 +/- 8 1.48 prior to and following raloxifene treatment, respectively (p < 0.001). Conclusion: Raloxifene significantly reduces CD34 and Ki-67 protein expression in breast carcinoma in postmenopausal women. Copyright (C) 2008 S. Karger AG, Basel

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.