Auxin Deprivation Induces Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells1

To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells.

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures1

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca2+-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca2+]cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca2+]cyt and inhibition of growth that was similar to the effect of the Ca2+ channel blocker La3+ and the Ca2+ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca2+ channel blockers verapamil and nifedipine did not induce a decrease in [Ca2+]cyt in these cells and also failed to inhibit growth. Al and La3+, but not verapamil or nifedipine, reduced the rate of Mn2+ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca2+- and Mn2+-permeable channels. These results suggest that Al may act to block Ca2+ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.

Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors.

Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases.

Tissue engineered perivascular endothelial cell implants regulate vascular injury.

Molecular biomaterial engineering permits in vivo transplantation of cells and tissues, offering the promise of restoration of physiologic control rather than pharmacologic dosing with isolated compounds. We engrafted endothelial cells on Gelfoam biopolymeric matrices with retention of viability, normal growth kinetics, immunoreactivity, and biochemical activity. The production of heparan sulfate proteoglycan and inhibition of basic fibroblast growth factor binding and activity by engrafted cells were indistinguishable from endothelial cells grown in culture. Perivascular implantation of Gelfoam-endothelial cell scaffolds around balloon-denuded rat carotid arteries reduced intimal hyperplasia 88.1%, far better than the isolated administration of heparin, the most effective endothelial mimic compound. In concert with a reduction in intimal area, cell proliferation was reduced by > 90%. To our knowledge, there have been no previous reports of extravascular cell implants controlling vasculoproliferative disease. Tissue engineered cells offer the potential for potent methods of vascular growth regulation and insight into the complex autocrine-paracrine control mechanisms within the blood vessel wall.

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.

Physical techniques in biological research,

Vol. 4-6 edited by William L. Nastuk.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

Os efeitos das queimadas na Amazônia em nível celular e molecular

The Amazon holds over half of the planet's remaining tropical forests and comprises the largest biodiversity in the world, accounting for approximately 60 % of the Brazilian territory. However, deforestation fires in the region causes serious problems to exposed human. The aim of this study was to evaluate the chemical compounds as well as the cellular and molecular effects after exposure to organic material extracted from particulate matter less than 10 µm (PM10) in the Amazon region. As for the chemical composition, n-alkanes analysis showed a prevalence of anthropogenic influence during the fires in the region. In addition, there was a predominance of monosaccharides from biomass burning markers. Also, the Polycyclic Aromatic Hydrocarbons (PAH) and their derivatives have also been identified in samples collected in the Amazon. By using the PAH concentrations was possible to calculate the BaP-equivalent and it was found that the dibenz(a) anthracene contributes with 83% to potential carcinogenic risk. As for the potential mutagenic risk, the benzo (a) pyrene is the HPA that has a major contribution in this analysis. It may be noted that the retene was the most abundant PAH. This compound was genotoxic and cause death by necrosis in the human lung cells. In biological tests, the data showed that organic PM10 is capable of causing genetic damage in both plant cells and in human lung cells. This damage cause an arrest in the G1 phase of the cell cycle exposed, increasing the expression of p53 and p21. Additionally, the PM10 caused cell death by apoptosis, increasing the foci of histone - H2AX. Given these results, it is important to emphasize the reduction and better control of biomass burning in the Amazon region thus improving the quality of health of the population being exposed. As clearly stated recently by the World Health Organization, the reduction of air pollution could save millions of lives annually.

Biologically Inspired Design of Protein-Silica Hybrid Nanoparticles for Drug Delivery Applications

The design and application of effective drug carriers is a fundamental concern in the delivery of therapeutics for the treatment of cancer and other vexing health problems. Traditionally utilized chemotherapeutics are limited in efficacy due to poor bioavailability as a result of their size and solubility as well as significant deleterious effects to healthy tissue through their inability to preferentially target pathological cells and tissues, especially in treatment of cancer. Thus, a major effort in the development of nanoscopic drug delivery vehicles for cancer treatment has focused on exploiting the inherent differences in tumor physiology and limiting the exposure of drugs to non-tumorous tissue, which is commonly achieved by encapsulation of chemotherapeutics within macromolecular or supramolecular carriers that incorporate targeting ligands and that enable controlled release. The overall aim of this work is to engineer a hybrid nanomaterial system comprised of protein and silica and to characterize its potential as an encapsulating drug carrier. The synthesis of silica, an attractive nanomaterial component because it is both biocompatible as well as structurally and chemically stable, within this system is catalyzed by self-assembled elastin-like polypeptide (ELP) micelles that incorporate of a class of biologically-inspired, silica-promoting peptides, silaffins. Furthermore, this methodology produces near-monodisperse, hybrid inorganic/micellar materials under mild reaction conditions such as temperature, pH and solvent. This work studies this material system along three avenues: 1) proof-of-concept silicification (i.e. the formation and deposition of silica upon organic materials) of ELP micellar templates, 2) encapsulation and pH-triggered release of small, hydrophobic chemotherapeutics, and 3) selective silicification of templates to potentiate retention of peptide targeting ability.

Proteomic profile of acute myeloid leukaemia: A review update

Proteome analysis is a complex and dynamic process that encompasses several analytical platforms that include protein sequencing, structural or expression proteomics, protein modification, sub-cellular protein localization, protein-protein interaction and biological functional proteomics. In fact, expression proteomics is extensively applied in a majority of biomarker detection studies because it provides a detailed overview of differentially expressed proteins in cellular pathways and disease processes. Proteomics are also effective and dynamic in protein-protein interactions and cross-talks between interacting molecules of the cell. Proteomics has evolved into a crucial tool used to investigate the biochemical changes that possibly lead to development of cancer biomarkers. This review draws attention to the progress and advancements in cancer proteomics technology with the aim of simplifying the understanding of the mechanisms underlying the disease and to contribute to detection of biomarkers in addition to the development of novel treatments. Given that proteome is a dynamic entity of cellular functions in health and disease, it is capable of reflecting the immediate environmental state of cells and tissues as shown in this review. The review shows the possibility of elucidating the pathophysiology of acute myeloid leukaemia (AML) through proteome expressions, thus confirming the viability of proteome analysis in profiling AML.

A particle based model to simulate microscale morphological changes of plant tissues during drying

Fundamental understanding on microscopic physical changes of plant materials is vital to optimize product quality and processing techniques, particularly in food engineering. Although grid-based numerical modelling can assist in this regard, it becomes quite challenging to overcome the inherited complexities of these biological materials especially when such materials undergo critical processing conditions such as drying, where the cellular structure undergoes extreme deformations. In this context, a meshfree particle based model was developed which is fundamentally capable of handling extreme deformations of plant tissues during drying. The model is built by coupling a particle based meshfree technique: Smoothed Particle Hydrodynamics (SPH) and a Discrete Element Method (DEM). Plant cells were initiated as hexagons and aggregated to form a tissue which also accounts for the characteristics of the middle lamella. In each cell, SPH was used to model cell protoplasm and DEM was used to model the cell wall. Drying was incorporated by varying the moisture content, the turgor pressure, and cell wall contraction effects. Compared to the state of the art grid-based microscale plant tissue drying models, the proposed model can be used to simulate tissues under excessive moisture content reductions incorporating cell wall wrinkling. Also, compared to the state of the art SPH-DEM tissue models, the proposed model better replicates real tissues and the cell-cell interactions used ensure efficient computations. Model predictions showed good agreement both qualitatively and quantitatively with experimental findings on dried plant tissues. The proposed modelling approach is fundamentally flexible to study different cellular structures for their microscale morphological changes at dehydration.

Glucosinolate-accumulating S-cells in Arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation

Plant secondary metabolites glucosinolates (GSL) have important functions in plant resistance to herbivores and pathogens. We identified all major GSL that are accumulated in S-cells in Arabidopsis by MALDI-TOF MS, and estimated by LC-MS that the total GSL concentration in these cells is above 130 mM. The precise locations of the S-cells outside phloem bundles in rosette and cauline leaves and in flower stalks were visualised using sulphur mapping by cryo-SEM/EDX. S-cells contain up to 40% of total sulphur in flower stalk tissues. S-cells in emerging flower stalks and developing leaf tissues show typical signs of Programmed Cell Death (PCD) or apoptosis, such as chromatin condensation in the nucleus and blebbing of the membranes. TUNEL staining for DNA double strand breaks confirmed PCD in S-cells in postmeristematic tissues in the flower stalk as well as in the leaf. Our results show that S-cells in postmeristematic tissues proceed to an extreme degree of metabolic specialisation besides PCD. Accumulation and maintenance of a high concentration of GSL in these cells are accompanied by degradation of a number of cell organelles. The substantial changes in the cell composition during S-cell differentiation indicate the importance of this particular GSL-based phloem defence system. The specific anatomy of the S-cells and ability to accumulate specialised secondary metabolites is similar to that of the non-articulated laticifer cells in latex plants and thus indicates a common evolutionary origin.

Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain

The purolindolines are small cysteine-rich proteins which are present in the grain of wheat. They have a major impact on the utilisation of the grain as they are the major determinants of grain texture, which affects both milling and baking properties. Bread and durum wheats were transformed with constructs comprising the promoter regions of the Puroindoline a (Pina) and Puroindoline b (Pinb) genes fused to the uidA (GUS) reporter gene. Nine lines showing 3:1 segregation for the transgene and comprising all transgene/species combinations were selected for detailed analysis of transgene expression during grain development. This showed that transgene expression occurred only in the starchy endosperm cells and was not observed in any other seed or vegetative tissues. The location of the puroindoline proteins in these cells was confirmed by tissue printing of developing grain, using a highly specific monoclonal antibody for detection and an antibody to the aleurone-localised 8S globulin as a control. This provides clear evidence that puroindolines are only synthesised and accumulated in the starchy endosperm cells of the wheat grain.

IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120 d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120 d a sustained production of cytokines (KC, IL-5, TNF-alpha, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(Pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A. (C) 2012 Elsevier Ltd. All rights reserved.

Nanomechanics to drive stem cells in injured tissues: insights from current research and future perspectives

Stem cells reside within tissue, ensuring its natural ability to repair an injury. They are involved in the natural repair of damaged tissue, which encompasses a complex process requiring the modulation of cell survival, extracellular matrix turnover, angiogenesis, and reverse remodeling. To date, the real reparative potential of each tissue is underestimated and noncommittal. The assessment of the biophysical properties of the extracellular environment is an innovative approach to better understand mechanisms underlying stem cell function, and consequently to develop safe and effective therapeutic strategies replacing the loss of tissue. Recent studies have focused on the role played by biomechanical signals that drive stem cell death, differentiation, and paracrinicity in a genetic and/or an epigenetic manner. Mechanical stimuli acting on the shape can influence the biochemistry and gene expression of resident stem cells and, therefore, the magnitude of biological responses that promote the healing of injured tissue. Nanotechnologies have proven to be a revolutionary tool capable of dissecting the cellular mechanosensing apparatus, allowing the intercellular cross-talk to be decoded and enabling the reparative potential of tissue to be enhanced without manipulation of stem cells. This review highlights the most relevant findings of stem cell mechanobiology and presents a fascinating perspective in regenerative medicine.