FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

Effect of β3-adrenoceptor on cardiac fibrosis in rat cardiac fibroblast cells and its potential mechanism

Purpose: To investigate the effect of β3-adrenoceptors (β3-AR) up-regulation on fibrosis in cardiac fibroblast cells in rats and its potential mechanism. Methods: Cardiac fibroblast cells (CFB) were isolated and identified from rats’ hearts. The β3-ARupregulated cardiac fibroblast cells were constructed by lentiviral transfection technology. Thereafter, Ang II was used to induce fibrosis in cardiac fibroblast cells, and subsequently, Western blot assay was performed to investigate fibrosis related marker proteins (TGF-β, Smad-2, p-Smad-2, Col-I and Col-III) in cardiac fibroblast cells. Results: β3-AR up-regulated cardiac fibroblast cells were successfully constructed. Furthermore, the results show that up-regulation of β3-AR increased the expressions of TGF-β, p-Smad-2, Col-I and Col- III proteins in Ang II treated cardiac fibroblast cells. Conclusion: The results suggest that up-regulation of β3-AR aggravates fibrosis of cardiac fibroblast cells. In other words, inhibition of β3-AR expressions in cardiac tissues would be beneficial for treating cardiac fibrosis and its related cardiac diseases.

Movement effects on the flow physics and nutrient delivery in engineered valvular tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-:τ: explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-:τ: environments.^

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

Toxicity Of Fungal-generated Silver Nanoparticles To Soil-inhabiting Pseudomonas Putida Kt2440, A Rhizospheric Bacterium Responsible For Plant Protection And Bioremediation.

Silver nanoparticles have attracted considerable attention due to their beneficial properties. But toxicity issues associated with them are also rising. The reports in the past suggested health hazards of silver nanoparticles at the cellular, molecular, or whole organismal level in eukaryotes. Whereas, there is also need to examine the exposure effects of silver nanoparticle to the microbes, which are beneficial to humans as well as environment. The available literature suggests the harmful effects of physically and chemically synthesised silver nanoparticles. The toxicity of biogenically synthesized nanoparticles has been less studied than physically and chemically synthesised nanoparticles. Hence, there is a greater need to study the toxic effects of biologically synthesised silver nanoparticles in general and mycosynthesized nanoparticles in particular. In the present study, attempts have been made to assess the risk associated with the exposure of mycosynthesized silver nanoparticles on a beneficial soil microbe Pseudomonas putida. KT2440. The study demonstrates mycosynthesis of silver nanoparticles and their characterisation by UV-vis spectrophotometry, FTIR, X-ray diffraction, nanosight LM20 - a particle size distribution analyzer and TEM. Silver nanoparticles obtained herein were found to exert the hazardous effect at the concentration of 0.4μg/ml, which warrants further detailed investigations concerning toxicity.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

Paracoccidioides brasilinsis-Induced Migration of Dendritic Cells and Subsequent T-Cell Activation in the Lung-Draining Lymph Nodes

Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response.

Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

Background: Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods: In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results: The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions: Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

In situ delivery of bone marrow cells and mesenchymal stem cells improves cardiovascular function in hypertensive rats submitted to myocardial infarction

This work aimed to evaluate cardiac morphology/function and histological changes induced by bone marrow cells (BMCs) and cultured mesenchymal stem cells (MSCs) injected at the myocardium of spontaneously hypertensive rats (SHR) submitted to surgical coronary occlusion. Female syngeneic adult SHR, submitted (MI) or not (C) to coronary occlusion, were treated 24 h later with in situ injections of normal medium (NM), or with MSCs (MSC) or BMCs (BM) from male rats. The animals were evaluated after 1 and 30 days by echocardiography, histology of heart sections and PCR for the Y chromosome. Improved ejection fraction and reduced left ventricle infarcted area were observed in MSC rats as compared to the other experimental groups. Treated groups had significantly reduced lesion tissue score, increased capillary density and normal (not-atrophied) myocytes, as compared to NM and C groups. The survival rate was higher in C, NM and MSC groups as compared to MI and BM groups. In situ injection of both MSCs and BMCs resulted in improved cardiac morphology, in a more physiological model of myocardial infarction represented by surgical coronary occlusion of spontaneously hypertensive rats. Only treatment with MSCs, however, ameliorated left ventricle dysfunction, suggesting a positive role of these cells in heart remodeling in infarcted hypertensive subjects.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition

P>During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response

Background: The transcription factors SREBP1 and SCAP are involved in intracellular cholesterol homeostasis. Polymorphisms of these genes have been associated with variations on serum lipid levels and response to statins that are potent cholesterol-lowering drugs. We evaluated the effects of atorvastatin on SREBF1a and SCAP mRNA expression in peripheral blood mononuclear cells (PBMC) and a possible association with gene polymorphisms and lowering-cholesterol response. Methods: Fifty-nine hypercholesterolemic patients were treated with atorvastatin (10 mg/day for 4 weeks). Serum lipid profile and mRNA expression in PBMC were assessed before and after the treatment. Gene expression was quantified by real-time PCR using GAPD as endogenous reference and mRNA expression in HepG2 cells as calibrator. SREBF1 -36delG and SCAP A2386G polymorphisms were detected by PCR-RFLP. Results: Our results showed that transcription of SREBF1a and SCAP was coordinately regulated by atorvastatin (r=0.595, p<0.001), and that reduction in SCAP transcription was associated with the 2386AA genotype (p=0.019). Individuals who responded to atorvastatin with a downregulation of SCAP had also a lower triglyceride compared to those who responded to atorvastatin with an upregulation of SCAP. Conclusion: Atorvastatin has differential effects on SREBF1a and SCAP mRNA expression in PBMC that are associated with baseline transcription levels, triglycerides response to atorvastatin and SCAP A2386G polymorphism. (c) 2008 Elsevier B.V. All rights reserved.

Effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the ability of Candida albicans to infect cells and induce inflammation

Vulvovaginal candidiasis, a high prevailing infection worldwide, is mainly caused by Candida albicans. Probiotic Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 have been previously shown to be useful as adjuvants in the treatment of women with VVC. In order to demonstrate and better understand the anti-Candida activity of the probiotic microorganisms in an in vitro model simulating vaginal candidiasis, a human vaginal epithelial cell line (VK2/E6E7) was infected with C. albicans 3153a and then challenged with probiotic L. rhamnosus GR-1 and/or L. reuteri RC-14 or their respective CFS (alone or in combination). At each time point (0, 6, 12 and 24 hr), numbers of yeast, lactobacilli and viable VK2/E6E7 cells were determined and, at 0, 6 and 12 hr, the supernatants were measured for cytokine levels. We found that C. albicans induced a significant increase in IL-1 alpha and IL-8 production by VK2/E6E7 cells. After lactobacilli challenge, epithelial cells did not alter IL-6, IL-1 alpha, RANTES and VEGF levels. However, CFS from the probiotic microorganisms up-regulated IL-8 and IP-10 levels secreted by VK2/E6E7 cells infected with C. albicans. At 24 hr of co-incubation, L. reuteri RC-14 alone and in combination with L. rhamnosus GR-1 decreased the yeast population recoverable from the cells. In conclusion, L. reuteri RC-14 alone and together with L. rhamnosus GR-1 have the potential to inhibit the yeast growth and their CFS may up-regulate IL-8 and IP-10 secretion by VK2/E6E7 cells, which could possibly have played an important role in helping to clear VVC in vivo.

Glycine metabolism by plant roots and its occurrence in Australian plant communities

Soluble organic nitrogen, including protein and amino acids, was found to be a ubiquitous form of soil N in diverse Australian environments. Fine roots of species representative of these environments were found to be active in the metabolism of glycine. The ability to incorporate [N-15]glycine was widespread among plant species from subantarctic to tropical communities. In species from subantarctic herbfield, subtropical coral cay, subtropical rainforest and wet heathland, [N-15]glycine incorporation ranged from 26 to 45% of (NH4+)-N-15 incorporation and was 2- to 3-fold greater than (NO3-)-N-15 incorporation. Most semiarid mulga and tropical savanna woodland species incorporated [N-15]glycine and (NO3-)-N-15 in similar amounts, 18-26% of (NH4+)-N-15 incorporation. We conclude that the potential to utilise amino acids as N sources is of widespread occurrence in plant communities and is not restricted to those from low temperature regimes or where N mineralisation is limited. Seedlings of Hakea (Proteaceae) were shown to metabolise glycine, with a rapid transfer of N-15 from glycine to serine and other amino compounds. The ability to take up and metabolise glycine was unaffected by the presence of equimolar concentrations of NO3- and NH4+. Isonicotinic acid hydrazide (INH) did not inhibit the transfer of N-15-label from glycine to serine indicating that serine hydroxymethyltransferase was not active in glycine catabolism. In contrast aminooxyacetate (AOA) strongly inhibited transfer of N-15 from glycine to serine and labelling of other amino compounds, suggesting that glycine is metabolised in roots and cluster roots of Hakea via an aminotransferase.