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  • Wnt signaling mediates self-organization and axis formation in embryoid bodies. 18983966

    Embryonic stem cells (ESCs) form descendants of all three germ layers when differentiated as aggregates, termed embryoid bodies. In vivo, differentiation of cells depends on signals and morphogen gradients that provide instructive and positional cues, but do such gradients exist in embryoid bodies? We report here the establishment of anteroposterior polarity and the formation of a primitive streak-like region in the embryoid body, dependent on local activation of the Wnt pathway. In this region, cells undergo an epithelial-to-mesenchymal transition and differentiate into mesendodermal progenitors. Exogenous Wnt3a protein posteriorizes the embryoid body, resulting in predominantly mesendodermal differentiation. Conversely, inhibiting Wnt signaling promotes anterior character and results in neurectodermal differentiation. The activation of Wnt signaling and primitive streak formation requires external signals but is self-reinforcing after initiation. Our findings show that the Wnt pathway mediates the local execution of a gastrulation-like process in the embryoid body, which displays an unexpected degree of self-organization.
    Document Type:
    Reference
    Product Catalog Number:
    AB2034
    Product Catalog Name:
    Anti-Laminin Antibody

  • Aerosolized ZnO nanoparticles induce toxicity in alveolar type II epithelial cells at the air-liquid interface. 21964423

    The majority of in vitro studies characterizing the impact of engineered nanoparticles (NPs) on cells that line the respiratory tract were conducted in cells exposed to NPs in suspension. This approach introduces processes that are unlikely to occur during inhaled NP exposures in vivo, such as the shedding of toxic doses of dissolved ions. ZnO NPs are used extensively and pose significant sources for human exposure. Exposures to airborne ZnO NPs can induce adverse effects, but the relevance of the dissolved Zn(2+) to the observed effects in vivo is still unclear. Our goal was to mimic in vivo exposures to airborne NPs and decipher the contribution of the intact NP from the contribution of the dissolved ions to airborne ZnO NP toxicity. We established the exposure of alveolar type II epithelial cells to aerosolized NPs at the air-liquid interface (ALI) and compared the impact of aerosolized ZnO NPs and NPs in suspension at the same cellular doses, measured as the number of particles per cell. By evaluating membrane integrity and cell viability 6 and 24 h post-exposure, we found that aerosolized NPs induced toxicity at the ALI at doses that were in the same order of magnitude as doses required to induce toxicity in submersed cultures. In addition, distinct patterns of oxidative stress were observed in the two exposure systems. These observations unravel the ability of airborne ZnO NPs to induce toxicity without the contribution of dissolved Zn(2+) and suggest distinct mechanisms at the ALI and in submersed cultures.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Secolignans with Antiangiogenic Activities from Peperomia dindygulensis. 21560234

    Two new secolignans, peperomins G and H (1 and 2, resp.), were isolated from the whole plant of Peperomia dindygulensis, together with five known secolignans, peperomin A (3), peperomin E (4), peperomin B (5), 2,3-trans-2-methyl-3-{(3-hydroxy-4,5-dimethoxyphenyl)[5-methoxy-3,4-(methylenedioxy)phenyl]methyl}butyrolactone (6), 2,3-cis-2-(hydroxymethyl)-3-{bis[5-methoxy-3,4-(methylenedioxy)phenyl]methyl}butyrolactone (7). Their structures and configurations were elucidated by spectroscopic methods including 2D-NMR techniques. Antiangiogenic effects of all compounds were evaluated using human umbilical vein endothelial cells (HUVEC) proliferation and tube-formation tests, with compounds 4 and 5 being active in the bioassay. Compounds 4 and 5 induced obvious cell toxicity to HUVEC with IC(50) values of 1.64±0.19 and 8.44±0.4 μM, respectively. Compounds 4 and 5 also exhibited significant HUVEC tube formation-inhibiting activity with IC(50) values of 3.13±0.09 and 6.24±0.12 μM, respectively.
    Document Type:
    Reference
    Product Catalog Number:
    ECM625
    Product Catalog Name:
    In Vitro Angiogenesis Assay Kit

  • Characterization of multifunctional nanosystems based on the avidin-nucleic acid interaction as signal enhancers in immuno-detection. 22414051

    The Avidin-Nucleic-Acids-Nano-Assembly (ANANAS) is a kind of soft poly avidin nanoparticle originating from the high affinity interaction between avidin and the nucleic acids. In this work we investigated the possibility of transforming ANANAS cores into stoichiometrically controlled multifunctional nanoparticles through a one-pot procedure, and we measured in a quantitative way their ability to work as reagents for enhanced immunodiagnostic detection. Initially, we measured the ANANAS loading capability for biotinylated proteins of different nature. About 200 molecules of biotin-horseradish-peroxidase (40KDa b-HRP) and 60 molecules of biotin-immunoglobulin-G (150KDa b-IgG) could be accommodated onto each nanoparticle, showing that steric limitations dictate the number of loadable entities. Stoichiometrically controlled functional assemblies were generated by mixing core particles with subsaturating amounts of b-HRP and b-IgG. When applied as detection reagents in an Enzyme-Linked-ImmunoSorbed-Assay (ELISA), these assemblies were up to two-orders of magnitude more sensitive than commercial HRP-based reagents. Assemblies of different composition displayed different efficacy, indicating that the system functionality can be fine-tuned. Within-assay variability (CV%), measured to assess if the assembly procedure is reproducible, was within 10%. Stability experiments demonstrated that the functionalyzed assemblies are stable in solution for more than one week. In principle, any biotinylated function can be loaded onto the core particle, whose high loading capacity and tunability may open the way toward further application in biomedicine.
    Document Type:
    Reference
    Product Catalog Number:
    AP124P
    Product Catalog Name:
    Goat Anti-Mouse IgG Antibody, Peroxidase Conjugated, H+L

  • Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development. 19085956

    The knowledge concerning fetal hepatic stellate cells (HSCs) is scarce, and their cell lineage and functions are largely unknown. The current study isolated fetal liver mesenchymal cells from a mouse expressing beta-galactosidase under the control of Msx2 promoter by fluorescence-activated cell sorting (FACS) and surveyed marker genes by microarray analysis. Based on the location and immunostaining with conventional and newly disclosed markers, we have identified three distinct populations of fetal liver mesenchymal cells expressing both desmin and p75 neurotrophin receptor (p75NTR): HSCs in the liver parenchyma; perivascular mesenchymal cells expressing alpha-smooth muscle actin (alpha-SMA); and submesothelial cells associated with the basal lamina beneath mesothelial cells and expressing activated leukocyte cell adhesion molecule (ALCAM) and platelet-derived growth factor receptor alpha. A transitional cell type from the submesothelial cell phenotype to fetal HSCs was also identified near the liver surface. Mesothelial cells expressed podoplanin and ALCAM. Ki-67 staining showed that proliferative activity of the submesothelial cells is higher than that of mesothelial cells and transitional cells. Using anti-ALCAM antibodies, submesothelial and mesothelial cells were isolated by FACS. The ALCAM(+) cells expressed hepatocyte growth factor and pleiotrophin. In culture, the ALCAM(+) cells rapidly acquired myofibroblastic morphology and alpha-SMA expression. The ALCAM(+) cells formed intracellular lipid droplets when embedded in collagen gel and treated with retinol, suggesting the potential for ALCAM(+) cells to differentiate to HSCs. Finally, we demonstrated that fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are all derived from mesoderm by using MesP1-Cre and ROSA26 reporter mice.Fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are mesodermal in origin, and ALCAM(+) submesothelial cells may be a precursor for HSCs in developing liver.
    Document Type:
    Reference
    Product Catalog Number:
    AB756P
    Product Catalog Name:
    Anti-Collagen Antibody, Type IV

  • Molecular and immunohistological characterization of human cartilage two years following autologous cell transplantation. 15634813

    BACKGROUND: There are only a few studies concerning the cellular, biochemical, and genetic processes that occur during the remodeling of graft tissue after autologous chondrocyte transplantation. The purpose of the present study was to quantify the expression of genes encoding extracellular matrix proteins and regulatory factors that are essential for cell differentiation in cartilage biopsy specimens from patients who had this treatment two years previously. METHODS: Two cartilage biopsy specimens from each of four patients who had been treated with autologous chondrocyte transplantation and from two multiorgan donors were used. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression of types I, II, and X collagen; aggrecan; cathepsin B; and early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for matrix proteins and regulatory proteins was carried out on paraffin-embedded sections. RESULTS: Type-I collagen mRNA was expressed in all of the samples evaluated. Type-II collagen was present in autologous chondrocyte transplantation samples but at lower levels than in the controls. Type-X collagen messenger was undetectable. Aggrecan mRNA was present in all of the samples at lower levels than in the controls, while cathepsin-B messenger levels were higher and Egr-1 and Sox-9 mRNAs were expressed at lower levels. The immunohistochemical analysis showed slight positivity for type-I collagen in all of the sections. Type-II collagen was found in all of the samples with positivity confined inside the cells, while the controls displayed a positivity that was diffuse in the extracellular matrix. Cathepsin B was slightly positive in all of the samples, while the controls were negative. Egr-1 protein was particularly evident in the areas negative for type-II collagen. Sox-9 was positive in all samples, with evident localization in the superficial and middle layers. CONCLUSIONS: In biopsy specimens from autologous chondrocyte transplantation tissue at two years, there is evidence of the formation of new tissue, which displays varying degrees of organization with some fibrous and fibrocartilaginous features. Long-term follow-up investigations are needed to verify whether, once all of the remodeling processes are completed, the newly formed tissue will acquire the more typical features of articular cartilage.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Synaptic connections of calbindin-immunoreactive cone bipolar cells in the inner plexiform layer of rabbit retina. 19937346

    In the mammalian retina, information concerning various aspects of an image is transferred in parallel, and cone bipolar cells are thought to play a major role in this parallel processing. We have examined the synaptic connections of calbindin-immunoreactive (IR) ON cone bipolar cells in the inner plexiform layer (IPL) of rabbit retina and have compared these synaptic connections with those that we have previously described for neurokinin 1 (NK1) receptor-IR cone bipolar cells. A total of 325 synapses made by calbindin-IR bipolar axon terminals have been identified in sublamina b of the IPL. The axons of calbindin-IR bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and are coupled to putative AII amacrine cells via gap junctions. The major output from calbindin-IR bipolar cells is to amacrine cell processes. These data resemble our findings for NK1 receptor-IR bipolar cells. However, the incidences of output synapses to ganglion cell dendrites of calbindin-IR bipolar cells are higher compared with the NK1-receptor-IR bipolar cells. On the basis of stratification level and synaptic connections, calbindin-IR ON cone bipolar cells might thus play an important role in the processing of various visual aspects, such as contrast, orientation, and approach sensing, and in transferring rod signals to the ON cone pathway.
    Document Type:
    Reference
    Product Catalog Number:
    AB5060
    Product Catalog Name:
    Anti-Substance P Receptor Antibody, pain

  • The effects of dexamethasone on insulin release and biosynthesis are dependent on the dose and duration of treatment. 11269888

    Complex results concerning the effect of glucocorticoids on insulin secretion have been reported. The aim of this study is to clarify the direct effects of glucocorticoids on pancreatic islets and to determine whether the effect of glucocorticoids on insulin biosynthesis or release is dependent on the dose and duration of treatment with glucocorticoid. Studies on insulin secretion and biosynthesis were performed with different concentrations (0, 1, 10, 100 nmol/l) and durations (1 and 6 h) of treatment with dexamethasone (dexa) in rat pancreatic islets. (1) One nmol/l dexa had no inhibitory effect on insulin secretion and biosynthesis. Ten and 100 nmol/l had an inhibitory effect on insulin secretion, which was mainly due to suppression of the first phase of insulin secretion. (2) Insulin content was significantly increased regardless of the concentration in 1-h treated islets. However, insulin content was markedly diminished with 100 nmol/l dexa in 6-h treated islets. (3) The preproinsulin mRNA expression of 6-h treated islets was suppressed in a dose-dependent manner. Our data revealed that, in the condition of short-term and low-dose glucocorticoid exposure, insulin secretion and biosynthesis are not affected. The secretory process of insulin seems to be the initial step of the inhibitory action of glucocorticoid. Both insulin release and biosynthesis are inhibited by chronic exposure to high dose dexamethasone. It can be concluded that glucocorticoid might be involved in the multisteps of insulin release and biosynthesis.
    Document Type:
    Reference
    Product Catalog Number:
    RI-13K
    Product Catalog Name:
    Rat Insulin RIA

  • Sensory and sympathetic nerve fibers undergo sprouting and neuroma formation in the painful arthritic joint of geriatric mice. 22548760

    Although the prevalence of arthritis dramatically increases with age, the great majority of preclinical studies concerning the mechanisms that drive arthritic joint pain have been performed in young animals. One mechanism hypothesized to contribute to arthritic pain is ectopic nerve sprouting; however, neuroplasticity is generally thought to be greater in young versus old nerves. Here we explore whether sensory and sympathetic nerve fibers can undergo a significant ectopic nerve remodeling in the painful arthritic knee joint of geriatric mice.Vehicle (saline) or complete Freund's adjuvant (CFA) was injected into the knee joint of 27- to 29-month-old female mice. Pain behaviors, macrophage infiltration, neovascularization, and the sprouting of sensory and sympathetic nerve fibers were then assessed 28 days later, when significant knee-joint pain was present. Knee joints were processed for immunohistochemistry by using antibodies raised against CD68 (monocytes/macrophages), PECAM (endothelial cells), calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kDa (NF200; sensory nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), and growth-associated protein 43 (GAP43; nerve fibers undergoing sprouting).At 4 weeks after initial injection, CFA-injected mice displayed robust pain-related behaviors (which included flinching, guarding, impaired limb use, and reduced weight bearing), whereas animals injected with vehicle alone displayed no significant pain-related behaviors. Similarly, in the CFA-injected knee joint, but not in the vehicle-injected knee joint, a remarkable increase was noted in the number of CD68+ macrophages, density of PECAM+ blood vessels, and density and formation of neuroma-like structures by CGRP+, NF200+, and TH+ nerve fibers in the synovium and periosteum.Sensory and sympathetic nerve fibers that innervate the aged knee joint clearly maintain the capacity for robust nerve sprouting and formation of neuroma-like structures after inflammation/injury. Understanding the factors that drive this neuroplasticity, whether this pathologic reorganization of nerve fibers contributes to chronic joint pain, and how the phenotype of sensory and sympathetic nerves changes with age may provide pharmacologic insight and targets for better controlling aging-related joint pain.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Anchorage-dependent cell growth: tyrosine kinases and phosphatases meet redox regulation. 15890002

    Recent data have provided new insight concerning the regulation of nontransformed cell proliferation in response to both soluble growth factors and adhesive cues. Nontransformed cells are anchorage-dependent for the execution of the complete mitotic program and cannot avoid the concomitant signals starting from mitogenic molecules, as growth factors, and adhesive agents belonging to the extracellular matrix. Protein tyrosine kinases (PTKs) and phosphotyrosine phosphatases (PTPs) together with soluble small molecules have been included among intracellular signal transducers of growth factor and extracellular matrix receptors. Reactive oxygen species retain a key role during both growth factor and integrin receptor signaling, and these second messengers are recognized to be a synergistic point of confluence for anchorage-dependent growth signaling. Redox-regulated proteins include PTPs and PTKs, although with opposite regulation of enzymatic activity. Transient oxidation of PTPs leads to their inactivation, through the formation of an intramolecular S-S bridge. Conversely, oxidation of PTKs leads to their activation, either by direct SH modification or, indirectly, by concomitant inhibition of PTPs that leads to sustained activation of PTKs. This review will focus on the redox regulation of PTPs and PTKs during anchorage-dependent cell growth and its implications for tumor biology.
    Document Type:
    Reference
    Product Catalog Number:
    05-947
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone PY20