AP132F Sigma-AldrichGoat Anti-Rabbit IgG Antibody, FITC conjugate

The intracellular concentration of chloride ([Cl(-)]i) determines the strength and polarity of GABA neurotransmission. STE20/SPS1-related proline/alanine-rich kinase (SPAK) is known as an indirect regulator of [Cl(-)]i for its activation of Na-K-2 Cl(-)co-transporters (NKCC) and inhibition of K-Cl(-)co-transporters (KCC) in many organs. NKCC1 or KCC2 expression changes have been demonstrated previously in the hippocampal neurons of mice with pilocarpine-induced status epilepticus (PISE). However, it remains unclear whether SPAK modulates [Cl(-)]i via NKCC1 or KCC2 in the brain. Also, there are no data clearly characterizing SPAK expression in cortical or hippocampal neurons or confirming an association between SPAK and epilepsy. In the present study, we examined SPAK expression and co-expression with NKCC1 and KCC2 in the hippocampal neurons of mice with PISE, and we investigated alterations in SPAK expression in the hippocampus of such mice. Significant increases in SPAK mRNA and protein levels were detected during various stages of PISE in the PISE mice in comparison to levels in age-matched sham (control) and blank treatment (control) mice. SPAK and NKCC1 expression increased in vitro, while KCC2 was down-regulated in hippocampal neurons following hypoxic conditioning. However, SPAK overexpression did not influence the expression levels of NKCC1 or KCC2. Using co-immunoprecipitation, we determined that the intensity of interaction between SPAK and NKCC1 and between SPAK and KCC2 increased markedly after oxygen-deprivation, whereas SPAK overexpression strengthened the relationships. The [Cl(-)]i of hippocampal neurons changed in a corresponding manner under the different conditions. Our data suggests that SPAK is involved in the plasticity of GABA signaling function in acquired epilepsy via adjustment of [Cl(-)]i in hippocampal neurons.

MSCs (mesenchymal stem cells) have attracted attention as a promising tool for regenerative medicine and transplantation therapy. MSCs exert neuroprotective effects by secreting a number of factors in vitro and in vivo. Similar characteristics are found in ADSCs (adipose-derived stem cells) and BMSCs (bone marrow stromal cells). Multipotent capability, easy accessibility and rapid proliferation of ADSCs have been established. Our main objective was to compare cell viability, growth rate, expression of neurotrophic factors and nestin genes in ADSCs and BMSCs. Cell doubling time and proliferation rate indicate that ADSCs has a higher proliferation rate than BMSCs. ADSCs and BMSCs express a similar pattern of CD71 and CD90 markers. Nestin immunostaining showed that ADSCs and BMSCs are immunopositive. The expression of neurotrophic factors genes in ADSCs proved similar to that of BMSCs genes. Thus adipose tissue stem cells with a high proliferation rate can express nestin and neurotrophic factor genes. Therefore ADSCs may be useful in future cell replacement therapies and help improve neurodegenerative diseases.

The markers of cell proliferation (Ki-67) and apoptosis [caspase-3, TdT-mediated biotin-dUTP nick-end labelling (TUNEL)] and the expression of syndecan-1 and heat shock protein 70 (Hsp70) were analyzed immunohistochemically in 11 developing human palates, from developmental weeks 6 to 10. During fusion of the primary palate, the proportion of proliferating cells decreased from 42 to 32% and the proportion of apoptotic cells decreased from 11 to 7% in the medial-edge epithelium. At later stages, the proportions of both types of cells decreased in the ectomesenchyme, except for proliferating cells in its non-condensing part. At developmental weeks 9-10, the epithelial seam in the secondary palate comprised 28% proliferative cells and 5% apoptotic cells. While condensing ectomesenchyme contained more apoptotic cells than proliferating cells, the opposite was observed for the non-condensing ectomesenchyme. Co-expression of syndecan-1 and Hsp70 was detected in cells budding from the epithelial seam. Our study indicates similar principles for human primary palate and secondary palate fusion, and parallel persistence of proliferation and apoptotic activity. While proliferation enables growth and fusion of different palatal primordia, apoptosis results in the removal of of large numbers epithelial cells at the fusion point. The disintegration of seam remnants seems to be executed through the processes of change in protein content and cell migration, probably leading to cell death as their final outcome.

Chronic inflammation is an important component of the fibroproliferative changes that characterise pulmonary hypertensive vasculopathy. Fibrocytes contribute to tissue remodelling in settings of chronic inflammation, including animal models of pulmonary hypertension (PH). We sought to determine whether circulating fibrocytes were increased in children and young adults with PH. 26 individuals with PH and 10 with normal cardiac anatomy were studied. Fresh blood was analysed by flow cytometry for fibrocytes expressing CD45 and procollagen. Fibrocyte numbers were correlated to clinical and haemodynamic parameters, and circulating CC chemokine ligand (CCL)2 and CXC chemokine ligand (CXCL)12 levels. We found an enrichment of circulating fibrocytes among those with PH. No differences in fibrocytes were observed among those with idiopathic versus secondary PH. Higher fibrocytes correlated to increasing mean pulmonary artery pressure and age, but not to length or type of treatment. Immunofluorescence analysis confirmed flow sorting specificity. Differences in plasma levels of CCL2 or CXCL12, which could mobilise fibrocytes from the bone marrow, were not found. We conclude that circulating fibrocytes are significantly increased in individuals with PH compared with controls. We speculate that these cells might play important roles in vascular remodelling in children and young adults with pulmonary hypertension.

Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate is a promising novel oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. These effects are presumed to originate from a combination of immunomodulatory and neuroprotective mechanisms. We aimed to clarify whether neuroprotective concentrations of dimethyl fumarate have immunomodulatory effects.We determined time- and concentration-dependent effects of dimethyl fumarate and its metabolite monomethyl fumarate on viability in a model of endogenous neuronal oxidative stress and clarified the mechanism of action by quantitating cellular glutathione content and recycling, nuclear translocation of transcription factors, and the expression of antioxidant genes. We compared this with changes in the cytokine profiles released by stimulated splenocytes measured by ELISPOT technology and analyzed the interactions between neuronal and immune cells and neuronal function and viability in cell death assays and multi-electrode arrays. Our observations show that dimethyl fumarate causes short-lived oxidative stress, which leads to increased levels and nuclear localization of the transcription factor nuclear factor erythroid 2-related factor 2 and a subsequent increase in glutathione synthesis and recycling in neuronal cells. Concentrations that were cytoprotective in neuronal cells had no negative effects on viability of splenocytes but suppressed the production of proinflammatory cytokines in cultures from C57BL/6 and SJL mice and had no effects on neuronal activity in multi-electrode arrays.These results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.

There are several analytical techniques currently in use in conservation science to identify proteins in artworks. However, as is often the case, the determination of the exact location of a protein in a complex layer structure is challenging due to difficulty in separating layers. Localization of the protein in a cross-section has been demonstrated through attenuated total reflectance-Fourier transform infrared mapping and imaging as well as chemiluminescent and fluorescent-labeled antibodies; however, these techniques either require expensive instrumental setups or produce results that can be challenging to interpret. This paper will present research using surface-enhanced Raman scattering (SERS) nanotags complexed to secondary antibodies in conjunction with primary antibodies for the localization of ovalbumin, collagen, and casein in cross-sections from replicas and artworks containing avian egg, animal glue, or casein binders. The advantages of this technique over the others are (1) the detection method is a Raman microscope, equipment found in several museum laboratories; (2) the distinctive SERS signal from the nanotag increases the detection limit of the protein and decreases the interference from other colorants present in the cross-section layers; and finally, (3) the large (120 nm) SERS-labeled antibodies do not appear to penetrate into the cross-section, eliminating the risk of spurious signal and misidentification. Any agglomerations due to surface texture are clearly visible under normal illumination and can be avoided easily during analysis or removed with a light polish. This technique not only allows protein localization in multilayered samples while preserving the stratigraphic information but also retains the protein specificity of the antibody approach.

Ciliogenesis in developing and post-natal human kidneys appears to influence cell proliferation and differentiation, apico-basal cell polarity, and tubular lumen formation. We have analyzed the appearance of primary cilia and differentiation of kidney cells in ten human conceptuses aged 6-22 weeks and in one 5-year-old kidney, using a double immunofluorescence labeling technique for α-tubulin, γ-tubulin, Oct-4, and Ki-67 and by electron microscopy. Immature forms of nephrons and ampullae were characterized by intense cell proliferation, which subsequently decreased during development. Primary cilia appeared on the surfaces of non-proliferating cells in developing nephrons, gradually increasing in length from 0.59 μm in renal vesicles to 0.81 μm in the S-forms of nephrons, ultimately reaching 3.04 μm in length in mature fetal and post-natal nephrons. Ciliary length increased from 0.59 μm in ampullae to 1.28 μm in post-natal collecting tubules. Mesenchymal to epithelial transformation of kidney cells coincided with the appearance of apico-basal polarity, both gap and tight junctions, and lumen formation. Up-regulation of Oct-4 expression correlated with the onset of kidney cell differentiation. Our results demonstrate the importance of proper primary cilia lengthening and Oct-4 expression for the normal development of fetal and post-natal kidneys and of apico-basal polarity for normal tubular lumen formation. Disturbances in these processes are associated with ciliopathies.

Collagen VI, one of the extracellular matrix proteins, has been implicated in regulating cell proliferation and reducing apoptosis in several different systems. However, the role of collagen VI in the central nervous system remains unclear. In this manuscript, we demonstrated that upon ultraviolet (UV) irradiation, mouse primary hippocampal neurons specifically up-regulate the expression of Col6a1, Col6a2, and Col6a3 mRNA and secreted collagen VI protein. Augmentation of collagen VI mRNA and protein after UV irradiation may have a neuroprotective role as suggested by the fact that extracellular supplying soluble collagen VI protein, but not other collagen proteins, reduced UV induced DNA damage, mitochondria dysfunction, and neurite shrinkage. We also tried to determine the signaling molecules that mediate the protective effect of collagen VI via Western blot and inhibitor analysis. After collagen VI treatment, UV irradiated neurons increased phosphorylation of Akt and decreased phosphorylation of JNK. Inhibiting Akt/Phosphatidylinositol 3-kinases (PI3K) pathway diminished the protective effect of collagen VI. Our study suggested a potential protective mechanism by which neurons up-regulate collagen VI production under stress conditions to activate Akt/PI3K anti-apoptotic signaling pathway.Copyright © 2011. Published by Elsevier Ltd.

Functional evidence of Na(+)-glucose cotransport in rat lung has been provided by Basset et al. (J. Physiol. 384:325-345, 1987). By autoradiography [(3)H]phloridzin binding has been found confined to alveolar epithelial type II cells in mouse and rabbit lungs (Boyd, J. Physiol. 422: 44P, 1990). In this research we checked by immunofluorescence whether Na(+)-glucose cotransporter (SGLT1) is also expressed in alveolar type I cells. Lungs of anesthetized rats and lambs were fixed by paraformaldehyde, perfused in pulmonary artery, or instilled into a bronchus, respectively. Tissue blocks embedded in paraffin or frozen were sectioned. Two specific anti-SGLT1 antibodies for rat recognizing aminoacid sequence 402-420, and 546-596 were used in both species. Bound primary antibody was detected by secondary antibody conjugated to fluorescein isothiocianate or Texas red, respectively. In some sections cellular nuclei were also stained. In rats alveolar type I cells were identified by fluorescent Erythrina cristagalli lectin. Sections were examined by confocal laser-scanning microscope. Both in rats and lambs alveolar epithelium was stained by either antibody; no labeling occurred in negative controls. Hence, SGLT1 appears to be also expressed in alveolar type I cells. This is functionally relevant because type I cells provide 95-97% of alveolar surface, and SGLT1, besides contributing to removal of lung liquid under some circumstances, keeps low glucose concentration in lining liquid, which is useful to prevent lung infection.

To investigate the spatial and temporal expression of proliferation Ki-67 marker, pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins during early development of the human tooth.