Slo1 caveolin-binding motif, a mechanism of caveolin-1-Slo1 interaction regulating Slo1 surface expression

The large conductance, voltage- and Ca2+-activated potassium (MaxiK, BK) channel and caveolin-1 play important roles in regulating vascular contractility. Here, we hypothesized that the MaxiK alpha-subunit (Slo1) and caveolin-1 may interact with each other. Slo1 and caveolin-1 physiological association in native vascular tissue is strongly supported by (i) detergent-free purification of caveolin-1-rich domains demonstrating a pool of aortic Slo1 co-migrating with caveolin-1 to light density sucrose fractions, (ii) reverse co-immunoprecipitation, and (iii) double immunolabeling of freshly isolated myocytes revealing caveolin-1 and Slo1 proximity at the plasmalemma. In HEK293T cells, Slo1-caveolin-1 association was unaffected by the smooth muscle MaxiK beta1-subunit. Sequence analysis revealed two potential caveolin-binding motifs along the Slo1 C terminus, one equivalent, 1007YNMLCFGIY1015, and another mirror image, 537YTEYLSSAF545, to the consensus sequence, varphiXXXXvarphiXXvarphi. Deletion of 1007YNMLCFGIY1015 caused approximately 80% loss of Slo1-caveolin-1 association while preserving channel normal folding and overall Slo1 and caveolin-1 intracellular distribution patterns. 537YTEYLSSAF545 deletion had an insignificant dissociative effect. Interestingly, caveolin-1 coexpression reduced Slo1 surface and functional expression near 70% without affecting channel voltage sensitivity, and deletion of 1007YNMLCFGIY1015 motif obliterated channel surface expression. The results suggest 1007YNMLCFGIY1015 possible participation in Slo1 plasmalemmal targeting and demonstrate its role as a main mechanism for caveolin-1 association with Slo1 potentially serving a dual role: (i) maintaining channels in intracellular compartments downsizing their surface expression and/or (ii) serving as anchor of plasma membrane resident channels to caveolin-1-rich membranes. Because the caveolin-1 scaffolding domain is juxtamembrane, it is tempting to suggest that Slo1-caveolin-1 interaction facilitates the tethering of the Slo1 C-terminal end to the membrane.     

Rapamycin Augments Immunomodulatory Properties of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis

The immunomodulatory and anti-inflammatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) have been considered as an appropriate candidate for treatment of autoimmune diseases. Previous studies have revealed that treatment with BM-MSCs may modulate immune responses and alleviate the symptoms in experimental autoimmune encephalomyelitis (EAE) mice, an animal model of multiple sclerosis. Therefore, the present study was designed to examine immunomodulatory effects of BM-MSCs in the treatment of myelin oligodendrocyte glycoprotein (MOG) 35-55-induced EAE in C57BL/6 mice. MSCs were obtained from the bone marrow of C57BL mice, cultured with DMEM/F12, and characterized with flow cytometry for the presence of cell surface markers for BM-MSCs. Following three passages, BM-MSCs were injected intraperitoneally into EAE mice alone or in combination with rapamycin. Immunological and histopathological effects of BM-MSCs and addition of rapamycin to BM-MSCs were evaluated. The results demonstrated that adding rapamycin to BM-MSCs transplantation in EAE mice significantly reduced inflammation infiltration and demyelination, enhanced the immunomodulatory functions, and inhibited progress of neurological impairments compared to BM-MSC transplantation and control groups. The immunological effects of rapamycin and BM-MSC treatments were associated with the inhibition of the Ag-specific lymphocyte proliferation, CD8+ cytolytic activity, and the Th1-type cytokine (gamma-interferon (IFN-γ)) and the increase of Th-2 cytokine (interleukin-4 (IL-4) and IL-10) production. Addition of rapamycin to BM-MSCs was able to ameliorate neurological deficits and provide neuroprotective effects in EAE. This suggests the potential of rapamycin and BM-MSC combined therapy to play neuroprotective roles in the treatment of neuroinflammatory disorders.     

An Early Ordovician Trilobite assemblage from the Lashkarak Formation, Damghan area, northern Iran

A low diversity Lower Ordovician trilobite assemblage is described from the middle part of the Lashkarak Formation at Simeh-Kuh, northwest of Damghan, northern Iran. Co-occurrence with conodonts of the lowermost Paroistodusproteus Biozone suggests an uppermost Tremadocian age. This assemblage includes four taxa, including one new genus and two new species, Damghanampyxginteri nov. gen. nov. sp. and Asaphellusfecundus nov. sp. The third diagnosable species from the formation is Taihungshaniamiqueli (Bergeron, 1894) which has previously been reported from southern France and the Turkish Taurides, suggesting north Gondwanan faunal affinities.     

Molecular and morphological evaluation of transgenic Persian walnut plants harboring Fld gene under osmotic stress condition

Molecular Biology Reports - Soil drought stress is a limiting factor of productivity in walnut (Juglans regia L). Ferredoxin (Fd) level decreases under adverse environmental stress. Functional...     

Alteration in genes expression patterns during in vitro differentiation of mouse spermatogonial cells into neuroepithelial-like cells

Pluripotent stem cells derived from testis is a new, natural, and unlimited source for cell therapy in regenerative medicine and represent a possible alternative to replacing of all cells in the body. Here, we designed a simple co-culture system of spermatogonia cells with Sertoli cells for the generation of embryonic stem-like cells from mouse testis. The importance of our simple method will be clear when we compared it with other complex and time-consuming methods. Embryonic stem-like colonies with sharp border confirmed by real-time PCR, immunocytochemistry and flow cytometry assessments. Embryonic stem-like colonies were immunopositive for pluripotency markers. Transition of spermatogonia cells to embryonic stem-like cells was accompanied by extensive changes in gene expression. These changes included significant increase in pluripotency genes expression and significant decrease in germ cell-specific genes expression. Also, we proved the differentiation capacity of embryonic stem-like cells to neuroepithelial-like cells which were immunoreactive to Nestin and Neurofilament 68. Evaluation of genes expression during in vitro differentiation into neuroepithelial-like cells showed high-level expression of Nestin whether this gene approximately has no expression in undifferentiated embryonic stem-like cells. Also, expression of pluripotency genes has significantly decreased in neuroepithelial-like cells compared with embryonic stem-like cells. This study shows that embryonic stem-like cells derived from testis are capable to differentiate into neuroepithelial-like cells that may provide a cellular reservoir usable for neurodegenerative disorders.     

Pathogen Presence in Wild Birds Inhabiting Landfills in Central Iran

EcoHealth - Wild birds are important in the transmission of many zoonotic pathogens such as salmonella and avian influenza virus (AIV). The current study investigated the presence of bacterial and...     

Localization of sarcolemmal proteins to lipid rafts in the myocardium

The localization of sarcolemmal proteins within the membrane can have a dramatic effect on excitation-contraction coupling. We examine the localization of the Na+-Ca2+ exchanger, the dihydropyridine receptor, and other proteins involved in excitation-contraction coupling in rat heart using biochemical and immunolocalization techniques. Specifically, we assess the distribution of proteins within the lipid raft fraction of the sarcolemma. We find that the distribution of proteins in lipid raft fractions is very dependent on the solubilization technique. A common technique using sodium carbonate/pH 11 to solubilize non-lipid raft proteins was inappropriate for use with sarcolemmal membranes. Use of Triton X-100 was more efficacious as a solubilization agent. A large majority of the Na+-Ca2+ exchanger, Na+/K+-ATPase, and plasma membrane Ca2+ pump are not present in lipid rafts. In contrast, most adenosine A1 receptors and dihydropyridine receptors were in lipid raft fractions. Most of the adenosine A1 receptors could be co-immunoprecipitated with caveolin indicating a localization to caveolae (a subclass of lipid rafts). In contrast, the dihydropyridine receptors could not be co-immunoprecipitated with caveolin. Most biochemical data were confirmed by high resolution immunolocalization studies. Using correlation analysis, only a small fraction of the Na+-Ca2+ exchangers colocalized with caveolin whereas a substantial fraction of dihydropyridine and adenosine A1 receptors did colocalize with caveolin. The most pertinent findings are that the Na+-Ca2+ exchanger and the dihydropyridine receptor are in separate sarcolemmal subcompartments. These spatial relationships may be relevant for understanding excitation-contraction coupling.     

Obolarina persica sp. nov., associated with dying Quercus in Iran

A new species, Obolarina persica, is described from Iran. It is widely associated with dying Quercus brantii, on which it produces charcoal-like stromata. The fungus described herein differs from the other described species, Ob. dryophila, primarily in its much larger ascospores.     

Partial purification and kinetic properties of three different d-glucosamine 6-P:N-acetyltransferase forms from human placenta

Three distinct forms of -glucosamine 6-P (Gm 6-P):N-acetyltransferases (EC 2.3.1.4) were partially purified from human placental homogenates by carboxy methyl-Sephadex chromatography. Purification of forms I and II were 13.5-fold, while that of form III was 114-fold. All three forms had a pH optimum value of 9.7 in glycine–NaOH buffer. Enzymes II and III had a K_m value for Gm 6-P of 3.0 mM, which was less than half of that observed for form I (7.1 mM). The corresponding K_m values for acetyl CoA were 0.157 (form I), 0.187 (form II) and 0.280 mM (form III), respectively. Activities of all three forms were inhibited at high concentrations of either substrate. These enzymes were inhibited from 82 to 92% by 2.5 mM p-chloromercuribenzoate. The inhibition was largely reversible by inclusion of 2.5 mM dithiothreitol in the incubation mixtures. There was no requirement for divalent cations, as demonstrated by lack of inhibition of enzyme activity by ethylene diamine tetraacetate. The results are discussed in terms of differences among the enzyme properties of human placental, rodent and porcine liver forms.     

Quantitative Determination of Spatial Protein-Protein Correlations in Fluorescence Confocal Microscopy

To quantify spatial protein-protein proximity (colocalization) in paired microscopic images of two sets of proteins labeled by distinct fluorophores, we showed that the cross-correlation and the autocorrelation functions of image intensity consisted of fast and slowly decaying components. The fast component resulted from clusters of proteins specifically labeled, and the slow component resulted from image heterogeneity and a broadly-distributed background. To better evaluate spatial proximity between the two specifically labeled proteins, we extracted the fast-decaying component by fitting the sharp peak in correlation functions to a Gaussian function, which was then used to obtain protein-protein proximity index and the Pearson's correlation coefficient. We also employed the median-filter method as a universal approach for background reduction to minimize nonspecific fluorescence. We illustrated our method by analyzing computer-simulated images and biological images.