Methyl-β-cyclodextrin is a useful compound for extraction and purification of prenylated enzymes from the retinal disc membrane

cGMP phosphodiesterase 6 (PDE6) and rhodopsin kinase (GRK1) are quantitatively minor prenylated proteins involved in vertebrate phototransduction. Here, we report that methyl-β-cyclodextrin (MCD), a torus-shaped oligosaccharide with a hydrophobic pore, can be used as a selective extractant for such prenylated proteins from frog retinal disc membranes, and that MCD makes it possible to purify frog PDE6 holoenzyme with very simple procedure. The EC50s of MCD for the extraction of GRK1 and PDE6 from the cytoplasmic surface of the disc membrane were 0.17 and 5.1 mM, respectively. By successive extraction of the membrane by 1 mM and then 20 mM MCD, we obtained crude GRK1 and PDE6, respectively. From the 20mM extract, we were able to purify the PDE6 holoenzyme using one-step anion-exchange column chromatography. From 1mM MCD extract, GRK1 was further purified by an affinity column. Following the removal of MCD by ultrafiltration, we were able to confirm integrity of these enzymes by reconstituting phototransduction system in vitro. We have therefore demonstrated that MCD is a useful compound for selective extraction and purification of prenylated peripheral membrane proteins from the cytoplasmic surface of biological membranes.     

Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury

The major heat shock protein, HSP70, is known to be involved in cytoprotection against environmental stresses mediated by their function as a "molecular chaperone". Monochloramine (NH(2)Cl) is a potent cytotoxic oxidant generated by neutrophil-derived hypochlorous acid and Helicobacter pylori urease-induced ammonia. In this study, to evaluate the cytoprotective effect of HSP70 against NH(2)Cl-induced gastric mucosal cell injury, rat gastric mucosal cells (RGM-1) were stably transfected with pBK-CMV containing the human HSP70 gene (7018-RGM-1) or pBK-CMV alone (pBK-CMV-12) as control cells. These cells were treated with various concentrations of NH(2)Cl. Cell Viability was determined by MTT assay and the direct plasma membrane damage was analyzed by lactate dehydrogenase (LDH) release assay. Apoptosis was determined by DNA fragmentation analysis. NH(2)Cl caused injury to pBK-CMV-12 cells in a concentration-dependent manner. NH(2)Cl-induced gastric cell injury was significantly diminished in HSP70 over-expressing cell line (7018-RGM-1) both necrosis and apoptosis compared to the control cell line (pBK-CMV-12) transfected with CMV vector alone. These result suggest that overexpression of HSP70 plays an important role in protecting gastric cells against NH(2)Cl-induced injury.     

Mediator subunits MED1 and MED24 cooperatively contribute to pubertal mammary gland development and growth of breast carcinoma cells

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.     

Dynamics of Mitotic Apparatus Formation and Tubulin Content during Oocyte Maturation in Starfish

To follow the topo-temporal behavior of structures containing tubulin and the change in tubulin content during oocyte maturation, starfish oocytes were extracted with a medium containing detergent so that morphological observation and biochemical analysis could be conducted on the same residual oocyte preparation simultaneously. Before 1-methyladenine (1-MeAde) stimulation, "pre-meiotic asters" were observed on the germinal vesicle at the animal pole. 1-MeAde caused the appearance of distinct asters at the position of the aster precursor. When germinal vesicle breakdown (GVBD) took place, chromosomes were condensed. Chromosome gathering was concurrent with a reduction in the size of nuclear matrix. The mitotic apparatus was first constructed parallel to the cortex and then changed its axis perpendicularly. Fluorescence of tubulin due to indirect immunofluorescence in the cytoplasm other than the mitotic apparatus decreased rapidly along the course of maturation at least up to the first metaphase. Despite these dynamic morphological change, the tubulin content in the whole oocyte and the residual structures, measured by SDS-PAGE and immunostaining, did not show remarkable (statistically significant) changes through the course of maturation, although the content tended to decrease a little before the second polar body formation and to increase thereafter in the latter.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.     

Comprehensive behavioural study of GluR4 knockout mice: implication in cognitive function

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA‐type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1–4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long‐term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T‐maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.     

The carboxy-terminal region of SMAP2 directs subcellular localization as well as Arf protein specificity

Small G proteins play a central role in the organization of secretory and endocytotic pathways. The recruitment of some effectors, including vesicle coat proteins, is mediated by the ADP-ribosylation factor (Arf) family. Arf proteins have distinct subcellular localizations. ArfGAPs (Arf GTPase-activating proteins) regulate Arf GTPase activity. Thus, each ArfGAP is distinctly localized to allow it to maintain a specific interaction with its target Arf(s). However, the domains that regulate the subcellular localization of ArfGAPs and the way in which these subcellular localizations affect the target specificities of ArfGAPs remain unclear. Recently, we identified two novel ArfGAPs, SMAP1 (Small ArfGAP protein 1) and SMAP2. In the current study, we identified sequences in the carboxy-terminal region of SMAP2 that are critical for its specific subcellular localization and its specificity for Arf proteins.     

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background

Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.

Principal Findings

Here, we demonstrate that CD4⁺CD25⁺CCR4⁺ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4⁺CD25⁺CCR4⁺ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4⁺CD25⁺CCR4⁺Foxp3⁻ T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.

Conclusions

We have defined a unique T cell subset—IFN-γ⁺CCR4⁺CD4⁺CD25⁺ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.