A dot-blot method for quantification of apurinic/apyrimidinic sites in DNA using an avidin plate and liposomes encapsulating a fluorescence dye

A dot-blot method for quantification of apurinic/apyrimidinic (AP) sites in genomic DNA (calf thymus DNA) is described using an avidin-modified glass slip and biotinylated liposomes containing sulforhodamine B as a fluorescence marker. Aldehyde reactive probe (ARP)-tagged DNA was found to be strongly adsorbed on an avidin slip, even if treated with ethanolamine and biotin, with an efficiency of 51% due to the positive surface charge of avidin, and unbound ARP was easily washed out of the surface with Milli-Q water. In the assay protocol, calf thymus DNA containing AP sites is reacted with ARP in solution and immobilized on an ethanolamine- and biotin-treated avidin slip (EAB-avidin slip), followed by incubation with streptavidin. The AP sites were finally quantified with biotinylated liposomes containing 1.5 mM sulforhodamine B as a fluorescence marker. The mean fluorescence intensity over the surface of the slip was an analytically relevant measure of the amount of AP sites in calf thymus DNA. By using the dot-blot assay, 1-5 AP sites per 10(4) nucleotides in 5 and 100 ng of DNA were quantified. The current dot-blot method has potential for quantification of AP sites in genomic DNA at a level of several nanograms.     

Acute effect of corticosterone on N-methyl-D-aspartate receptor-mediated Ca2+ elevation in mouse hippocampal slices

We examined the rapid effects of corticosterone (CORT) on N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ signals in adult mouse hippocampal slices by using Ca2+ imaging technique. Application of NMDA caused a transient elevation of intracellular Ca2+ concentration followed by a decay to a plateau within 150s. The 30min preincubation of CORT induced a significant decrease of the peak amplitude of NMDA-induced Ca2+ elevation in the CA1 region. The rapid effect of CORT was induced at a stress-induced level (0.4-10microM). Because the membrane non-permeable bovine serum albumin-conjugated CORT also induced a similar rapid effect, the rapid effect of CORT might be induced via putative surface CORT receptors. In contrast, CORT induced no significant effects on NMDA-induced Ca2+ elevation in the dentate gyrus. In the CA3 region, CORT effects were not evaluated, because the marked elevation of NMDA-induced Ca2+ signals was not observed there.     

Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site

Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.     

Parkin potentiates ATP-induced currents due to activation of P2X receptors in PC12 cells

Loss-of-function mutations of the parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP). Parkin was shown to function as a RING-type E3 ubiquitin protein ligase. However, the function of parkin in neuronal cells remains elusive. Here, we show that expression of parkin-potentiated adenosine triphosphate (ATP)-induced currents that result from activation of the P2X receptors which are widely distributed in the brain and involved in neurotransmission. ATP-induced inward currents were measured in mock-, wild-type or mutant (T415N)-parkin-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in wild-type parkin-transfected cells. However, the immunocytochemical study showed no apparent increase in the number of P2X receptors or in ubiquitin levels. The increased currents were attenuated by inhibition of cAMP-dependent protein kinase (PKA) but not protein kinase C (PKC) or Ca2+ and calmodulin-dependent protein kinase (CaMKII). ATP-induced currents were also regulated by phosphatases and cyclin-dependent protein kinase 5 (CDK5) via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32), though the phosphorylation at Thr-34 and Thr-75 were unchanged or rather attenuated. We also tried to investigate the effect of alpha-synuclein, a substrate of parkin and also forming Lysine 63-linked multiubiquitin chains. Expression of alpha-synuclein did not affect the amplitude of ATP-induced currents. Our finding provides the evidence for a relationship between parkin and a neurotransmitter receptor, suggesting that parkin may play an important role in synaptic activity.     

DOCK2 is required in T cell precursors for development of Valpha14 NK T cells

Mouse CD1d-restricted Valpha14 NKT cells are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster myoblast city, is critical for lymphocyte migration and regulates T cell responsiveness through immunological synapse formation, yet its role in Valpha14 NKT cells remains unknown. We found that DOCK2 deficiency causes marked reduction of Valpha14 NKT cells in the thymus, liver, and spleen. When alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14 NKT cells, was administrated, cytokine production was scarcely detected in DOCK2-deficient mice, suggesting that DOCK2 deficiency primarily affects generation of Valpha14 NKT cells. Supporting this idea, staining with CD1d/alpha-GalCer tetramers revealed that CD44- NK1.1- Valpha14 NKT cell precursors are severely reduced in the thymuses of DOCK2-deficient mice. In addition, studies using bone marrow chimeras indicated that development of Valpha14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. These results indicate that DOCK2 is required for positive selection of Valpha14 NKT cells in a cell-autonomous manner, thereby suggesting that avidity-based selection also governs development of this unique subset of lymphocytes in the thymus.     

Chlorophyll metabolism

Since the 1970s, researchers have proposed several regulatory pathways governing chlorophyll metabolism, but only recently have the underlying molecular mechanisms been elucidated. The recent data indicate that such regulatory systems are more complex than originally anticipated. For instance, the pathways involve a series of protein-protein interactions, including complex formation, the dual localization of enzymes within chloroplasts, and a novel protein degradation mechanism that is triggered by pigments. Furthermore, several lines of evidence suggest that chlorophyll metabolism might not only significantly impact the assembly of photosynthetic machineries but also influence processes such as programmed cell death, the 'stay-green' phenomenon, and chloroplast-nucleus communication.     

Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.     

Leu309 plays a critical role in the encapsulation of substrate protein into the internal cavity of GroEL

In the crystal structure of the native GroEL.GroES.substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL.GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K).substrate complex failed to bind GroES to produce a stable GroEL(L309K).GroES.substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K).GroES.substrate complex was stabilized with BeF(x), but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu(309)), appears to play a critical role in encapsulation of the substrate.     

Fgf16 is essential for pectoral fin bud formation in zebrafish

Zebrafish pectoral fin bud formation is an excellent model for studying morphogenesis. Fibroblast growth factors (Fgfs) and sonic hedgehog (shh) are essential for pectoral fin bud formation. We found that Fgf16 was expressed in the apical ectodermal ridge (AER) of fin buds. A knockdown of Fgf16 function resulted in no fin bud outgrowth. Fgf16 is required for cell proliferation and differentiation in the mesenchyme and the AER of the fin buds, respectively. Fgf16 functions downstream of Fgf10, a mesenchymal factor, signaling to induce the expression of Fgf4 and Fgf8 in the AER. Fgf16 in the AER and shh in the zone of polarizing activity (ZPA) interact to induce and/or maintain each other's expression. These findings have revealed that Fgf16, a newly identified AER factor, plays a crucial role in pectoral fin bud outgrowth by mediating the interactions of AER-mesenchyme and AER-ZPA.