Structure and function of the PsbP protein of Photosystem II from higher plants

PsbP is a membrane extrinsic subunit of Photosystem II (PS II), which is involved in retaining Ca²⁺ and Cl⁻, two inorganic cofactors for the water-splitting reaction. In this study, we re-investigated the role of N-terminal region of PsbP on the basis of its three-dimensional structure. In previous paper [Ifuku and Sato (2002) Plant Cell Physiol 43: 1244–1249], a truncated PsbP lacking 19 N-terminal residues (Δ19) was found to bind to NaCl-washed PS II lacking PsbP and PsbQ without activation of oxygen evolution at all. Three-dimensional (3D) structure of PsbP suggests that deletion of 19 N-terminal residues would destabilize its protein structure, as indicated by the high sensitivity of Δ19 to trypsin digestion. Thus, a truncated PsbP lacking 15 N-terminal residues (Δ15), which retained core PsbP structure, was produced. Whereas Δ15 was resistant to trypsin digestion and bound to NaCl-washed PS II membranes, it did not show the activation of oxygen evolution. This result indicated that the interaction of 15-residue N-terminal flexible region of PsbP with PS II was important for Ca²⁺ and Cl⁻ retention in PS II, although the 15 N-terminal residues were not essential for the binding of PsbP to PS II. The possible N-terminal residues of PsbP that would be involved in this interaction are discussed.     

Involvement of calcium-sensing receptor in osteoblastic differentiation of mouse MC3T3-E1 cells

We have previously shown that the extracellular calcium-sensing receptor (CaR) is expressed in various bone marrow-derived cell lines and plays an important role in stimulating their proliferation and chemotaxis. It has also been reported that the CaR modulates matrix production and mineralization in chondrogenic cells. However, it remains unclear whether the CaR plays any role in regulating osteoblast differentiation. In this study, we found that mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium (2.8 and 3.8 mM) or a specific CaR activator, NPS-R467 (1 and 3 microM). Next, we stably transfected MC3T3-E1 cells with either a CaR antisense vector (AS clone) or a vector containing the inactivating R185Q variant of the CaR (DN clone) that has previously been shown to exert a dominant negative action. Alkaline phosphatase activities were decreased compared with controls in both the AS and DN clones. However, the levels of type I procollagen and osteopontin mRNA in the AS clone, as detected by Northern blotting, were almost the same as in the controls. On the other hand, the expression of osteocalcin, which is expressed at a later stage of osteoblastic differentiation, was significantly reduced in both the AS and DN clones. Mineralization was also decreased in both clones. In conclusion, this study showed that the abolition of CaR function results in diminishing alkaline phosphatase activity, osteocalcin expression, and mineralization in mouse osteoblastic cells. This suggests that the CaR may be involved in osteoblastic differentiation.     

The role of terminal-link stimuli in concurrent-chain schedules: revisited using a behavioral-history procedure

A behavioral-history procedure was used to study the function of terminal-link stimuli as conditioned reinforcers in multiple concurrent-chain schedules of reinforcement. First, three pigeons were exposed to multiple concurrent-chain schedules in which the two multiple-schedule components were correlated with a blue and a white stimulus, respectively. In each component the initial links were equal independent variable-interval (VI) 15 s schedules. A fixed-interval (FI) 10 s schedule operated on the red key in one terminal link while extinction operated on the green key in the alternative terminal link. When large preferences for the red stimulus had been established, two tests were conducted. In the terminal-link test, under new initial-link stimuli--purple and brown--an FI 10 s schedule operated for both the red and green terminal-link stimuli. In the subsequent initial-link test, the blue and white initial-link stimuli were reintroduced, and, as in the terminal-link test, FI 10s operated for both the red and the green terminal-link stimuli. In the terminal-link test, the three pigeons showed no preference for the terminal links with the red stimulus, but showed clear and consistent preferences for the red stimulus when blue and white stimuli were reintroduced as initial-link stimuli in the initial-link test. This suggests that there are multiple sources of control over initial-link response allocation in concurrent-chains, including control by both terminal- and initial-link stimuli.     

Leupaxin binds to PEST domain tyrosine phosphatase PEP

PEST domain tyrosine phosphatase (PEP) is an intracellular protein tyrosine phosphatase and characterized by PEST motifs and proline-rich domains in the carboxyl terminal half. PEP is primarily expressed in hematopoietic cells, and together with PEP-binding Csk, may act as a negative regulator of antigen receptor signaling in lymphocytes. Here, we show the binding capability of PEP for leupaxin, which is preferentially expressed in hematopoietic cells and a comparatively new member of the paxillin family characterized by two protein-protein interaction modules, LIM domains and LD motifs. These results suggested that leupaxin might participate in the regulation of the signaling cascade through the binding to PEP in lymphocytes.     

Higher frequency of premature stop codon mutations at vpu gene of human immunodeficiency virus type 1 CRF01_AE compared with those of other subtypes

Our previous study demonstrated the anti-apoptosis function of the human immunodeficiency virus type 1 (HIV-1) vpu gene product in normal CD4+ T lymphocytes. In this study, using sequences obtained from the HIV sequence database, we compared vpu sequences from 184 preparations of various subtypes of HIV-1 from diverse geographical regions. Our analysis revealed that CRF01_AE isolates had premature stop codon mutations at the vpu gene at a much higher rate (36%) than other subtypes (0-9%). The premature stop codon mutations in vpu existed mostly at two amino acid residues: the methionine initiation codon and the boundary between the transmembrane (TM) and cytoplasmic domains. The mutations at the latter site were more often detected in CRF01_AE. The higher mutation rates at vpu in CRF01_AE were confirmed by sequence comparison of polymerase chain reaction products newly obtained directly from the DNA extracted from peripheral blood mononuclear cells (PBMCs), but not from the RNA from the plasma, in CRF01_AE- and subtype B-infected individuals. This finding may indicate the possibility that the more abundant population of HIV-1 CRF01_AE is able to induce apoptosis in CD4+ T lymphocytes than the populations of other subtypes.     

Cooperative effect of hydrophobic and electrostatic forces on alcohol-induced alpha-helix formation of alpha1-acid glycoprotein

Alpha1-acid glycoprotein (AGP) is a serum glycoprotein that mainly binds basic drugs. Previous reports have shown that AGP converts from a beta-sheet to an alpha-helix upon interaction with biomembranes. In the current studies, we found that alkanols, diols, and halogenols all induce this conformational change. Increased length and bulkiness of the hydrocarbon group and the presence of a halogen atom promoted this conversion, whereas the presence of a hydroxyl group inhibited it. Moreover, the effect was dependent on the hydrophobic and electrostatic properties of the alcohols. These results indicate that, in a membrane environment, hydrophobic and electrostatic factors cooperatively induce the transition of AGP from a beta-sheet to an alpha-helix.     

Application of QSAR analysis to organic anion transporting polypeptide 1a5 (Oatp1a5) substrates

Organic anion transporting polypeptide 1a5, Slco1a5 (previously called Oatp3, Slc21a7) is a multispecific transmembrane transport protein that belongs to the OATP/SLCO superfamily of solute carriers. It is expressed in several epithelial barriers such as the small intestine and the choroid plexus where it might play an important role in the disposition of numerous endogenous and exogenous organic compounds. Since the molecular basis of the multispecificity of Oatp1a5 is not known and the three-dimensional structure not solved yet, we used three-dimensional quantitative structure-activity relationship (3D-QSAR) techniques to obtain topological information on the substrate binding site of the protein. We aligned a heterogeneous data set of 18 Oatp1a5 substrates using the Genetic Algorithm Similarity Program (GASP) and performed comparative molecular field analysis (CoMFA) using this alignment. This resulted in a reasonable QSAR model including steric and electrostatic fields with a leave-one-out cross-validated r(cv)2 value of 0.705 and a no-cross-validated regression coefficient r2 value of 0.949. Based on the derived model we identified new potential Oatp1a5 substrates and confirmed their predicted apparent affinity values experimentally.