Important role of methionine 145 in dimerization of bovine β-lactoglobulin

β-Lactoglobulin (LG) contains nine β-strands (strands A-I) and one α-helix. Strands A-H form a β-barrel. At neutral pH, bovine LG (BLG) forms a dimer and the dimer interface consists of AB-loops and the I-strands of two subunits. On the other hand, equine LG (ELG) is monomeric. The residues 145-153 of BLG, which compose a dimer interface, are entirely different from those of ELG. The difference in the association states between BLG and ELG can be attributed to the residues 145-153. To confirm this, we constructed a chimeric LG, ImBLG (I-strand mutated BLG), in which the residues 145-153 were replaced with those of ELG. Gel-filtration chromatography and analytical ultracentrifugation revealed that ImBLG existed as a monomer. To identify the residues important for dimerization, we constructed several revertants and investigated their association. This experiment revealed that, in addition to the interface residues (Ile147, Leu149 and Phe151), Met145 is critical for dimerization. Although Met145 does not contact with the other protomer, it seems to be important in determining the backbone conformation of the I-strand. This was supported by the fact that all Met145-containing mutants showed circular dichroism spectra similar to BLG but different from ImBLG.     

Synthesis and biological evaluation of molecular probes based on the 9-methylstreptimidone derivative DTCM-glutarimide

Molecular probes based on 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide) were synthesized and assessed for inhibitory activity against LPS-induced NO production. Among the probes examined, several derivatives exhibited potential for use in determining the target proteins of DTCM-glutarimide.     

Enhancement of optical properties of dyes for bioprobes by freezing effect of molecular motion using POSS-core dendrimers

We demonstrate that the POSS-core dendrimer induced various kinds of favourable properties of trisvinyl-pyridinium triphenylamine (TP3PY) as a bioprobe. By using the amphiphilicity of the POSS core, the complexes of TP3PY with G2 POSS-core dendrimer were prepared, and the series of properties were investigated for the application as a bioprobe. Initially, it was shown that the adsorption of TP3PY onto the vessels was highly prohibited by the complex formation with the dendrimers. The solution states of the dendrimer complexes were maintained at least for 7 days. Moreover, it was found that the improvement of quantum yields and the elongation of fluorescent lifetimes were observed by the complexation with the dendrimers. Similar photochemical properties were obtained in a glassy state of 2-methyltetrahydrofuranat -196°C. The molecular rotations occurring at the excited state could be restricted by the complex formation with dendrimers. These characteristics induced by the complexation with the POSS-core dendrimer are of significance to improve the signal to noise ratio and the accuracy on the detection as a bioprobe.     

The mutated S1-haplotype in sour cherry has an altered S-haplotype-specific F-box protein gene

Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S(1)-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Ujfehértói Fürt?s carrying the mutated S(1)-haplotype (S(1)'S(4)S(d)S(null)) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S(1)-haplotype revealed that the mutated S(1)-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S(1)-haplotype pollen part mutant, termed S(1)', contains a 615-bp Ds-like element within the S(1)-haplotype-specific F-box protein gene (SFB(1)'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB(1) containing only 75 of the 375 amino acids present in the wild-type SFB(1). S(1)' along with 2 other previously characterized Prunus S-haplotype mutants, S(f) and S(6m), illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.     

MHC class I-like MILL molecules are beta2-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression

MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta2-microglobulin (beta2m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta2m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.     

Measurements of fast neutrons in Hiroshima by use of 39Ar

The survivors of the A-bomb explosions over Hiroshima and Nagasaki were exposed to a mixed neutron and gamma radiation field. To validate the high-energy portion of the neutron field and thus the neutron dose to the survivors, a method is described that allows retrospective assessment of the fast neutrons from the A-bombs. This is accomplished by the extraction of the noble gas argon from biotites separated from Hiroshima granite samples, and then the detection of the (39)Ar activity that was produced by the capture of the fast neutrons on potassium. Adjusted to the year 1945, activities measured in the first samples taken at distances of 94, 818, 992, and 1,173 m from the hypocenter were 6.9+/-0.2, 0.32+/-0.01, 0.14+/-0.02, and 0.09+/-0.01 mBq/g K, respectively. All signals were significantly above detector background and show low uncertainties. Considering their uncertainties they agree with the calculated (39)Ar activation in the samples, based on the most recent dosimetry system DS02. It is concluded that this method can be used to investigate samples obtained from large distances in Hiroshima, where previous data on fast neutrons are characterized by considerable uncertainties. Additionally, the method can be used to reconstruct the fast neutron fluence in Nagasaki, where no experimental data exist.     

Transforming growth factor-beta in the brain regulates fat metabolism during endurance exercise

We have previously reported that the concentration of transforming growth factor-beta (TGF-beta) increases in the cerebrospinal fluid of rats during exercise and that there is an increase in whole body fat oxidation following the intracisternal administration of TGF-beta. These results led us to postulate that TGF-beta in the brain regulates the enhancement of fatty acid oxidation during exercise. To test this hypothesis, we carried out respiratory gas analysis during treadmill running following the inhibition of TGF-beta activity in rat brain by intracisternal administration of anti-TGF-beta antibody or SB-431542, an inhibitor of the type 1 TGF-beta receptor. We found that each reagent partially blocked the increase in the fatty acid oxidation. We also compared the plasma concentrations of energy substrates in the group administered anti-TGF-beta antibody and the control group during running. We found that the plasma concentrations of nonesterified fatty acids and ketone bodies in the group administered anti-TGF-beta antibody were lower than in the control group at the end of running. In the same way, we carried out respiratory gas analysis during treadmill running after depressing corticotropin-releasing factor activity in the brain using intracisternal administration of astressin, an inhibitor of the corticotropin-releasing factor receptor. However, there were no significant differences in respiratory exchange ratio or oxygen consumption in moderate running (60% maximum oxygen consumption). These results suggest that brain TGF-beta has a role in enhancing fatty acid oxidation during endurance exercise and that this regulation is executed at least partly via the type 1 TGF-beta receptor signal transduction system.     

Application of HSVtk suicide gene to X-SCID gene therapy: ganciclovir treatment offsets gene corrected X-SCID B cells

Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human gamma c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the gamma c chain, the cells were treated with ganciclovir (GCV). The gamma c chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the gamma c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.