Location of the Bombyx mori 175kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin

To identify and gain a better understanding of the cadherin-like receptor-binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin-like protein-binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175kDa cadherin-like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, amino peptidaseN1, from Bo.mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N-(9-acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175-binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175-binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo.mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.     

Length-dependent activation and auto-oscillation in skeletal myofibrils at partial activation by Ca2+

The length-dependent activation of skeletal myofibrils was examined at the single-sarcomere level with phase-contrast microscopy at sarcomere length (SL) >2.2 μm. At the maximal activation by Ca²⁺ (pCa 4.5) the active force linearly decreased with increasing SL, while at partial activation by Ca²⁺ (pCa 6.1-6.5) the larger active force was generated at longer SL. Throughout these experiments, the distribution of SL was kept homogeneous upon activation. In addition, we found that the spontaneous oscillation of force and SL frequently occurs in the SL range 2.2-2.6 μm at pCa 6.1-6.2. Either changes in [Ca²⁺] or osmotic compression of the myofilament lattice induced by the addition of dextran T-500, affected both the length dependence of activation and the occurrence of auto-oscillation. These results suggest that the force-generating properties of sarcomeres in striated muscle are determined not only by [Ca²⁺], but also by the lattice spacing as a function of SL.     

Preparation and Isolation of Oligogalacturonic Acids and Their Biological Effects in Cockscomb (Celosia argentea L.) Seedlings

A mixture of oligogalacturonic acids, the partial degradation substances of polygalacturonic acid, promoted shoot growth in cockscomb (Celosia argentea L.) seedlings, which generally had a high sensitivity for growth-promoting substances. The effect of the mixture of oligogalacturonic acids on shoot growth of cockscomb was higher than that of the polygalacturonic acid at concentrations above 30 ppm. These oligomers were loaded onto an anion exchange column, DEAE Sephadex A-25, and separated into individual oligomer sizes using the NH4HCO3 eluent system. This separation method has the advantage of using NH4HCO3 as the eluent solution; NH4HCO3 in the sample solution is effectively removed by lyophilization. Each of the isolated oligogalacturonic acids gave a single band on a fluorophore-assisted carbohydrate electrophoresis (FACE), and they showed the m/z value which corresponded to their molecular ion peaks [M-H]- on a fast atom bombardment mass spectrometry (FAB-MS) analysis. These results showed that the successive chromatography method used in this study is well suited for the preparation of oligogalacturonic acid for the plant growth test. Furthermore, we showed that the effective degree of polymerization (DP) of oligogalacturonic acid was around 8 on shoot growth of cockscomb seedlings, and the effects of both smaller and larger oligogalacturonic acids were slightly lower than that of octa-galacturonide. Octa-galacturonide promoted shoot growth of cockscomb at concentrations above 10 mM, and showed a 66% promotion at the most effective concentration of 300 mM. Root growth was slightly inhibited at concentrations above 300 mM. These results suggest that DP around 8 of oligogalacturonic acids has the function to control shoot growth in cockscomb as a growth-promoting substance.     

Ophthalmologic changes related to radiation exposure and age in adult health study sample, Hiroshima and Nagasaki

A 2-year ophthalmologic study of age and radiation-related ophthalmologic lesions among the atomic bomb survivors in Hiroshima and Nagasaki was conducted in 1978-80. The study sample in both cities was composed of all persons exposed to 100+ rad, their controls, and all other persons with a previous record of axial opacities or posterior subcapsular changes. Most of the losses were due to persons who refused to participate or for whom it was not possible to arrange for an ophthalmologic examination at the time of the regularly scheduled medical examination. It should be emphasized, however, that the loss of persons in both the control and the 100+ rad groups did not change systematically with increasing age by city. Increased lenticular opacities, other lens changes, and loss of visual acuity and accommodation occurred with increasing age in both exposed and control subjects as manifestations of the normal aging process. A highly significant excess risk for all age categories in the 300+ rad group in comparison to those in the control group was observed for both axial opacities and posterior subcapsular changes in Hiroshima, but not in Nagasaki. A stronger radiosensitive aging effect for persons who were under 15 years old at the time of the bombing (ATB) was observed for both axial opacities and posterior subcapsular changes in Hiroshima.     

Structure-activity relationship of clovamide and its related compounds for the inhibition of amyloid β aggregation

Alzheimer’s disease (AD), a neurodegenerative disorder, is characterized by aggregation of amyloid β-protein (Aβ). Aβ aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Inhibition of Aβ aggregation by naturally occurring compounds is thus a promising strategy for the treatment of AD. We have already reported that caffeoylquinic acids and phenylethanoid glycosides, which possess two or more catechol moieties, strongly inhibited Aβ aggregation. Clovamide (1) containing two catechol moieties, isolated from cacao beans (Theobroma cacao L.), is believed to exhibit preventive effects on Aβ aggregation. To investigate the structure-activity relationship of clovamide (1) for the inhibition of Aβ aggregation, we synthesized 1 and related compounds 2–11 through reaction between l-DOPA, d-DOPA, l-tyrosine, or l-phenylalanine and caffeic acid, p-coumaric acid, or cinnamic acid, and compounds 12 and 13 were derived from 1. Among tested compounds 1–13, those containing one or two catechol moieties exhibited potent anti-aggregation activity, whereas the non-catechol-type related compounds showed little or no activity. This suggests that at least one catechol moiety is essential for inhibition of Aβ42 aggregation, and this activity increases depending on the number of catechol moieties. Consequently, clovamide (1) and its related compounds may be a promising therapeutic option for inhibiting Aβ-mediated pathology in AD.     

Vinyldiaminotriazine-acridine conjugate as G-quadruplex alkylating agent

Higher-order structures of nucleic acids have become widely noted for their biological consequences and the discovery of an alkylating small molecule for these structures has been of interest due to its therapeutic potential. We previously developed the vinyldiaminotriazine (VDAT)-acridine conjugate as a T-T mismatch alkylating agent. In this report, we focused on the finding of the alkylation to the G-quadruplex (G4) DNA with VDAT-acridine conjugates. The VDAT-acridine conjugates exhibited a considerable alkylation ability to G4 under mild conditions. Moreover, the investigation of properties with the alkylated G4 revealed that alkylation by this conjugate significantly increased the stability of the G4 structure. This study provides a starting point in the further development of selective G4 alkylating small molecules.