Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties

Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site. 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein. To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of [3H]myristic acid. 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound [3H]myristic acid at the NH2-terminal glycine. In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of [35S]methionine-labeled protein on two-dimensional gels. Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated. Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties. In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.     

Evaluation of fatigue crack growth behavior of GLARE3 fiber/metal laminates using a compliance method

The objectives of this study were to investigate the effectiveness of a compliance method for analyzing the fatigue crack growth of GLARE3 fiber/metal laminates. The materials tested were GLARE3-5/4 (2.6 mm thick) and GLARE3-3/2 (1.4 mm thick). Centrally notched specimens with two kinds of notch length and two kinds of fiber orientation were fatigue tested under constant amplitude loading. The expression of the experimental stress intensity factor, K_exp, for the 2024-T3 aluminum-alloy layers of a GLARE3 is formulated and K_exp were obtained from the relationship between crack length and specimen compliance. The test results clarified the following: (1) da/dN–ΔK_exp relationships roughly show the linear relationship independent of the maximum stress level, specimen thickness, notch length, and fiber orientations, (2) the da/dN–ΔK_exp relationships approximately agree with the linear part and its extension of Paris–Erdogan’s law obtained for the da/dN–ΔK relationship of the 2024-T3 aluminum-alloy, (3) the compliance method is effective for analyzing fatigue crack growth in GLARE3 laminates.     

Reduction of lipophilicity at the lipophilic domain of RXR agonists enables production of subtype preference: RXRalpha-preferential agonist possessing a sulfonamide moiety

Retinoid X receptor agonists (RXR agonists, rexinoids) are interesting candidates for the treatment of cancers such as tamoxifen-resistant breast cancer and taxol-resistant lung cancer. However, well-known RXR agonists possess a strong lipophilic character. In addition, although RXR has three subtypes, no subtype-selective RXR agonists are known. Thus we aimed to produce less-lipophilic and subtype-selective RXR agonists. By designing sulfonamide-type RXR agonists, 4-[N-methanesulfonyl-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)amino]benzoic acid (8 a) was found to prefer RXRalpha over RXRbeta and RXRgamma, although the potency is less than the potencies of well-known RXR pan-agonists. Moreover, our results suggest that the reduction of lipophilicity at the hydrophobic interaction region of RXR agonists enables production of RXR subtype preference. Our finding will be useful for the creation of more potent and less-lipophilic subtype-selective RXR agonists aimed at the reduction of undesirable side effects.     

Detection of Lymph Node Metastases in Human Colorectal Cancer by Using 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence with Spectral Unmixing

Accurate evaluation of metastatic lymph nodes (LNs) is indispensable for adequate treatment of colorectal cancer (CRC) patients. Here, we demonstrate detection of metastases of human CRC in removed fresh LNs using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence. A spectral unmixing method was employed to reduce the overlap of collagen autofluorescence on PpIX fluorescence. A total of 17 surgery patients with advanced CRC were included in this study. After 5-ALA at a dose of 15 mg/kg of body weight was applied orally 2 h prior to surgery, 87 LNs were subjected to spectral fluorescence imaging and histopathological diagnosis, and statistical analysis was performed. No apparent side effect was observed to be associated with 5-ALA administration. The spectral unmixing fluorescence intensity of PpIX in metastatic LNs was 10.2-fold greater than that in nonmetastaic LNs. The receiver-operating-characteristic (ROC) analysis showed that the area under the curve (AUC) was calculated as 0.95. Our results show the potential of 5-ALA-induced PpIX fluorescence processed by spectral unmixing for detecting metastases in excised fresh LNs from patients with CRC, suggesting that this rapid and feasible method is applicable to gross evaluation of resected LN samples in pathology laboratories.     

Solution to the boundary-value problems of non-uniform axial mixing in tapered aeration

The effect of axial mixing in an aeration tank of activated sludge process operating with a mode of tapered aeration has been studied from the view point of a heterogenous system. The dispersion model is applied to represent the flow system with tapered aeration, by which a formation of non-uniform axial mixing with an abrupt change of mixing intensity at the section-boundary is assumed.A numerical technique for solving the multi-point boundary-value problem associated with the system-model of tapered aeration is specifically introduced. By using this computational scheme, several simulations with different operating conditions resulting in various patterns of axial mixing; heterogenous, homogeneous, non-uniform and uniform axial mixing, are carried out.