Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

Attachment strength of an adhesive nuisance mussel,limnoperna fortunei,against water flow

The attachment strength of the freshwater mussel Limnoperna fortunei against water flow was studied. Newton's expression successfully described the hydrodynamic drag force acting on the mussel with a drag coefficient value of 1.03. The drag‐resistant force (defined as hydrodynamic drag force at mussel detachment) was smaller than the detachment force measured using a tensile load test. A fairly good correlation was obtained between the drag‐resistant force and the number of secreted threads. The drag‐resistant force divided by the number of threads increased with shell size, suggesting that byssal thread strength increased with mussel growth. For the mussel specimens obtained from a water transmission pipe, thread width increased with shell size. However, thread width was not dependent on current velocity. There was no correlation between the number of secreted threads and shell length, which indicated that the number of secreted threads did not change with mussel size. Therefore, the water velocity needed to detach mussels increases with shell size of the mussel when the number of secreted threads is constant. The increases in the water velocity to detach mussels with larger shells suggests that the mussel becomes more resistant to water flow as it grows. It is estimated that a flow velocity of around lms^‐1 is critical for attachment/detachment of a juvenile mussel with a shell length of a few millimeters and one hundred byssal threads.     

Studies on fouling by the freshwater mussel limnoperna fortunei and the antifouling effects of low energy surfaces

Attachment of the freshwater mussel, Limnoperna fortunei, was tested using non‐treated surfaces, viz. glass, nylon, rubber, silicone and Teflon, together with glass surfaces modified with nine kinds of silane coupling agents. Among the surfaces tested, the mussel avoided attaching to Teflon, silicone, and glass modified with 3‐bromopropyltrimethoxysilane or 3,3,3‐(trifluo‐ropropyl)‐trimethoxysilane. With respect to the relationship between the percentage attachment and the surface free energy (sfe) of the substrates, it was found that attachment was considerably reduced on the substrates which exhibited relatively low sfe, as above. The mean number of secreted byssuses per attaching mussel also decreased with decreasing substrate sfe. Furthermore, when the sfe was divided into the dispersion and polar components, the percentage mussel attachment was related to the polar component. These results suggest that effective antifouling towards L. fortunei is achieved on substrates with a low sfe polar component.     

Activation of a mechanosensitive BK channel by membrane stress created with amphipaths

Some BK channels are activated in response to membrane stretch. However, it remains largely unknown which membrane component transmits forces to the channel and which part of the channel senses the force. Recently, we have shown that a BK channel cloned from chick heart (named SAKCa channel) is a stretch activated channel, while deletion of a 59 amino acids splice insert (STREX) located in the cytoplasmic side, abolishes its stretch-sensitivity. This finding raised a question whether stress in the bilayer is crucial for the mechanical activation of the channel. To address this question we examined the effects of membrane perturbing amphipaths on the stretch activation of the SAKCa channel and its STREX-deletion mutant. We found that both anionic amphipath trinitrophenol (TNP) and cationic amphipath chlorpromazine (CPZ) could dose-dependently activate the channel by leftward shifting the voltage activation curve when applied alone. In contrast, TNP and CPZ compensated each other's effect when applied sequentially. These results can be understood in the framework of the bilayer couple hypothesis, suggesting that stress in the plasma membrane can activate the SAKCa channel. Interestingly, the STREX-deletion mutant channel has much less sensitivity to the amphipaths, suggesting that STREX acts as an intermediate structure that can indirectly convey stress in the membrane to the gate of the SAKCa channel via an unidentified membrane associated protein(s) that can detect or transmit stress in the membrane.     

Discovery and structure–activity relationship of thienopyridine derivatives as bone anabolic agents

A cell-based assay was performed for the discovery of novel bone anabolic agents. Alkaline phosphatase (ALPase) activity of ST2 cells was utilized as an indicator of osteoblastic differentiation, and thienopyridine derivative 1 was identified as a hit compound. 3-Aminothieno[2,3-b]pyridine-2-carboxamide was confirmed to be a necessary core structure for the enhancement of ALPase activity, and then optimization of the C4-substituent on the thienopyridine ring was carried out. Introduction of cyclic amino groups to the C4-position of the thienopyridine ring improved the activity. Especially, N-phenyl-homopiperazine derivatives were found to be strong enhancers of ALPase among this new series. Furthermore, 3-amino-4-(4-phenyl-1,4-diazepan-1-yl)thieno[2,3-b]pyridine-2-carboxamide (15k) was orally administered to ovariectomized (OVX) rats over 6 weeks for evaluating the effects on areal bone mineral density (aBMD), and statistically significant improvements in aBMD were observed from the dosage of 10 mg/kg/day.     

Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.