Identification of novel short peptide inhibitors of soluble 37/48 kDa oligomers of amyloid β42

Since the soluble oligomers of 42-residue amyloid β (Aβ42) might be neurotoxins at an early stage of Alzheimer's disease (AD), inhibition of soluble Aβ42 oligomerization should be effective in the treatment of AD. We have found by phage display that a 7-residue peptide, SRPGLRR, exhibited inhibitory activity against soluble 37/48 kDa oligomers of Aβ42. In the present study, we newly prepared 3- and 4-residue random peptides libraries and performed pannings of them against soluble Aβ42 to search for important factors in the inhibition of Aβ42 oligomerization. After the fifth round, arginine-containing peptides were enriched in both libraries. SDS-PAGE and size-exclusion chromatography indicated that the inhibitory activities of 4-residue peptides against the soluble 37/48 kDa oligomers of Aβ42 were higher than those of the 3-residue peptides, and RFRK exhibited strong inhibitory activity as well as SRPGLRR. These short peptides should be useful for the suppression of soluble Aβ42 oligomer formation.     

Cellular DBP and E4BP4 proteins are critical for determining the period length of the circadian oscillator

The phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts.     

Magnetic resonance imaging of progressive cardiomyopathic changes in the db/db mouse

The db/db mouse is a well-established model of diabetes. Previous reports have documented contractile dysfunction (i.e., cardiomyopathy) in these animals, although the extant literature provides limited insights into cardiac structure and function as they change over time. To better elucidate the natural history of cardiomyopathy in db/db mice, we performed cardiac magnetic resonance (CMR) scans on these animals. CMR imaging was conducted with a 4.7-T magnet on female db/db mice and control db/+ littermates at 5, 9, 13, 17, and 22 wk of age. Gated gradient echo sequences were used to obtain cineographic short-axis slices from apex to base. From these images left ventricular (LV) mass (LVM), wall thickness, end-diastolic volume (LVEDV), and ejection fraction (LVEF) were determined. Additionally, cardiac [(18)F]fluorodeoxyglucose ([(18)F]FDG) PET scanning, pressure-volume loops, and real-time quantitative PCR on db/db myocardium were performed. Relative to control, db/db mice developed significant increases in LVM and wall thickness as early as 9 wk of age. LVEDV diverged slightly later, at 13 wk. Interestingly, compared with the baseline level, LVEF in the db/db group did not decrease significantly until 22 wk. Additionally, [(18)F]FDG metabolic imaging showed a 40% decrease in glucose uptake in db/db mice. Furthermore, contractile dysfunction was observed in 15-wk db/db mice undergoing pressure-volume loops. Finally, real-time quantitative PCR revealed an age-dependent recapitulation of the fetal gene program, consistent with a myopathic process. In summary, as assessed by CMR, db/db mice develop characteristic structural and functional changes consistent with cardiomyopathy.     

Potent anticancer activities of novel aminophenol analogues against various cancer cell lines

Novel aminophenol analogues were synthesized based on the structure of fenretinide (N-(4-hydroxyphenyl)retinamide, 5), which is a potent anticancer agent. Our findings showed that the anticancer activities of 5 were due to the side chain attached to the aminophenol moiety. A p-octylaminophenol (p-OAP) provided the most potent anticancer activity among p-alkylaminophenols examined. In this study, we investigated anticancer activities against various cancer cell lines by the new aminophenols, p-dodecylaminophenol (1), p-decylaminophenol (2), N-(4-hydroxyphenyl)dodecananamide (3), and N-(4-hydroxyphenyl)decananamide (4), which exhibits a side chain as long as 5. Cell growth of breast cancer (MCF-7, MCF-7/Adr(R)), prostate cancer (DU-145), and leukemia (HL60) cells was suppressed by 1 and 2 in a fashion dependent on the length of the alkyl chain attached to the aminophenol. In contrast, 3 and 4 were extremely weak. Compound 5 was less potent than 1. Cell growth of liver cancer (HepG2) was not markedly affected by these compounds. In addition, apoptosis of HL60 cells was induced by 1 and 2 in a chain length-dependent manner, but not by 3 and 4. Incorporation of compounds into HL60 cells was in the order 1>2=3>4. These results indicated that anticancer activities for 1 and 2 are correlated with their incorporation into cancer cells and their capability to induce apoptosis, but not for 3 and 4. Compound 1, a potent anticancer agent with potency strikingly greater than 5, may potentially be useful in clinic.     

Electrochemical consideration on the optimum pH of bilirubin oxidase

Steady-state current-potential curves were obtained for the direct electron transfer (DET) of bilirubin oxidase (BOD) at a highly oriented pyrolytic graphite electrode, and the theoretical analysis based on nonlinear regression enabled us to determine the formal redox potential (E degrees') of BOD in a wide pH range of 2.0 to 8.5. Cyclic voltammetric measurements were also performed for substrates, including p-phenylenediamine (PPD), o-aminophenol (OAP), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and their E degrees ' values or the anodic peak potentials (for OAP) were determined at various pH values. The difference in the redox potentials between BOD and substrates (DeltaE degrees') showed a maximum at pH 6.5 to 8.0, pH 6.5 to 8.0, and pH 3.5 to 4.5 for PPD, OAP, and ABTS, respectively. These pH ranges should be thermodynamically most favorable for the electron transfer between BOD and the respective substrates. In practice, the pH ranges showing a maximum DeltaE degrees' corresponded well with the optimum pH values for the O(2) reduction activity of BOD: pH 6.5 to 7.5, pH 8.0 to 8.5, and pH 4.0 for PPD, OAP, and ABTS, respectively. Thus, it was suggested that DeltaE degrees ' should be one of the primary factors determining the activity of BOD with the substrates.     

The Expression of Fn14 via Mechanical Stress-activated JNK Contributes to Apoptosis Induction in Osteoblasts

Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca²⁺ influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.     

Rab17‐mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion

Autophagy plays a crucial role in host defence by facilitating the degradation of invading bacteria such as Group A Streptococcus (GAS). GAS‐containing autophagosome‐like vacuoles (GcAVs) form when GAS‐targeting autophagic membranes entrap invading bacteria. However, the membrane origin and the precise molecular mechanism that underlies GcAV formation remain unclear. In this study, we found that Rab17 mediates the supply of membrane from recycling endosomes (REs) to GcAVs. We showed that GcAVs contain the RE marker transferrin receptor (TfR). Colocalization analyses demonstrated that Rab17 colocalized effectively with GcAV. Rab17 and TfR were visible as punctate structures attached to GcAVs and the Rab17‐positive dots were recruited to the GAS‐capturing membrane. Overexpression of Rab17 increased the TfR‐positive GcAV content, whereas expression of the dominant‐negative Rab17 form (Rab17 N132I) caused a decrease, thereby suggesting the involvement of Rab17 in RE–GcAV fusion. The efficiency of GcAV formation was lower in Rab17 N132I‐overexpressing cells. Furthermore, knockdown of Rabex‐5, the upstream activator of Rab17, reduced the GcAV formation efficiency. These results suggest that Rab17 and Rab17‐mediated REs are involved in GcAV formation. This newly identified function of Rab17 in supplying membrane from REs to GcAVs demonstrates that RE functions as a primary membrane source during antibacterial autophagy.     

Development of Implant/Interconnected Porous Hydroxyapatite Complex as New Concept Graft Material

Background

Dental implant has been successfully used to replace missing teeth. However, in some clinical situations, implant placement may be difficult because of a large bone defect. We designed novel complex biomaterial to simultaneously restore bone and place implant. This complex was incorporated implant into interconnected porous calcium hydroxyapatite (IP-CHA). We then tested this Implant/IP-CHA complex and evaluated its effect on subsequent bone regeneration and implant stability in vivo.

Methodology/Principal Findings

A cylinder-type IP-CHA was used in this study. After forming inside of the cylinder, an implant was placed inside to fabricate the Implant/IP-CHA complex. This complex was then placed into the prepared bone socket in the femur of four beagle-Labrador hybrid dogs. As a control, implants were placed directly into the femur without any bone substrate. Bone sockets were allowed to heal for 2, 3 and 6 months and implant stability quotients (ISQ) were measured. Finally, tissue blocks containing the Implant/IP-CHA complexes were harvested. Specimens were processed for histology and stained with toluidine blue and bone implant contact (BIC) was measured. The ISQs of complex groups was 77.8±2.9 in the 6-month, 72.0±5.7 in the 3-month and 47.4±11.0 in the 2-month. There was no significant difference between the 3- or 6-month complex groups and implant control groups. In the 2-month group, connective tissue, including capillary angiogenesis, was predominant around the implants, although newly formed bone could also be observed. While, in the 3 and 6-month groups, newly formed bone could be seen in contact to most of the implant surface. The BICs of complex groups was 2.18±3.77 in the 2-month, 44.03±29.58 in the 3-month, and 51.23±8.25 in the 6-month. Significant difference was detected between the 2 and 6-month.

Conclusions/Significance

Within the results of this study, the IP-CHA/implant complex might be able to achieve both bone reconstruction and implant stability.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.     

Phosphorylation of protein phosphatase 2Czeta by c-Jun NH2-terminal kinase at Ser92 attenuates its phosphatase activity

The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Czeta, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH 2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser (92), Thr (202), and Thr (205) of PP2Czeta. Phosphorylation of these Ser and Thr residues of PP2Czeta ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Czeta by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser (92) but not at Thr (202) or Thr (205) suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Czeta at Ser (92) by stress-activated JNK attenuates its phosphatase activity in cells.