Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.     

Study on Tribological Properties of Polytetrafluoroethylene Drawn Uniaxially at Different Temperature

Summary: Wear behavior correlations with morphology have been established from polytetrafluoroethylene (PTFE) drawn at 200, 327, and 375 °C with draw ratio about 4. The friction coefficient and wear rate for PTFE drawn at 327 °C are lower and the wear rate is lower than that of undrawn PTFE by about 30%. The structures of samples were characterized by scanning electron microscope (SEM), DSC, and wide angle X‐ray diffraction (WAXD). Results indicate that the debris morphologies of samples are different. The differences in the tribological behavior of undrawn and drawn samples were attributed to the improvement of the degree of the crystalline, fibrillation, and orderliness by drawing, especially, for PTFE drawn at 327 °C. The orderliness of molecular arrangement along the drawn direction is also higher for PTFE drawn at 327 °C than those of PTFE drawn at 200 and 375 °C, respectively. Therefore, the intensity of covalent bond along drawn direction is higher. The shear resistance and the deformability of the material are greatly improved and the size of the wear breakage unit decreases, which results in a good tribological property for PTFE drawn at 327 °C.

SEM morphology of fractured surface perpendicular to the draw direction for PTFE drawn at 327 °C.     

大豆蛋白/PVA复合纤维细观形态结构

通过扫描电镜、光学显微镜等方法对大豆蛋白/PVA复合纤维的微细结构做了分析和比较,结果表明,大豆蛋白/PVA复合纤维有比较明显的皮芯结构,皮层厚度约为1.8μm,芯层厚度约为5.8μm。大豆蛋白/PVA复合纤维有明显的孔洞和表面皱缩。通过对断口的观察,发现大豆蛋白/PVA复合纤维有原纤结构。     

Reg-1α, a New Substrate of Calpain-2 Depending on Its Glycosylation Status

Reg-1α/lithostathine, a protein mainly associated with the digestive system, was previously shown to be overexpressed in the pre-clinical stages of Alzheimer’s disease. In vitro, the glycosylated protein was reported to form fibrils at physiological pH following the proteolytic action of trypsin. However, the nature of the protease able to act in the central nervous system is unknown. In the present study, we showed that Reg-1α can be cleaved in vitro by calpain-2, the calcium activated neutral protease, overexpressed in neurodegenerative diseases. Using chemical crosslinking experiments, we found that the two proteins can interact with each other. Identification of the cleavage site using mass spectrometry, between Gln⁴ and Thr⁵, was found in agreement with the in silico prediction of the calpain cleavage site, in a position different from the one reported for trypsin, i.e., Arg¹¹-Ile¹² peptide bond. We showed that the cleavage was impeded by the presence of the neighboring glycosylation of Thr⁵. Moreover, in vitro studies using electron microscopy showed that calpain-cleaved protein does not form fibrils as observed after trypsin cleavage. Collectively, our results show that calpain-2 cleaves Reg-1α in vitro, and that this action is not associated with fibril formation.     

不同大豆原料形成蛋白纤维聚合物的比较

4种通过不同加工处理得到的大豆蛋白,在低pH条件下90℃加热10h所形成的聚合物形态存在很大不同。利用硫磺素T（Th T）荧光强度、差量扫描仪（DSC）和SDS-PAGE凝胶电泳分析了来自不同原料大豆蛋白的聚合动力学和组成差异。结果表明,4种大豆原料因其加工工艺不同,蛋白质组成存在差异,在本实验的条件下,11S的存在尤其是碱性亚基会抑制纤维聚合物的形成。此外,离子强度也是纤维形成的一个必要条件。      

竹原纤维纱线直径系数的测定

竹纤维是一种前景广阔的新型绿色环保凉爽型纤维,用竹纤维制作的服装和服饰越来越受到人们的青睐,但缺少设计必须的直径系数数据。因此,文章采用显微镜投影法及电脑图像分析软件,对常用的竹原纤维纱线直径进行了测定,并阐述了不同张力及加捻系数对直径系数的影响,得到竹原纤维纱线的直径系数平均值为0．0355,可供纺织产品设计开发人员参考。     

Effects of pepsin hydrolysis on the soy β‐conglycinin aggregates formed by heat treatment at different pH

The effects of pepsin hydrolysis on the β‐conglycinin aggregates formed by heat treatment at different pH were investigated. Results showed that fibrils were still observed, whereas the random aggregates were easily to be digested in the simulated gastric fluid. Electrophoresis and molecular weight analysis indicated that large aggregates still existed after pepsin treatment for fibrils. Hydrolysis resulted in changes in the apparent viscosity (ηapp) of 6% fibril solutions. The ηapp at the shear rate range (0–30 s^?1) increased in the order of fibrils < fibrils with pepsin for 60 min < fibrils with pepsin for 30 min. Smaller peptide/fibril fragments were generated, and additional aggregates were reformed during the hydrolysis process, as evidenced by thioflavin T and atomic force microscopy images. The native β‐conglycinin hydrolysates comprised a mixture of polypeptides enriched in about 47 kDa. These findings would provide valuable information about effects of enzymatic hydrolysis on plant oligometric globulin aggregates.     

The nanometre-scale physiology of bone: steric modelling and scanning transmission electron microscopy of collagen�Cmineral structure

The nanometre-scale structure of collagen and bioapatite within bone establishes bone''s physical properties, including strength and toughness. However, the nanostructural organization within bone is not well known and is debated. Widely accepted models hypothesize that apatite mineral (‘bioapatite’) is present predominantly inside collagen fibrils: in ‘gap channels’ between abutting collagen molecules, and in ‘intermolecular spaces’ between adjacent collagen molecules. However, recent studies report evidence of substantial extrafibrillar bioapatite, challenging this hypothesis. We studied the nanostructure of bioapatite and collagen in mouse bones by scanning transmission electron microscopy (STEM) using electron energy loss spectroscopy and high-angle annular dark-field imaging. Additionally, we developed a steric model to estimate the packing density of bioapatite within gap channels. Our steric model and STEM results constrain the fraction of total bioapatite in bone that is distributed within fibrils at less than or equal to 0.42 inside gap channels and less than or equal to 0.28 inside intermolecular overlap regions. Therefore, a significant fraction of bone''s bioapatite (greater than or equal to 0.3) must be external to the fibrils. Furthermore, we observe extrafibrillar bioapatite between non-mineralized collagen fibrils, suggesting that initial bioapatite nucleation and growth are not confined to the gap channels as hypothesized in some models. These results have important implications for the mechanics of partially mineralized and developing tissues.     

The effect of limited proteolysis by different proteases on the formation of whey protein fibrils

Four proteases: trypsin, protease A, pepsin, and protease M were selected to modify whey protein concentrate (WPC) at a low degree of hydrolysis (0.1, 0.2, and 0.3%) before adjusting to pH 2.0 and heating at 90°C to gain insight into the influence of proteolysis on fibril formation. The kinetics of fibril formation were performed on native and modified WPC using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and far-UV circular dichroism spectroscopy for the morphological and secondary structural analyses. The change in surface hydrophobicity and content of free sulfhydryl groups were also observed during the formation of fibrils for the native and modified WPC. The content of aggregation and thioflavin T kinetic data indicated that the ability of fibril formation was apparently different for WPC modified by the 4 proteases. Whey protein concentrate modified by trypsin aggregated more during heating and the fibril formation rate was faster than that of the native WPC. Whey protein concentrate modified by the other proteases showed slower aggregation with worse amyloid fibril morphology. Compared with the native WPC, the structure of WPC changed differently after being modified by proteases. The state of α-helix structure for modified WPC played the most important role in the formation of fibrils. Under the mild conditions used in this work, the α-helix structure of WPC modified by trypsin caused little destruction and resulted in fibrils with good morphology; the content of α-helices for WPC modified by other proteases decreased to 36.19 to 50.94%; thus, fibril formation was inhibited. In addition, it was beneficial for the modified WPC to form fibrils such that the surface hydrophobicity increased and the content of free sulfhydryl groups slightly decreased during heating.     

Effect of CaCl2 on 2 heat-induced whey protein concentrate fibrillation pathways: Spontaneous and nuclear induction

Amyloid fibrils have many excellent functional properties that facilitate their applications in the food industry. There are 2 pathways for whey protein concentrate (WPC) to form amyloid fibril aggregates: spontaneous pathway and nuclear induction pathway. Low ionic strength is a necessary condition for the spontaneous pathway to proceed successfully. In this paper, the effect of salt ions on 2 WPC fibrillation pathways was investigated by adding CaCl₂. The results demonstrated WPC fibrils were unable to form normally through spontaneous pathway as adding CaCl₂; but still could form through nuclear induction pathway with 20 to 30 mM CaCl₂, the nuclei accelerated the fibrillation process led to the resistance to the disordered aggregation brought by CaCl₂. Moreover, divalent cations (Ca²⁺, Mg²⁺) had much stronger effects than monovalent cations (Na⁺) on fibril formation, and the results of X-ray photoelectron spectrum together with Fourier-transform infrared spectroscopy suggested that Ca²⁺ had a greater effect on the fibril formation than Cl^?.