A nanoporous reactor for efficient proteolysis

A nanoreactor based on mesoporous silicates is described for efficient tryptic digestion of proteins within the mesochannels. Cyano-functionalized mesoporous silicate (CNS), with an average pore diameter of 18 nm, is a good support for trypsin, with rapid in situ digestion of the model proteins, cytochrome c and myoglobin. The generated peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Proteolysis by trypsin-CNS is much more efficient than in-solution digestion, which can be attributed to nanoscopic confinement and concentration enrichment of the substrate within the mesopores. Proteins at concentrations of 2 ng muL(-1) were successfully identified after digestion for 20 min. A biological complex sample extracted from the cytoplasm of human liver tissue was digested by using the CNS-based reactor. Coupled with reverse-phase HPLC and MALDI-TOF MS/MS, 165 proteins were identified after standard protein data searching. This nanoreactor combines the advantages of short digestion time with retention of enzymatic activity, providing a promising way to advance the development of proteomics.     

The method of combined inspection

For costly and/or destructive inspections for a complex population, the sum of observations, the test statistic in common use, is shown to be inefficient in risk control. For the complex population consisting of Bernoulli distributions, the method of combined inspection is proposed in this paper, Evaluation based on examples and simulation results is conducted according to the method of limited classification. It is concluded that the new method is efficient and applicable.     

Small amplitude, free longitudinal vibrations of a load on a finitely deformed stress-softening spring with limiting extensibility

A constitutive theory for a general class of incompressible, isotropic stress-softening, limited elastic rubberlike materials is introduced. The model is applied to study the small amplitude, free longitudinal vibrational frequency of a load about a suspended static equilibrium stretch of a finitely deformed, stress-softening spring with limiting extensibility. A number of physical results, including bounds on the frequency, are reported. It is proved, for example, that the normalized vibrational frequency for the ideally elastic neo-Hookean oscillator is a lower bound for the normalized frequency of every incompressible, isotropic stress-softening, limited elastic oscillator within the general class. All results are illustrated for the special limited elastic Gent and the purely elastic Demiray biomaterial models, both with stress-softening characterized by a Zú?iga–Beatty front factor damage function. The results for both stress-softening models are compared with experimental data for several gum rubbers and thoracic aortic tissue provided by others; and, overall, it is found that the stress-softening, limited elastic Gent model best characterizes the data.     

二次损失下G-M模型中可估函数的条件Minimax估计与性质

在二次损失下关于任意矩阵V对G-M模型讨论了齐次线性估计类中可估函数的条件Mimimax估计与性质。     

Comparison of Operating Mechanisms for the Poly(N-vinylcarbazole) Based Non-Volatile Memory Devices

Summary: The organic non-volatile memory devices (NVMs) based on poly(N-vinylcarbazole) (PVK) with the different structures and compositions were fabricated and evaluated. The resistance states in the devices were controlled by the external electric field and exhibited the distinctive properties; the device with a single PVK layer was a write-once read-many-times memory by the field induced filament as a conduction path and its memory properties depended on the PVK thickness, the PVK/Al/PVK structured device was operated by a space charge limited current model and was sensitive to preparing condition of the internal Al layer, and the device performances with the PVK based charge transfer complex depended on the composition of the CT materials and the surface condition of the bottom electrode.     

牛天然纤溶酶原环状结构域K1—3的获得

采用Ｌｙｓ－Ｓｅｐｈｅｒｏｓｅ４Ｂ亲和层析,从新生牛血清中分离提纯的牛纤溶酶原（ｐａｌｓｍｉｎｏｇｅｎ,ｐｇｎ）。利用ＳＤＳ－聚丙烯酰胺凝胶电泳分析,对其纯度和相对分子质量进行了鉴定。利用猪胰弹性蛋白酶酶切纤溶酶原获得了环状结构域Ｋ１－３,采用Ｌｙｓ－Ｓｅｐｈｅｒｏｓｅ４Ｂ亲和层析对该片段进行了分离提取。     

Detection of major xylanase-containing cellulose-binding domain from Penicillium verruculosum by combination of chromatofocusing and limited proteolysis

Adsorption on microcrystalline cell ulose of enzyme components of cellulase complex from Penicillium verruculosum was studied by chromatofocusing on a Mono P column. The most strongly adsorbed and major component was identified as xylanase (XYN) with MW 65 k Da and pl 4.5. The high adsorption degree of XYN on cellulose indicated the possible presence of a cellulose-binding domain in the molecular sturcture. Limited proteolysis of XYN with papain was carried out. Kinetics of proteolysis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and measuring activities toward insoluble xylan and 4-methylumbelliferyl-β-d-lactoside (MUF-LAC). During the proteolysis, formation of two polypeptides with MW 51 and 14k Da was observed. No loss of activity toward thesolu blesubstrate was observed, wherease the activity toward xylan decreased rapidly. Adsorption distribution coefficient (K _d) of the core protein separated by gel-filtration was found to be 15 times lower than the K _d for the initial nondigested XYN (0.02 and 0.29 L/g, respectively). The activity of core protein toward insoluble xylan was close to zero, whereas the activity toward MUF-LAC was close to that exhibited by the original enzyme. The results presented indicate a bifunctional organization of XYN, where one domain acts as a binding anchor for insoluble substrates and the other, localized in the core protein, contains the active site.     

Salivary peptidome in type 1 diabetes mellitus

Diabetic patients show a high susceptibility to oral diseases of inflammatory, catabolic and chronic nature with potential impact on saliva composition. In this study, our purpose was to characterize type 1 diabetes‐induced alterations in the salivary peptidome aiming to find prospective biomarkers for type 1 diabetes oral health evaluation. Peptidomic analysis of saliva from controls (n = 5) and type 1 diabetic patients (n = 5) were performed by liquid chromatography followed by mass spectrometry. The proteolytic activity and metalloproteinases expression was accessed by zymography and slot blot analysis, respectively. Data evidenced a significant increase in the percentage of peptides in diabetic patients paralleled by a higher proteolytic activity, compared with healthy individuals. The nonsalivary gland protein fragments identified in saliva were mainly derived from collagen and extracellular matrix proteins, namely collagen type I. The cleavage site frequency analysis showed significant differences between healthy and type 1 diabetic individuals, highlighting the activity of proteases such as matrix metalloproteinase‐9 and cathepsin D. Our results highlight salivary collagen fragments as potential biomarkers to follow up diabetes‐related oral damage. Copyright © 2011 John Wiley & Sons, Ltd.     

Lactobacillus plantarum amylase acting on crude starch granules

The microheterogeneous native amylolytic complex secreted by the isolate A6 of Lactobacillus plantarum revealed a selective enzyme specificity loss when submitted to a limited proteolysis under a suboptimum pH condition. A clear electrophoretic profile change toward just one shorter, more acidic, and equally active polypeptide fragment resulted from the pronase E pretreatment. Although the whole enzyme activity remained apparently unaffected for soluble starch, the native parallel activity on intact and nongelatinized starch granules either from cereals or tubers was dramatically reduced. This phenomenon was more clearly documented by scanning electron microscopy using the easiest accessible native substrate: wheat starch granules. The anion-exchange-purified native enzymes from L. plantarum displayed a different optimum pH curve when compared with the thermotolerant α-amylase from Bacillus licheniformis. The α-amylases from the lactic-acid-producing A6 isolate presented an electrophoretic profile easily distinguishable from those from B. liqueniformis and B. subtilis species.