多孔微载体无血清培养rCHO细胞生产u-PA

在30L搅拌式反应器中无血清培养分泌尿激酶型纤溶酶原激活剂(u-PA)的DNA重组CHO细胞,定期部分更换Cytopore多孔微载体,使生长在多孔微载体中的细胞不断更新繁殖,解决大规模细胞培养中的细胞凋亡问题。在91d连接换液培养过程中,细胞密度可维持在(1.3～2.6)×107/mL,活细胞比率维持在90%以上。在7.5L搅拌罐中培养细胞,利用外部周期性压力振荡刺激并结合载体更新技术,可减轻密度效应对细胞生长和表达的影响,在一定程度上提高细胞在高密度培养条件下的表达水平。在67d连续换液培养中,细胞最高密度为2.64×10⁷/mL,活细胞比率维持在95%以上。与稳压操作相比,利用周期变压刺激技术可提高产量10%～20%,且可降低葡萄糖厌氧代谢生成乳酸的转化率,利用4步纯化工艺,从含u-PA约135g的2100 L上清中获得约80guPA(单链比例约为90%)。     

Corrections of the periodic boundary conditions with rectangular simulation boxes on the diffusion coefficient,general aspects

It is generally known that the effect of the periodic boundary conditions using cubic boxes manifests itself as the underestimation for the self-diffusion coefficient and the difference between the experimental value and the simulation is proportional to the reciprocal of system size. Rather than the cubic boxes, the effect of the rectangular boxes, which has been normally ignored, however, exhibits some distinct features, according to the hydrodynamics theory on molecular diffusion. In particular, the diffusion coefficient along the direction of the short side length of the box is always larger than that along the larger side length. What’s unusual is that some components or even all three components of the diffusion coefficient may exceed that of an infinite system. Through the comparisons of the flow patterns excited by the diffusing particles, we find that the positive and negative corrections are linked to the local and global vortex of fluid, respectively. It appears that the box shape affects the diffusion by modifying the flow pattern. The theoretical predictions are further tested by molecular dynamics simulations.     

Permanence and Positive Periodic Solutions for Kolmogorov Competing Species Systems with Periodic Forced Terms

IIntroductionhnsldertheKolmogorovtypeequationswithforcedtermsforn－spedeswheremevectorfunctionIscontinuousandT－periodicInthet—variable（T＞0）andP。（t）IS。COntlnuOuST－period1CfUnCtlol（l—1，2，…，n），。nddoeSn。tCh。ngeitsSignSlfl［0，Inthispaper，wesaythatthesystem（1．1）lspermanent，Ifthereexiststwoconstantssuchthatanysolutionexistsgloballyon［0，＋OO）andthatthereexistsTO＞0suchthatPe。anenceofconslde。dspeczeshasbeenw；delydlscu。edInllte。ture（see［1，2．3Jandtherefer…     

The molecular mode of action and species specificity of canakinumab,a human monoclonal antibody neutralizing IL-1β

Interleukin-1β (IL-1β) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-IL-1β antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with IL-1β from mouse, rat, rabbit, or macaques. The crystal structure of the canakinumab Fab bound to human IL-1β was determined in an attempt to rationalize the species specificity. The X-ray analysis reveals a complex surface epitope with an intricate network of well-ordered water molecules at the antibody-antigen interface. The canakinumab paratope is largely pre-organized, as demonstrated by the structure determination of the free Fab. Glu 64 of human IL-1β is a pivotal epitope residue explaining the exquisite species specificity of canakinumab. We identified marmoset as the only non-human primate species that carries Glu 64 in its IL-1β and demonstrates full cross-reactivity of canakinumab, thereby enabling toxicological studies in this species. As demonstrated by the X-ray structure of the complex with IL-1β, canakinumab binds IL-1β on the opposite side with respect to the IL-1RAcP binding site, and in an approximately orthogonal orientation with respect to IL-1RI. However, the antibody and IL-1RI binding sites slightly overlap and the V_H region of canakinumab would sterically interfere with the D1 domain of IL-1RI, as shown by a structural overlay with the IL-1β:IL-1RI complex. Therefore, direct competition with IL-1RI for IL-1β binding is the molecular mechanism of neutralization by canakinumab, which is also confirmed by competition assays with recombinant IL-1RI and IL-1RII.     

A determination of liquid–vapour interfacial properties for methanol using a linear-combination-based isotropic periodic sum

We performed molecular dynamics simulations of the methanol liquid–vapour interfacial systems for a wide temperature range of 200–350 K. The linear-combination-based isotropic periodic sum (LIPS) method was used for estimating Coulombic interactions of the systems. The temperature dependence of the liquid and vapour phase densities, surface thickness and surface tension using the LIPS method was almost equal to that of the Ewald sum method. The temperature dependence of the electrostatic potential profile using the LIPS method was very similar to that of the Ewald sum method. We conclude that the LIPS method provides a reasonable accuracy in determining the liquid–vapour interfacial properties of polar molecules for many temperature conditions.     

LC–MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences

Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4–8 glucose units on Tyr 195 of glycogenin-1, but only 0–4 glucose units on Tyr-228 of glycogenin-2. The glucosylation of glycogenin-2 was enhanced to 2–4 glucose units by the co-presence of enzymatically active glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive glycogenin-1, at least not an enzymatically inactivated Thr83Met glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.     

Comparsion of staining characteristics of Toto bodies

Toto bodies are eosinophilic structures that resemble the cells of the superficial cell layer of the oral epithelium. Toto bodies commonly are associated with inflammatory gingival and other mucosal lesions including pyogenic granuloma, irritational fibroma, epulis fissuratum, peripheral giant cell granuloma and inflammatory hyperplastic gingivitis. We evaluated staining characteristics of Toto bodies to establish their origin and to identify their significance in lesions. We investigated pyogenic granuloma, fibroma and leukoplakia with epithelium that exhibited Toto bodies after hematoxylin and eosin (staining. Sections were stained with Alcian blue, periodic acid-Schiff and Ayoub-Shklar stains to evaluate staining intensity and distribution. More Toto bodies were found in pyogenic granuloma than in fibroma and leukoplakia. PAS and Alcian blue staining exhibited mild intensity and did not establish the origin of Toto bodies. High staining intensity and diffuse distribution of stain was observed using Ayoub-Shklar staining, which indicated that Toto bodies originate from keratin.     

Repeated unit cell (RUC) approach for pure bending analysis of coronary stents

Flexibility is one of the key properties of coronary stents. The objective of this paper is to characterize the bending behaviour of stents through finite element analysis with repeated unit cell (RUC) models. General periodic boundary conditions for the RUC under the pure bending condition are formulated. It is found that the proposed RUC approach can provide accurate numerical results of bending behaviour of stents with much less computational costs. Bending stiffness, post-yield bending behaviour and the relationship between moment and bending curvature are investigated for Palmaz–Schatz stents and stents with the V- and S-shaped links. It is found that the effect of link geometry on the bending behaviour of stent is significant. The behaviour of stents subjected to cyclic bending is also investigated.     

Asymptotic behaviour of the non-autonomous competing two-species Lotka–Volterra models with impulsive effect

In this paper, the nonautonomous competing two-species Lotka–Volterra models with impulsive effect are considered, where all the parameters are time-dependent and asymptotically approach the corresponding periodic functions. Under some conditions, it is shown that the semi-trivial positive solutions of the models asymptotically approach the semi-trivial positive periodic solutions of the corresponding periodic system. It is also shown that the positive solution of the models asymptotically approach the positive periodic solution of the corresponding periodic system.