Artificial Cytoskeleton Assembly for Synthetic Cell Motility

Imitation of cellular processes in cell-like compartments is a current research focus in synthetic biology. Here, a method is introduced for assembling an artificial cytoskeleton in a synthetic cell model system based on a poly(N-isopropyl acrylamide) (PNIPAM) composite material. Toward this end, a PNIPAM-based composite material inside water-in-oil droplets that are stabilized with PNIPAM-functionalized and commercial fluorosurfactants is introduced. The temperature-mediated contraction/release behavior of the PNIPAM-based cytoskeleton is investigated. The reversibility of the PNIPAM transition is further examined in bulk and in droplets and it could be shown that hydrogel induced deformation could be used to controllably manipulate droplet-based synthetic cell motility upon temperature changes. It is envisioned that a combination of the presented artificial cytoskeleton with naturally occurring components might expand the bandwidth of the bottom-up synthetic biology.     

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.     

Potential Vasculoprotective Effects of Blackcurrant (Ribes nigrum) Extract in Diabetic KK-Ay Mice

Polyphenols are bioactive compounds found naturally in fruits and vegetables; they are widely used in disease prevention and health maintenance. Polyphenol-rich blackcurrant extract (BCE) exerts beneficial effects on vascular health in menopausal model animals. However, the vasculoprotective effects in diabetes mellitus (DM) and atherosclerotic vascular disease secondary to DM are unknown. Therefore, we investigated whether BCE is effective in preventing atherosclerosis using KK-A^y mice as a diabetes model. The mice were divided into three groups and fed a high-fat diet supplemented with 1% BCE (BCE1), 3% BCE (BCE2), or Control for 9 weeks. The mice in the BCE2 group showed a considerable reduction in the disturbance of elastic lamina, foam cell formation, and vascular remodeling compared to those in the BCE1 and Control groups. Immunohistochemical staining indicated that the score of endothelial nitric oxide synthase staining intensity was significantly higher in both BCE2 (2.9) and BCE1 (1.9) compared to that in the Control (1.1). Furthermore, the score for the percentage of alpha-smooth muscle actin was significantly lower in the BCE2 (2.9%) than in the Control (2.1%). Our results suggest that the intake of anthocyanin-rich BCE could have beneficial effects on the blood vessels of diabetic patients.     

Purification and Biochemical Characterization of Extracellular β-Glucosidases from the Hypercellulolytic Pol6 Mutant of <Emphasis Type="Italic">Penicillium occitanis</Emphasis>

The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and β-glucosidase production with high cellulose degradation efficiency (Jain et al., Enzyme Microb Technol, 12:691–696, 1990; Hadj-Taieb et al., Appl Microbiol Biotechnol, 37:197–201, 1992; Ellouz Chaabouni et al., Enzyme Microb Technol, 16:538–542, 1994; Ellouz Chaabouni et al., Appl Microbiol Biotechnol, 43:267–269, 1995). In this work, two forms of β-glucosidase (β-glu 1 and β-glu 2) were purified from the culture supernatant of the Pol6 strain by gel filtration, ion exchange chromatography, and preparative anionic native electrophoresis. These enzymes were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis. However, both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW, 98 kDa), shared the same amino acid composition, carbohydrate content (8%), and kinetic properties. Moreover, they strongly cross-reacted immunologically. They were active on cellobiose and pNPG with Km values of 1.43 and 0.37 mM, respectively. β-glu 1 and β-glu 2 were competitively inhibited by 1 mM of glucose and 0.03 mM of δ-gluconolactone. They were also significantly inhibited by Hg²⁺ and Cu² at 2 mM. The addition of purified enzymes to the poor β-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H₃P0₄ swollen cellulose but had no effect with P. occitanis crude extract. Besides their hydrolytic activities, β-glu 1 and β-glu 2 were endowed with trans-glycosidase activity at high concentration of glucose.     

Optogenetic Engineering: Light‐Directed Cell Motility

Genetically encoded, light‐activatable proteins provide the means to probe biochemical pathways at specific subcellular locations with exquisite temporal control. However, engineering these systems in order to provide a dramatic jump in localized activity, while retaining a low dark‐state background remains a significant challenge. When placed within the framework of a genetically encodable, light‐activatable heterodimerizer system, the actin‐remodelling protein cofilin induces dramatic changes in the F‐actin network and consequent cell motility upon illumination. We demonstrate that the use of a partially impaired mutant of cofilin is critical for maintaining low background activity in the dark. We also show that light‐directed recruitment of the reduced activity cofilin mutants to the cytoskeleton is sufficient to induce F‐actin remodeling, formation of filopodia, and directed cell motility.     

Microcalorimetric Investigations of K+- and Mg2+-Induced Polymerization of Actin at Temperatures from 293.15 K to 310.15 K

Microcalorimetric measurements of the polymerization of actin in the presence of 100 mM KCl and 2 mM MgCl₂were carried out with a Calvet MS-80 microcalorimeter at temperatures from 293.15 to 310.15 K. It was observed that the polymerization of actin was endothermic and the enthalpy change for actin polymerization was temperature-dependent. The enthalpy change ΔH^owas fitted to ΔH^o(kJ mol^-1)=434.0-1.16 (T/K) and the change in heat capacity ΔC_p ^ocalculated from ΔH^owas -1.16 kJ (mol K)^-1in the above range of temperatures. The direct calorimetry results showed that the enthalpy and entropy change for actin polymerization could not be obtained from measurements of the critical concentration and the only way to assess the enthalpy change for the polymerization of actin and similar reactions lies in the use of calorimetry. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional proteomics,human genetics and cancer biology of GIPC family members

GIPC1, GIPC2 and GIPC3 consist of GIPC homology 1 (GH1) domain, PDZ domain and GH2 domain. The regions around the GH1 and GH2 domains of GIPC1 are involved in dimerization and interaction with myosin VI (MYO6), respectively. The PDZ domain of GIPC1 is involved in interactions with transmembrane proteins [IGF1R, NTRK1, ADRB1, DRD2, TGFβR3 (transforming growth factorβ receptor type III), SDC4, SEMA4C, LRP1, NRP1, GLUT1, integrin α5 and VANGL2], cytosolic signaling regulators (APPL1 and RGS19) and viral proteins (HBc and HPV-18 E6). GIPC1 is an adaptor protein with dimerizing ability that loads PDZ ligands as cargoes for MYO6-dependent endosomal trafficking. GIPC1 is required for cell-surface expression of IGF1R and TGFβR3. GIPC1 is also required for integrin recycling during cell migration, angiogenesis and cytokinesis. On early endosomes, GIPC1 assembles receptor tyrosine kinases (RTKs) and APPL1 for activation of PI3K–AKT signaling, and G protein-coupled receptors (GPCRs) and RGS19 for attenuation of inhibitory Gα signaling. GIPC1 upregulation in breast, ovarian and pancreatic cancers promotes tumor proliferation and invasion, whereas GIPC1 downregulation in cervical cancer with human papillomavirus type 18 infection leads to resistance to cytostatic transforming growth factorβ signaling. GIPC2 is downregulated in acute lymphocytic leukemia owing to epigenetic silencing, while Gipc2 is upregulated in estrogen-induced mammary tumors. Somatic mutations of GIPC2 occur in malignant melanoma, and colorectal and ovarian cancers. Germ-line mutations of the GIPC3 or MYO6 gene cause nonsyndromic hearing loss. As GIPC proteins are involved in trafficking, signaling and recycling of RTKs, GPCRs, integrins and other transmembrane proteins, dysregulation of GIPCs results in human pathologies, such as cancer and hereditary deafness.     

漆树漆酶两种同工酶分离纯化与特性研究

应用柱层析和HPLC方法从漆树漆液中分离提纯漆酶的两种同工酶L1和L2 .两者均为低温酶 ,最适的温度分别为 2 0℃和 13℃ ,pH值分别为 6.7和 7.2 ,等电点分别为 8.6和 9.1.通过十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 ,测得它们的分子量分别为 1.2× 10 5和 1.0 5× 10 5.两种酶均不能催化氧化间氨基苯甲酸 ,对 3 氨基酪氨酸等有很高的反应活性