G蛋白亲和色谱法纯化动物血清、抗体效果比较

比较 Hitrap G蛋白 Sepharose亲和色谱系统对不同动物血清或腹水的纯化效果 ,为抗体纯化提供依据。结果显示 ,G蛋白对不同动物血清 Ig G吸附能力不同 ,体现在单位体积抗体回收量明显不同 ,这一特点与 A蛋白亲和色谱系统相似。该方法简便快速 ,纯化抗体纯度高 ,免疫活性好 ,色谱柱可反复使用多次     

Hypoxia-induced acute lung injury in murine models of sickle cell disease

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.     

乳酸氧化酶研究进展

乳酸氧化酶能够氧化乳酸生成丙酮酸,由于反应过程中不需要外源辅酶作为电子受体,而具有较好的应用前景。乳酸氧化酶和许多黄素蛋白酶相比较,具有明显的共性,因此可视为黄素蛋白家族中的一员。乳酸单加氧酶的催化机理和乳酸氧化酶相似,但产物不同,这主要是由于中间产物复合体稳定性的差别。乳酸单加氧酶催化形成的中间复合体EFMNH2pyruvate很稳定,在氧的作用下,生成EFMNpyruvateH2O2中间体,继续反应形成乙酸,CO2和H2O;乳酸氧化酶生成的EFMNH2pyruvate复合体不稳定,丙酮酸很快从复合体上分离下来,还原型中间体EFMNH2被氧氧化,同时形成过氧化氢。     

The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1

Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.     

Highly branched HK peptides are effective carriers of siRNA

BACKGROUND: Both viral and nonviral carriers have been used to carry small interfering RNA molecules (siRNA) to their cytosolic mRNA target. To date, few peptide carriers have been developed that have proved effective for siRNA delivery. Our previous branched carriers composed of histidine and lysine were useful for transfection of plasmids. In this study, we determined if these and more highly branched HK polymers were effective carriers of siRNA. METHODS: Several branched polymers were synthesized on a Ranin Voyager synthesizer. These polymers were then screened for their ability to transfer siRNA into SVR-bag4 cells, MDA-MB-435 cells, and C6 cells. After one polymer, H3K8b, was identified as an effective carrier of siRNA, additional polymers were synthesized to determine the essential domains for siRNA transport. The size/zeta-potential of HK : siRNA complexes were measured with the N4 submicron particle size analyzer and the Delsa 440 SX zeta-potential analyzer, respectively. Toxicity of the highly branched polymers in complex with siRNA was investigated by flow cytometry. RESULTS: In an endothelial cell line (SVR-bag4) that stably expressed beta-galactosidase (beta-gal), an siRNA in complex with the H3K8b polymer inhibited beta-gal expression by more than 80%. In contrast, the polymer H2K4b, which was an effective carrier of plasmids, was not an efficient carrier of siRNA. The size and surface charge did not distinguish effective from ineffective HK carriers of siRNA. By modifying H3K8b, we then determined what properties of H3K8b augmented siRNA delivery. The histidine-rich domain and the length of the terminal arms of H3K8 were important for siRNA delivery. The modestly more effective analog of H3K8b containing an integrin ligand, H3K8b(+RGD), was able to inhibit markedly intracellular beta-gal expression. Furthermore, we determined that H3K8b(+RGD) in complex with a luciferase-targeting siRNA inhibited luciferase expression in MDA-MB-435 cells. At its optimal concentration for inhibiting its target, H3K8b(+RGD) : siRNA complex had minimal toxicity. In contrast, carriers of siRNA such as Oligofectamine and Lipofectamine 2000 were significantly more toxic. CONCLUSIONS: Both the degree of complexity and the sequence specificity are important factors to be considered for developing the HK carrier of siRNA. In particular, we found that certain branched HK polymers (H3K8b, H3K8b(+RGD), and similar structural analogs) with eight terminal branches and a histidine-rich domain were effective carriers of siRNA.     

Calcium binding sequences in calmyrin regulates interaction with presenilin-2

Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.     

Thermodynamics of microbial growth and metabolism: an analysis of the current situation

This paper attempts to review in how far thermodynamic analysis can be used to understand and predict the performance of microorganisms with respect to growth and bio-product synthesis. In the first part, a simple thermodynamic model of microbial growth is developed which explains the relationship between the driving force for growth in terms of Gibbs energy dissipation and biomass yield. From the currently available literature, it appears that the Gibbs energy dissipation per C-mol of biomass grown, which represents the driving force for chemotrophic growth, may have been adapted by evolutionary processes to strike a reasonable compromise between metabolic rate and growth efficiency. Based on empirical correlations of the C-molar Gibbs energy dissipation, the wide variety of biomass yields observed in nature can be explained and roughly predicted. This type of analysis may be highly useful in environmental applications, where such wide variations occur. It is however not able to predict biomass yields in very complex systems such as mammalian cells nor is it able to predict or to assess bio-product or recombinant protein yields. For this purpose, a much more sophisticated treatment that accounts for individual metabolic pathways separately is required. Based on glycolysis as a test example, it is shown in the last part that simple thermodynamic analysis leads to erroneous conclusions even in well-known, simple cases. Potential sources for errors have been analyzed and can be used to identify the most important needs for future research.     

The HARP-like Domain-Containing Protein AH2/ZRANB3 Binds to PCNA and Participates in Cellular Response to Replication Stress

Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in?vitro and its function in?vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrates the in?vivo functions of AH2, but also reveals a common feature of this new subfamily of proteins with annealing activity.     

Altered leaf colour is associated with increased superoxide‐scavenging activity in aureusidin‐producing transgenic plants

The health‐promoting property of diets rich in fruits and vegetables is based, in part, on the additive and synergistic effects of multiple antioxidants. In an attempt to further enhance food quality, we introduced into crops the capability to synthesize a yellow antioxidant, aureusidin, that is normally produced only by some ornamental plants. For this purpose, the snapdragon (Antirrhinum majus) chalcone 4′‐O‐glucosyltransferase (Am4’CGT) and aureusidin synthase (AmAs1) genes, which catalyse the synthesis of aureusidin from chalcone, were expressed in tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa) plants that displayed a functionally active chalcone/flavanone biosynthetic pathway. Leaves of the resulting transgenic plants developed a yellow hue and displayed higher superoxide dismutase (SOD) inhibiting and oxygen radical absorbance capacity (ORAC) activities than control leaves. Our results suggest that the nutritional qualities of leafy vegetables can be enhanced through the introduction of aurone biosynthetic pathways.