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11.
Yongbo Hu Erin Webb Jasbir Singh Barry A Morgan James A Gainor Thomas D Gordon Teruna J Siahaan 《The Journal of biological chemistry》2002,277(10):8366-8371
The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest. 相似文献
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13.
Promotion of indole alkaloid production in Catharanthus roseus cell cultures by rare earth elements 总被引:3,自引:0,他引:3
Production of the indole alkaloids, ajmalicine or catharanthine, in cell suspension cultures of Catharanthus roseus was enhanced by cerium (CeO2 and CeCl3), yttrium (Y2O3) and neodymium (NdCl3). The yield of ajmalicine in these treated-cultures reached 51 mg l–1 (CeO2), 40 mg l–1 (CeCl3), 41 mg l–1 (Y2O3) and 49 mg l–1 (NdCl3) while catharanthine production reached to 36 mg l–1 (CeO2) and 31 mg l–1 (CeCl3). A major portion of increased alkaloids was released into medium in these treatments. But Sm2O3, SmCl3, La2O3, LaCl3, complex of chromium (III)-titanium (IV) and NaSeO4 treatments had little effect on alkaloid production of C. roseus cell cultures. 相似文献
14.
Laixing Hu Maureen L. Kully David W. Boykin Norman Abood 《Bioorganic & medicinal chemistry letters》2009,19(5):1292-1295
A new class of novel bis-benzimidazole diamidine compounds have been synthesized and evaluated for in vitro antibacterial activities, including drug-resistant bacterial strains. Anti-MRSA and anti-VRE activities of the most potent compound 1 were more active than Vancomycin. The mechanism of action for this class of compounds appears to be different from existing antibiotics. Bis-benzimidazole diamidine compounds have potential for further investigation as a new class of potent anti-MRSA and anti-VRE agents. 相似文献
15.
Yan-yiu Yu Chelsea Bahney Diane Hu Ralph S. Marcucio Theodore Miclau III 《Journal of visualized experiments : JoVE》2012,(62)
Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice. 相似文献
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18.
Zac Yates Kannan Gunasekaran Hongxing Zhou Zhonghua Hu Zhi Liu Randal R. Ketchem Boxu Yan 《The Journal of biological chemistry》2010,285(24):18662-18671
Hydroxyl radicals induce hinge cleavage in a human IgG1 molecule via initial radical formation at the first hinge Cys231 followed by electron transfer to the upper hinge residues. To enable engineering of a stable monoclonal antibody hinge, we investigated the role of the hinge His229 residue using structure modeling and site-directed mutagenesis. Direct involvement of His229 in the reaction mechanism is suggested by a 75–85% reduction of the hinge cleavage for variants in which His229 was substituted with either Gln, Ser, or Ala. In contrast, mutation of Lys227 to Gln, Ser, or Ala increased hinge cleavage. However, the H229S/K227S double mutant shows hinge cleavage levels similar to that of the single H229S variant, further revealing the importance of His229. Examination of the hinge structure shows that His229 is capable of forming hydrogen bonds with surrounding residues. These observations led us to hypothesize that the imidazole ring of His229 may function to facilitate the cleavage by forming a transient radical center that is capable of extracting a proton from neighboring residues. The work presented here suggests the feasibility of engineering a new generation of monoclonal antibodies capable of resisting hinge cleavage to improve product stability and efficacy. 相似文献
19.
The MIND multiprotein complex is a conserved, essential component of eukaryotic kinetochores and is a constituent of the tripartite KMN network that directly attaches the kinetochore to the mitotic spindle. The primary microtubule-binding complex in this network, NDC80, has been extensively characterized, but very little is known about the structure or function of the MIND complex. In this study, we present biochemical, hydrodynamic, electron microscopy, and small-angle x-ray scattering data that provide insight into the overall architecture and assembly of the MIND complex and the physical relationship of the complex with other components of the KMN network. We propose a model for the overall structure of the complex and provide data on the interactions with NDC80, Spc105p, and thus the mitotic spindle. 相似文献
20.
Houchun H. Hu Hee‐Won Kim Krishna S. Nayak Michael I. Goran 《Obesity (Silver Spring, Md.)》2010,18(4):841-847
The ability to accurately and noninvasively quantify fatty infiltration in organs such as the liver and the pancreas remains a critical component in understanding the link between obesity and its comorbidities such as type 2 diabetes and fatty liver disease. Single‐voxel (1H) proton magnetic resonance spectroscopy (MRS) has long been regarded as the gold‐standard noninvasive technique for such measurements. Recent advances in three‐dimensional fat–water magnetic resonance imaging (MRI) methods have led to the development of rapid, robust, and quantitative approaches that can accurately characterize the proportion of fat and water content in underlying tissues across the full imaging volume, and hence entire organs of interest. One such technique is called IDEAL (Iterative Decomposition with Echo Asymmetry and Least squares estimation). This article prospectively compares three‐dimensional (3D) IDEAL‐MRI and single‐voxel MRS in the assessment of hepatic (HFF) and pancreatic fat fraction (PFF) in 16 healthy subjects. MRS acquisitions took 3–4 min to complete whereas IDEAL acquisitions were completed in 20‐s breath‐holds. In the liver, there was a strong correlation (slope = 0.90, r2 = 0.95, P < 0.001) between IDEAL and MRS‐based fat fractions. In the pancreas, a poorer agreement between IDEAL and MRS was observed (slope = 0.32, r2 = 0.51, P < 0.02). The discrepancy of PFF is likely explained by MRS signal contamination from surrounding visceral fat, presumably during respiratory motion. We conclude that IDEAL is equally accurate in characterizing hepatic fat content as MRS, and is potentially better suited for fat quantification in smaller organs such as the pancreas. 相似文献