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61.
Goel A Beresford GW Colcher D Pavlinkova G Booth BJ Baranowska-Kortylewicz J Batra SK 《Journal of biochemistry》2000,127(5):829-836
Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications. 相似文献
62.
William E. Dale Peter S. Batra Edward H. Blaine 《In vitro cellular & developmental biology. Animal》1998,34(10):805-812
Summary We have used in vivo balloon catheterization in combination with in vitro organ culture to develop a model system for vascular neointima formation. A Fogarty balloon catheter was used to deendothelialize
and rupture the internal elastic lamina of aortae in adult rabbits. After three d of recovery, aortae were harvested, divided
into segments, and placed into organ culture. We obtained a daily index of cell proliferation in cultured vessels using [3H]thymidine incorporation into DNA. Also, segments were collected and processed for routine histology or immunohistochemistry.
Aortic segments that had undergone ballooning 3 d before harvest and then cultured exhibited diffuse neointimal growth after
several d in vitro, whereas those from sham-operated (nonballooned) rabbits showed generally only a single endothelial cell layer that is characteristic
of normal intima. Aortae that were harvested, balloon-damaged in vitro, and then cultured exhibited no neointimal growth. The neointima that developed in cultured segments from in vivo ballooned rabbits was primarily of smooth muscle cell origin as determined by positive immunostaining for α-smooth muscle
actin. The intima:media thickness ratios were significantly higher in aortic segments from ballooned rabbits at harvest and
after 4 or 7 d in culture compared with those from nonballooned rabbits. Also, the [3H]thymidine index was higher in the in vivo ballooned aorta compared to non-ballooned or in vitro ballooned vessel. We conclude that ballooning in vivo followed by exposure to blood-borne elements produces an enhanced proliferative response in cultured vessels that is distinct
from other in vitro models of neointimal growth. 相似文献
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65.
Saara Laulumaa Tuomo Nieminen Mari Lehtim?ki Shweta Aggarwal Mikael Simons Michael M. Koza Ilpo Vattulainen Petri Kursula Francesca Natali 《PloS one》2015,10(6)
Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations. 相似文献
66.
Shweta Tripathi Guangshun Wang Mitchell White Li Qi Jeffery Taubenberger Kevan L. Hartshorn 《PloS one》2015,10(4)
Human LL-37, a cationic antimicrobial peptide, was recently shown to have antiviral activity against influenza A virus (IAV) strains in vitro and in vivo. In this study we compared the anti-influenza activity of LL-37 with that of several fragments derived from LL-37. We first tested the peptides against a seasonal H3N2 strain and the mouse adapted H1N1 strain, PR-8. The N-terminal fragment, LL-23, had slight neutralizing activity against these strains. In LL-23V9 serine 9 is substituted by valine creating a continuous hydrophobic surface. LL-23V9 has been shown to have increased anti-bacterial activity compared to LL-23 and we now show slightly increased antiviral activity compared to LL-23 as well. The short central fragments, FK-13 and KR-12, which have anti-bacterial activity did not inhibit IAV. In contrast, a longer 20 amino acid central fragment of LL-37 (GI-20) had neutralizing activity similar to LL-37. None of the peptides inhibited viral hemagglutination or neuraminidase activity. We next tested activity of the peptides against a strain of pandemic H1N1 of 2009 (A/California/04/09/H1N1 or “Cal09”). Unexpectedly, LL-37 had markedly reduced activity against Cal09 using several cell types and assays of antiviral activity. A mutant viral strain containing just the hemagglutinin (HA) of 2009 pandemic H1N1 was inhibited by LL-37, suggested that genes other than the HA are involved in the resistance of pH1N1. In contrast, GI-20 did inhibit Cal09. In conclusion, the central helix of LL-37 incorporated in GI-20 appears to be required for optimal antiviral activity. The finding that GI-20 inhibits Cal09 suggests that it may be possible to engineer derivatives of LL-37 with improved antiviral properties. 相似文献
67.
68.
Alok Ranjan Singh Shweta Joshi Rahul Arya Arvind Mohan Kayastha Jitendra Kumar Saxena 《European biophysics journal : EBJ》2010,39(2):289-297
Guanidine hydrochloride and urea-induced unfolding of B. malayi hexokinase (BmHk), a tetrameric protein, was examined in detail by using various optical spectroscopic techniques, enzymatic
activity measurements, and size-exclusion chromatography. The equilibrium unfolding of BmHk by guanidine hydrochloride (GdmCl)
and urea proceeded through stabilization of several unique oligomeric intermediates. In the presence of low concentrations
of GdmCl, stabilization of an enzymatically active folded dimer of BmHk was observed. However an enzymatically inactive dimer
of BmHk was observed for urea-treated BmHk. This is the first report of an enzymatically active dimer of hexokinase from any
human filarial parasite. Furthermore, although complete recovery of the native enzyme was observed on refolding of BmHk samples
denatured by use of low concentrations of GdmCl or urea, no recovery of the native enzyme was observed for BmHk samples denatured
by use of high concentrations of GdmCl or urea. 相似文献
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70.
Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the
same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification
is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex
PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification
control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target
PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able
to detect as low as 101–102 organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone
water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based
dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous,
rapid and reliable detection of enterotoxic B. cereus group organisms. 相似文献