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31.
Subedi GP Satoh T Hanashima S Ikeda A Nakada H Sato R Mizuno M Yuasa N Fujita-Yamaguchi Y Yamaguchi Y 《Protein expression and purification》2012,82(1):197-204
Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed "pCold-PDI" vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains. 相似文献
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CS Bedetti NC Jorge FCG Trigueiro GP Bragança LV Modolo 《Biotechnic & histochemistry》2018,93(2):149-154
We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones. 相似文献
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Yagya Prasad Subedi Madher N. Alfindee Jaya P. Shrestha Greg Becker Michelle Grilley Jon Y. Takemoto Cheng-Wei Tom Chang 《Bioorganic & medicinal chemistry letters》2018,28(18):3034-3037
Phosphonates, azoles and quinones are pharmacophores found in bioactive compounds. A series of phosphonates conjugated to azoles and quinones with variable carbon chain lengths were synthesized in 3–4 steps with good yield. Antifungal assay of these compounds showed that ethyl protected phosphates have excellent inhibitory activity against phytopathogenic fungus Fusarium graminearum, and the free-base phosphates have good activity against human pathogenic fungi Aspergillus flavus and Candida albicans. Structure- activity relationship (SAR) studies showed activity increases with longer carbon chain length between phosphonate and anthraquinone analogs consisting of azole and quinone moieties. These newly synthesized compounds also have mild antibacterial activities to Gram positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytotoxicity analysis of these compounds against HeLa cells reveals that the phosphoric acid analogs are less toxic compared to ethyl protected phosphonates. Three leads compounds have been identified with prominent antifungal activity and low cytotoxicity. 相似文献
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Response of wheat genotypes to sowing date and boron fertilization aimed at controlling sterility in a rice-wheat rotation in Nepal 总被引:2,自引:0,他引:2
Spikelet sterility in wheat (Triticum aestivum L.) is emerging as a production threat in different parts of Nepal. This study was aimed at determining the effects of sowing date and boron application in controlling spikelet sterility in four different genotypes of spring wheat in a rice-wheat system in the western hills of Nepal. Four genotypes of known different responses to boron were planted on 21 November, 6 December and 21 December, 1994 with or without boron application at 1 kg B ha-1 (i.e. 9 kg borax ha-1) on a soil that was known to be deficient in boron.The effect of sowing date was significant for the phenology, yield components, percentage sterility and grain yield. Sterility was significantly increased in the crop planted on 21 December, which had also the lowest 1000 seed weight and grain yield; there was an almost 50% grain yield reduction compared to the crop planted on 21 November. Terminal moisture stress (i.e. lack of moisture during the later part of the development) was observed in the late sown crop which also amplified the extent of sterility associated with boron deficiency. Genotypes differed in response to sowing dates and boron treatment for all of the phenological events measured, yield components, grain yield and percentage sterility. SW-41 and BL-1022 had significantly higher sterility at all sowing dates. BL-1249 showed a consistently lower% sterility over all sowing dates and boron treatments. The addition of boron significantly increased the number of grains set per spike thereby decreasing the total sterility in boron responsive genotypes SW-41 and BL-1022 while those not susceptible did not respond. The boron concentration in the flag leaf at anthesis was increased in treatments with added B in the soil but genotypes did not differ in boron concentration for any soil treatment. 相似文献
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In vertebrates and the cephalochordate, amphioxus, the closest vertebrate
relative, Hox genes are linked in a single cluster. Accompanying the
emergence of higher vertebrates, the Hox gene cluster duplicated in either
a single step or multiple steps, resulting in the four-cluster state
present in teleosts and tetrapods. Mammalian Hox clusters (designated A, B,
C, and D) extend over 100 kb and are located on four different chromosomes.
Reconstructing the history of the duplications and its relation to
vertebrate evolution has been problematic due to the lack of alignable
sequence information. In this study, the problem was approached by
conducting a statistical analysis of sequences from the fibrillar-type
collagens (I, II, III, and IV), genes closely linked to each Hox cluster
which likely share the same duplication history as the Hox genes. We find
statistical support for the hypothesis that the cluster duplication
occurred as multiple distinct events and that the four-cluster situation
arose by a three- step sequential process.
相似文献
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The alcohol dehydrogenase (Adh) gene was isolated from Drosophila simulans
and D. mauritiana, and the DNA sequence of a 4.6-kb region, containing the
structural gene and flanking sequence, was determined for each. These
sequences were compared with the Adh region of D. melanogaster to
characterize changes that occur in the Drosophila genome during evolution
and to identify conserved sequences of functional importance. Drosophila
simulans and D. mauritiana Adh are organized in a manner similar to that of
D. melanogaster Adh, including the presence of two promoters for the single
Adh gene. This study identified conserved flanking elements that, in
conjunction with other studies, suggest regions that may be involved in the
control of Adh expression. Inter- and intraspecies comparisons revealed
differences in the kinds of sequence changes that have accumulated.
Sequence divergence in and around the Adh gene was used to assess inter-
and intraspecies evolutionary relationships. Finally, there appears to be
an unrelated structural gene located directly 3' of the Adh transcribed
region.
相似文献
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Central CCL2 signaling onto MCH neurons mediates metabolic and behavioral adaptation to inflammation 下载免费PDF全文
Ophélia Le Thuc Céline Cansell Miled Bourourou Raphaël GP Denis Katharina Stobbe Nadège Devaux Alice Guyon Julie Cazareth Catherine Heurteaux William Rostène Serge Luquet Jean‐Louis Nahon Carole Rovère 《EMBO reports》2016,17(12):1738-1752
Sickness behavior defines the endocrine, autonomic, behavioral, and metabolic responses associated with infection. While inflammatory responses were suggested to be instrumental in the loss of appetite and body weight, the molecular underpinning remains unknown. Here, we show that systemic or central lipopolysaccharide (LPS) injection results in specific hypothalamic changes characterized by a precocious increase in the chemokine ligand 2 (CCL2) followed by an increase in pro‐inflammatory cytokines and a decrease in the orexigenic neuropeptide melanin‐concentrating hormone (MCH). We therefore hypothesized that CCL2 could be the central relay for the loss in body weight induced by the inflammatory signal LPS. We find that central delivery of CCL2 promotes neuroinflammation and the decrease in MCH and body weight. MCH neurons express CCL2 receptor and respond to CCL2 by decreasing both electrical activity and MCH release. Pharmacological or genetic inhibition of CCL2 signaling opposes the response to LPS at both molecular and physiologic levels. We conclude that CCL2 signaling onto MCH neurons represents a core mechanism that relays peripheral inflammation to sickness behavior. 相似文献
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