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1.
The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA', which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules. 相似文献
2.
Cardiac glycoside transport was investigated on the organ and whole plant level. Uptake experiments were carried out with shoot and root cultures of Digitalis lanata. In both systems primary cardenolides, i.e., those with a terminal glucose in their oligosaccharide side chain, were taken up against their concentration gradient, whereas the glucose-free secondary cardenolides were not. Active uptake of primary cardenolides was further evidenced by KCN inhibition of uptake. Using plantlets grown in vitro the long-distance transport of primary cardenolides from the leaves to the roots was demonstrated. Cardenolides were also detected in etiolated leaves, induced on plants with green leaves, which are supposed to be unable to synthezise cardenolides de novo, providing further evidence for long-distance transport. Several primary cardenolides were detected in the honeydew excreted by aphids fed on Digitalis lanata leaves, indicating that the phloem is a transporting tissue for cardenolides. On the other hand, the xylem sap obtained by applying the pressure-chamber technique was cardenolide-free. It was concluded that in Digitalis primary cardenolides serve as both the transport and the storage form of cardenolides. After their synthesis they are either stored in the vacuoles of the source tissue or loaded into the sieve tubes, from which they are unloaded at other sites where they are trapped in the vacuoles of the respective sink tissue. 相似文献
3.
Alexander Christmann Jacqueline Christmann Petra Schiller Burkhard Frenzel 《Trees - Structure and Function》1996,10(5):331-338
Levels of indole-3-acetic acid (IAA) were determined in needles from silver fir (Abies alba Mill.) trees in the northern Black Forest. IAA was quantified by gas chromatography (GC) as 1-heptafluorobutyryl-IAA-methylester
(HFB-IAA-ME) using electron capture detection. Prior to GC analysis, extensive purification of needle extracts was performed
employing two HPLC steps. Peak identity of HFB-IAA-ME was confirmed by combined gas chromatography-mass spectrometry in selected
samples. Levels of IAA in needles belonging to different needle age-classes exhibited a cyclic seasonal pattern with highest
concentrations in winter and lowest levels in spring when bud-break occurred. Such a cyclic seasonal pattern of IAA levels
was also observed in needles from declining fir trees or fir trees suffering from a strong sulfur impact (S-impact) in the
field due to a local SO2 source. Levels of IAA increased with increasing needle age. This age dependency of IAA concentrations was most pronounced
in late autumn when IAA levels were high and nearly disappeared in spring when IAA levels reached their minimum. In needles
from declining fir trees or fir trees suffering from a strong S-impact in the field, IAA levels hardly increased with increasing
needle age. It is suggested that in healthy trees high levels of IAA protect older needles from abscission and that the considerable
losses of older needles of declining fir trees or of fir trees under S-impact are a consequence of the low levels of IAA found
in older needles of such trees.
Received: 4 May 1995 / Accepted: 29 August 1995 相似文献
4.
Alexander Akermann Jens Weiermüller Jens Christmann La Guirande Gregor Glaser Annette Knaus Roland Ulber 《Engineering in Life Science》2020,20(5-6):168-180
Brewers’ spent grain (BSG) is a low‐cost by‐product of the brewing process. BSG liquor names the liquid components of BSG, mainly glucose, maltose, and long‐chain α‐1,4‐glycosidic bond glucose oligomers. These substances should be separated in existing BSG biorefineries, as they might lead to an increased formation of microbe‐inhibiting compounds in well‐established hydrothermal/enzymatic saccharification processes. In most cases, this liquid fraction is discarded. The present study presents for the first time an optimized process with BSG liquor for the purpose of producing bulk chemicals (e.g., lactate) in relevant concentrations. The process comprises the application of yeast extract, produced from own brewing processes, as the sole supplemented complex constituent in a simultaneous fermentation and saccharification process. Kinetic parameters for the final optimized process conditions with the organism Lactobacillus delbrueckii subsp. lactis were: maximum specific growth rate µmax = 0.47 h?1, maximum lactate concentration cLac, max = 79.06 g L?1, process yield YPS = 0.89 gLac gSugar?1, lactate production rate qP = 4.18 gLac gCDW?1 h?1, and productivity P 15 h = 4.93 gLac L?1 h?1. BSG liquor, linked with yeast extract from Brewers’ yeast, can be a promising substrate for further bioprocess engineering tasks and contribute to a holistic and sustainable usage of Brewers’ spent grain. 相似文献
5.
BS Sabna Thankappan Bency Mahendran Ramasamy Muthusamy Gayathri Femil selta Daniel Raja Angayarkanni Jayaraman 《Probiotics and antimicrobial proteins》2021,13(4):993-1004
Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of... 相似文献
6.
MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents 总被引:4,自引:0,他引:4
O(6)-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against alkylating agents that generate, among other lesions, O(6)-alkylguanine in DNA (collectively termed O(6)-alkylating agents [O(6)AA]). The defense is highly important, since O(6)AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O(6)AA induced DNA damage is subject to repair, which is executed by MGMT, AlkB homologous proteins (ABH) and base excision repair (BER). Although this review focuses on MGMT, the mechanism of repair by ABH and BER will also be discussed. Experimental systems, in which MGMT has been modulated, revealed that O(6)-methylguanine (O(6)MeG) and O(6)-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O(6)MeG-induced clastogenicity and cell death require MutS alpha-dependent mismatch repair (MMR), whereas O(6)-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O(6)MeG to provoke cytotoxicity. In MGMT depleted cells, O(6)MeG induces apoptosis almost exclusively, barely any necrosis, which is presumably due to the remarkable ability of secondarily formed DNA double-strand breaks (DSBs) to trigger apoptosis via ATM/ATR, Chk1, Chk2, p53 and p73. Depending on the cellular background, O(6)MeG activates both the death receptor and the mitochondrial apoptotic pathway. The inter-individual expression of MGMT in human lymphocytes is highly variable. Given the key role of MGMT in cellular defense, determination of MGMT activity could be useful for assessing a patient's drug sensitivity. MGMT is expressed at highly variable amounts in human tumors. In gliomas, a correlation was found between MGMT activity, MGMT promoter methylation and response to O(6)AA. Although the human MGMT gene is inducible by glucocorticoids and genotoxins such as radiation and alkylating agents, the role of this induction in the protection against carcinogens and the development of chemotherapeutic alkylating drug resistance are still unclear. Modulation of MGMT expression in tumors and normal tissue is currently being investigated as a possible strategy for improving cancer therapy. 相似文献
7.
Extracts of mature silver fir (Abies alba Mill.) needles, current-year, and one-year old (A) and seven to nine-year old (B), were purified by reversed and normal phase HPLC. Gibberellin (GA)-like compounds were detected by the Tan-ginbozu dwarf rice micro-drop bioassay and corresponding fractions were analyzed by GC-MS. GA9 was present in small amounts, while a major component was a cellulase-hydrolysable GA9 conjugate which was assumed to be GA9 glucosyl ester. It is proposed that GA9 glucosyl ester plays a key role in the regulation of endogenous GA levels in silver fir needles. 相似文献
8.
Anderson O. Lobo Erica F. Antunes Mariana BS Palma Cristina Pacheco‐Soares Vladimir J. Trava‐Airoldi Evaldo J. Corat 《Cell biology international》2010,34(4):393-398
Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications. 相似文献
9.
10.