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51.
Synthesis and in vitro bioactivity of C-terminal deleted analogs of human growth hormone-releasing factor 总被引:4,自引:0,他引:4
N Ling A Baird W B Wehrenberg N Ueno T Munegumi P Brazeau 《Biochemical and biophysical research communications》1984,123(2):854-861
A series of C-terminal deleted analogs of human growth hormone-releasing factor (hGRF) with either an amidated or a free carboxylic acid C-terminus were synthesized by solid phase methodology. Their capacity to release growth hormone was tested on rat anterior pituitary cells in monolayer culture. A gradual decrease of bioactivity down to 23% relative to hGRF was noted when the C-terminal amino acids were deleted to hGRF (1-34)OH. Further deletions, however, did not decrease the bioactivity because the potencies of the fragments, hGRF(1-31)NH2, (1-30)NH2 and (1-29)NH2 remained at about 50% of that of hGRF. Continual deletion of residues to hGRF(1-23)NH2, (1-22)NH2 and (1-21)NH2 still yielded bioactive fragments with full intrinsic activity despite very low potency. Only with the deletion down to hGRF(1-19)NH2 did the bioactivity completely disappear. Thus, together with the data published in a previous paper (1), the minimal biologically active core of hGRF with full intrinsic activity comprises the fragment (3-21). 相似文献
52.
To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen. 相似文献
53.
C. Steven McDaniel Taghi Manshouri M. Zouhair Atassi 《Journal of Protein Chemistry》1987,6(5):455-461
A peptide corresponding to residues 26–41 of α-bungarotoxin, and closed by a disulfide bond between two cysteine residues
at the amino and C terminal ends of the peptide, was synthesized and the monomeric form was purified. The peptide, which represents
the exposed part of the long central loop of the toxin molecule, was examined for binding to acetylcholine receptor. The peptide
was shown by radiometric titrations to bind radiolabeled receptor, and radiolabeled peptide was bound by receptor. The specificity
of the binding was confirmed by inhibition with the parent toxin. A synthetic analog of the peptide in which Trp-28 was replaced
by glycine had very little (10%) of the original activity. Succinylation of the amino groups of the peptide resulted in virtually
complete (98%) loss of the binding activity. These results indicate that a shortened loop peptide corresponding to the region
26–41 of α-bungarotoxin exhibits binding activities mimicking those of the parent molecule. In this region, Trp-28, and one
or both of Lys-26 and Lys-38, are essential contact residues in the binding to receptor. 相似文献
54.
55.
A M O'Brien C O'Fágáin P F Nielsen K G Welinder 《Biotechnology and bioengineering》2001,76(4):277-284
The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 A of the EGNHS crosslinker. 相似文献
56.
Pavel Veprek Jan Jezek Jirí Velek Hatem Tallima Mona Montash Rashika El Ridi 《Journal of peptide science》2004,10(6):350-362
Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail. 相似文献
57.
A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH2= Z-Gly-Phe-NH2>Z-Ser-Leu-NH2>Z-Gly-Leu-NH2>Z-Gly-Gly-NH2. A linear correlation was found between the respective HPLC retention time parameterk for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH2, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.Abbreviations Z
carbobenzoxy
- DEPE
dielaidoylphosphatidylethanolamine
- DSC
differential scanning calorimetry
- HPLC
high pressure liquid chromatography
- CPE
cytopathic effect
To whom correspondence should be addressed. 相似文献
58.
The study was composed of 27 persons that displayed vague symptoms similar to those of the victims of Minamata and Iraq. Skew
distributions of mercury were observed in individual erythrocytes and neutrophil granulocytes when measured by PIXE. Mercury
could not be detected in the platelets. The erythrocytes also displayed lowered concentrations of magnesium and zinc, together
with increased concentrations of calcium and strontium. The neutrophils displayed decreased concentrations of magnesium and
zinc and increased concentrations of calcium, strontium, and iron. The presence of mercury and the altered elemental profiles
in the erythrocytes and the neutrophil granulocytes are suggested as early signs of exposure. 相似文献
59.
A particle-induced X-ray emission (PIXE) analysis method is presented, which allows measurement of eight elements (i.e., K,
Ca, Mn, Fe, Cu, Zn, Se, and Rb) in human brain samples of only a few mg dry weight. The precision and accuracy of the method
were investigated by analyzing animal brain matter with both PIXE and instrumental neutron activation analysis (INAA). The
method was applied to measure the 8 elements in 46 different regions of 3 human brains. The sections analyzed originated from
either the left or the right cerebral hemisphere, brain stem, and cerebellum. For one of the brains, sections were also analyzed
from 26 corresponding regions of both hemispheres. For all elements, similar concentrations were found in the corresponding
areas of the left and right sides of the brain. The concentrations (in μg/g dry weight) of the elements K, Fe, Cu, Zn, Se,
and Rb were consistently higher in cortical structures than in white matter. Deep nuclei and brain stem, which have a mixed
composition, showed intermediate values for K, Zn, Se, and Rb. A hierarchical cluster analysis indicated that the various
brain regions clustered into two large groups, one comprising gray and mixed matter regions and the other, white and mixed
matter brain areas. 相似文献
60.
A. Tyagi J. Hermans J. Steppuhn Ch. Jansson F. Vater R. G. Herrmann 《Molecular & general genetics : MGG》1987,207(2-3):288-293
Summary Several cDNA clones encoding the 33 kDa protein associated with the photosynthetic water oxidation activity of spinach were sequenced. A 1208 bp insert of one of the clones encodes the entire 331 amino acid residues of the precursor protein including 84 amino acids (8.5 kDa) of the amino-terminal transit peptide, 49 bp of the 5 and 111 bp of the 3 untranslated segment of the mRNA. The 3 poly(A) tail starts 19 bp downstream from a putative polyadenylation signal, TATAAA. The hydrophilic mature protein consists of 247 amino acid residues corresponding to an Mr of 26.5 kDa, which is 6.5 kDa smaller than the value determined by SDS-polyacrylamide gel electrophoresis (33–34 kDa), and shows a certain degree of conservation with the putative Mn-complexing active sites of bacterial Mn-dependent superoxide dismutases. The anatomy of the unusually long transit sequence is discussed with regard to current concepts of protein import into and protein routein within the organelle. 相似文献