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131.
Immune response to human influenza virus hemagglutinin expressed in Escherichia coli 总被引:9,自引:0,他引:9
Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine. 相似文献
132.
Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III 总被引:57,自引:0,他引:57
We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway. 相似文献
133.
The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes. 相似文献
134.
Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing 总被引:1,自引:0,他引:1
B E Magun S R Planck L M Matrisian J S Finch 《Biochemical and biophysical research communications》1982,108(1):299-306
When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium 125iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized 125I-labelled molecules revealed processing of all the 125I-EGF species to macromolecules with more acidic isoelectric points. The 125I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure. 相似文献
135.
D. Sömjen G.J. Sömjen A. Harell G.L. Mechanic I. Binderman 《Biochemical and biophysical research communications》1982,106(2):644-651
Cytosol preparations and cells from 6-day old cultured differentiating chick limb-bud mesenchyme, which consist of a high proportion of chondrocytes, were shown to specifically bind 24R,25 dihydroxycholecalciferol. Nuclei from identical cultures also showed specific binding for 24R,25 dihydroxycholecalciferol. On the contrary, similar preparations of limb-bud mesenchyme cells (6-day old cultures) pretreated on day one by 5-bromodesoxyuridine which induced a fibroblast phenotypic expression, failed to show any specific binding for either 24R,25 or 1α,25 dihydroxycholecalciferol. Pronase treatment of the cytosol indicated that the receptor was protein-like in nature. The chromatographic properties of the protein-receptor on diethylaminoethyl cellulose and Sephadex G-100 columns were similar to those of the protein receptor found for 1α,25 dihydroxycholecalciferol. This report is the first demonstration that a cytosol protein receptor for 24R,25 dihydroxycholecalciferol exists in developing skeletal tissue. 24,25 dihydroxyvitamin D3 but not any of the other metabolites was shown to induce DNA synthesis after 24 h by almost two-fold and protein synthesis after 5 h by 240%. These results suggest an important physiological role for 24R,25 dihydroxyvitamin D3 in the development of skeletal tissue. 相似文献
136.
1,6-Diamino-2,5-anhydro-1,6-dideoxy-dl-glucitol dihydrochloride and some derivatives were synthesized from 3,5-di-O-acetyl-1,6-dibromo-1,6-dideoxy-d-mannitol. Introduction of the 2,5-anhydro ring and subsequent replacement of the terminal bromine atoms by azide gave low yields of the diazide; therefore, a reverse reaction-sequence was applied. The azido groups were reduced with hydrogen sulfide-pyridine, and the amino groups formed were methylated by using formaldehyde-formic acid and subsequently treating with borohydride. According to 13C-n.m.r. investigations, the symmetrically substituted, 2,5-anhydroglucitol derivatives are present mainly in the 4T3 [“north” (N) type of twist] conformation, whereas the analogous l-iditol derivatives mainly adopt the 3T4 [“south” (S)] type. The different quaternary salts obtained on methylation of the corresponding 1,6-bis(dimethylamino)derivatives with methyl iodide (aiming at the structure of epi-muscarine) showed no muscarine-like, biological activity. 相似文献
137.
138.
Two formerly-uncharacterized subunits of 9 kDa and 14 kDa were found in spinach PSI complex. The 9 kDa subunit was released upon removal of antenna chlorophyll complex, whereas the 14 kDa subunit was tightly bound to the core complex. We determined the N-terminal amino acid sequence of the 9 kDa, and an internal sequence of the 14 kDa subunit after protease treatment, since the N-terminus of the latter protein was blocked. These partial sequences suggested that both subunits are new PSI components. 相似文献
139.
We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems. 相似文献
140.
The dark-relaxation kinetics of variable fluorescence, Fv, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t1/2 = 1 s) and slow (t1/2 = 7–8 s) phases in fv dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of Fv dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on Fv dark decay indicates that the slow phase develops when a fraction of QA
– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700+ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. Fv rise appears which is prevented by far-red (PSI-absorbed) light. This transient fv rise reflects the accumulation of QA
– in the dark. The observation of this transient Fv rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in Fv dark-decay kinetics represents the reoxidation of QA
– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of QA
– with the donor side of PSII.Abbreviations CCCP
carbonylcyanide m-chlorophenylhydrazone
- FO
initial fluorescence level
- Fv
variable fluorescence
- P700
primary electron donor of PSI
- PSI, II
photosystem I, II
- QA (QA
–) QB (QB
–)
primary and secondary stable electron acceptor of PSII in oxidized (reduced) state
Supported by grant B6.1/88 DST, Govt. of India. 相似文献