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排序方式: 共有590条查询结果,搜索用时 31 毫秒
91.
92.
O P Kuipers J Kerver J van Meersbergen R Vis R Dijkman H M Verheij G H de Haas 《Protein engineering》1990,3(7):599-603
Residue 31 of porcine pancreatic phospholipase A2 (PLA2) is located at the entrance to the active site. To study the role of residue 31 in PLA2, six mutant enzymes were produced by site-directed mutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Ser or Gly. Direct binding studies indicated a three to six times greater affinity of the Trp31 PLA2 for both monomeric and micellar substrate analogs, relative to the wild-type enzyme. The other five mutants possess an unchanged affinity for monomers of the product analog n-decylphosphocholine and for micelles of the diacyl substrate analog rac-1,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine. The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholine were decreased 9-20 times for these five mutants. Kinetic studies with monomeric substrates showed that the mutants have Vmax values which range between 15 and 70% relative to the wild-type enzyme. The Vmax values for micelles of the zwitterionic substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3-50 times. The Km values for the monomeric substrate and the Km values for the micellar substrate were hardly affected in the case of five of the six mutants, but were considerably decreased when Trp was present at position 31. The results of these investigations point to a versatile role for the residue at position 31: involvement in the binding and orientating of monomeric substrate (analogs), involvement in the binding of the enzyme to micellar substrate analogs and possibly involvement in shielding the active site from excess water. 相似文献
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96.
Dongdong Mu Manuel Montalbán-López Yoshimitsu Masuda Oscar P. Kuipers 《Applied and environmental microbiology》2013,79(14):4503-4508
Here, we report a new zinc-inducible expression system for Lactococcus lactis, called Zirex, consisting of the pneumococcal repressor SczA and PczcD. PczcD tightly regulates the expression of green fluorescent protein in L. lactis. We show the applicability of Zirex together with the nisin-controlled expression system, enabling simultaneous but independent regulation of different genes. 相似文献
97.
Characterization and localization of repetitive DNA sequences in the ornamental Alstroemeria aurea Graham 总被引:1,自引:0,他引:1
M. J. De Jeu J. Lasschuit A. G. J. Kuipers S. A. Kamstra R. G. F. Visser 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,94(8):982-990
Three repetitive DNA sequences were isolated from a genomic DNA library of the ornamental Alstroemeria aurea Graham. Two repeats, A001-I and A001-II, were quite homologous and highly A. aurea-specific. A001-I was a 217-bp sequence with several telomeric TTTAGGG repeats at the 5′ end and a unique sequence of 98 bp
at the other end. The third repeat, A001-IV, was a 840-bp sequence which contained two sub-sequences of 56 and 74 bp respectively,
previously found in chloroplast (cp) DNA of tobacco and spinach and to a lesser extent in the cpDNA of maize and rice. Repeat
A001-IV was not species-specific and its hybridization signal was weaker than the other repeats. Fluorescence in situ hybridization
(FISH) revealed the A. aurea-specific repeats to be located in the heterochromatic regions of all A. aurea chromosomes. The differences in FISH pattern make them useful tools for karyotype analysis. The non-species-specific sequence
A001-IV gave a dispersed signal over all the Alstroemeria chromosomes in an interspecific hybrid. The potential use of these repetitive DNA sequences for the study of phylogenetic
relationships within the genus Alstroemeria is discussed.
Received: 24 November 1996/Accepted: 20 December 1996 相似文献
98.
Nathan T Kuipers Charity L Sauder Chester A Ray 《Journal of applied physiology》2007,102(4):1410-1415
Static and pulsed magnetic fields have been reported to have a variety of physiological effects. However, the effect of static magnetic fields on pain perception and sympathetic function is equivocal. To address this question, we measured pain perception during reproducible noxious stimuli during acute exposure to static magnets. Pain perception, muscle sympathetic nerve activity, mean arterial pressure, heart rate, and forearm blood velocity were measured during rest, isometric handgrip, postexercise muscle ischemia, and cold pressor test during magnet and placebo exposure in 15 subjects (25 +/- 1 yr; 8 men and 7 women) following 1 h of exposure. During magnet exposure, subjects were placed on a mattress with 95 evenly spaced 0.06-T magnets imbedded in it. During placebo exposure, subjects were placed on an identical mattress without magnets. The order of the two exposure conditions was randomized. At rest, no significant differences were noted in muscle sympathetic nerve activity (8 +/- 1 and 7 +/- 1 bursts/min for magnet and placebo, respectively), mean arterial pressure (91 +/- 3 and 93 +/- 3 mmHg), heart rate (63 +/- 2 and 62 +/- 2 beats/min), and forearm blood velocity (3.0 +/- 0.3 and 2.6 +/- 0.3 cm/s). Magnets did not alter pain perception during the three stimuli. During all interventions, no significant differences between exposure conditions were found in muscle sympathetic nerve activity and hemodynamic measurements. These results indicate that acute exposure to static magnetic fields does not alter pain perception, sympathetic function, and hemodynamics at rest or during noxious stimuli. 相似文献
99.
Charge modification of plasma and milk proteins results in antiviral active compounds. 总被引:5,自引:0,他引:5
P J Swart M C Harmsen M E Kuipers A A Van Dijk B W Van Der Strate P H Van Berkel J H Nuijens C Smit M Witvrouw E De Clercq M P de Béthune R Pauwels D K Meijer 《Journal of peptide science》1999,5(12):563-576
Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti-HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available epsilonNH2 groups were anionized yielded the most potent anti-HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti-HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120-CD4 mediated virus/cell binding. The relative contribution of these effects to the anti-HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti-HIV activity but a clearly increased anti-HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti-HIV and anti-HCMV activity, respectively. The N-terminal portion of Lf appeared essential for its anti-HCMV effect: N-terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications. 相似文献
100.