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1.
K Rippe N B Ramsing R Klement T M Jovin 《Journal of biomolecular structure & dynamics》1990,7(6):1199-1209
DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix. 相似文献
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Tissue-specific expression of the chicken alpha A-crystallin gene in cultured lens epithelia and transgenic mice 总被引:10,自引:0,他引:10
J F Klement E F Wawrousek J Piatigorsky 《The Journal of biological chemistry》1989,264(33):19837-19844
The present experiments show that the single gene for the lens-specific protein alpha A-crystallin of chickens and mice uses a different subset of cis- and trans-acting regulatory elements for expression in transfected embryonic chicken lens epithelial cells. A chicken alpha A-crystallin-chloramphenicol acetyltransferase (CAT) fusion gene required 162 base pairs whereas the murine alpha A-crystallin-CAT fusion gene required only 111 base pairs of 5'-flanking sequences for efficient tissue-specific expression in the transfected chicken lens cells. Gel retardation and competition experiments were performed using embryonic chicken lens nuclear extract and oligodeoxynucleotides identical to the 5'-flanking region of the chicken (-170/-111) and murine (-111/-88 and -88/-55) alpha A-crystallin gene. The results indicated that these homologous promoters use different nuclear factors for function. Methylation interference analysis identified a dyad of symmetry (CTGGTTCCCACCAG) at position -153 to -140 in the chicken alpha A-crystallin promoter which binds one or more lens nuclear factors. Gel mobility shift experiments using nuclear extracts of brain, reticulocytes, and muscle of embryonic chickens or HeLa cells suggested that the factor(s) binding to the chicken alpha A-crystallin gene promoter sequences are not lens specific. Despite differences in the functional and protein-binding properties of the alpha A-crystallin gene promoter of chickens and mice, expression of the chicken alpha A-crystallin-CAT fusion gene in transgenic mice was lens specific, consistent with a common underlying mechanism for expression of the alpha A-crystallin gene in chickens and mice. 相似文献
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Bovine ephemeral fever (BEF) is an economically important viral vector-borne cattle disease. Several live-attenuated, inactivated and recombinant vaccines have been tested, demonstrating varying efficacy. However, to the best of our knowledge, duration of immunity conferred by an inactivated vaccine has never been reported. In the last decade, Israel has faced an increasing number of BEF outbreaks. The need for an effective vaccine compatible with strains circulating in the Middle East region led to the development of a MONTANIDE™ ISA 206 VG (water-in-oil-in-water), inactivated vaccine based on a local strain. We tested the safety, immunogenicity and duration of immunity conferred by this vaccine. The induced neutralizing antibody (NA) response was followed for 493 days in 40 cows vaccinated by different protocols. The vaccine did not cause adverse reactions or a decrease in milk production. All cows [except 2 (6.7%) which did not respond to vaccination] showed a significant rise in NA titer of up to 1:256 following the second, third or fourth booster vaccination. Neutralizing antibody levels declined gradually to 1:16 up to 120 days post vaccination. This decline continued in cows vaccinated only twice, whereas cows vaccinated 3 or 4 times showed stable titers of approximately 1:16 for up to 267 days post vaccination. At least three vaccinations with the inactivated BEF vaccine were needed to confer long-lasting immunity. These results may have significant implications for the choice of vaccination protocol with inactivated BEF vaccines. Complementary challenge data should however be added to the above results in order to determine what is the minimal NA response conferring protection from clinical disease. 相似文献
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Pipecolic acid naturally occurs in microorganisms, plants, and animals, where it plays many roles, including the interactions between these organisms, and is a key constituent of many natural and synthetic bioactive molecules. This article provides a review of current knowledge on the natural occurrence of pipecolic acid and the known and potential significance of its L‐ and D‐enantiomers in different scientific disciplines. Knowledge gaps with perspectives for future research identified within this article include the roles of the L‐ versus the D‐enantiomer of pipecolic acid in plant resistance, nutrient acquisition, and decontamination of polluted soils, as well as rhizosphere ecology and medical issues. Chirality 25:823–831, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
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Varga B Barabás O Kovári J Tóth J Hunyadi-Gulyás E Klement E Medzihradszky KF Tölgyesi F Fidy J Vértessy BG 《FEBS letters》2007,581(24):4783-4788
Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus. 相似文献
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Compelling evidence suggests that the heterotrimeric G protein G(i3) specifically transmits the antiautophagic effects of insulin and amino acids in the liver. This points to a previously unrecognized cross talk between the insulin receptor tyrosine kinase and G(i3). Interestingly, G(i3) is localized not only to plasma membranes but also to membranes of the autophagosomal compartment. Furthermore, as part of insulin's or phenylalanine's actions to inhibit autophagy, G(i3) is redistributed away from autophagosomes. Therefore, endomembrane-associated rather than plasma membrane-localized G(i3) may serve as the target of insulin's endocrine and metabolic actions. We therefore propose that the function and regulation of organelle-associated heterotrimeric G proteins may be different from their roles at the plasma membrane where they act as signal transducers of seven-transmembrane receptors. Here, we discuss recent findings and propose a function for G(i3) in mTOR-dependent signaling pathways. We hypothesize that G(i) family members may have tissue-specific roles in the regulation of autophagy under different physiological and pathological conditions. 相似文献
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Alpha-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant alpha-synuclein (alpha-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of alpha-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of alpha-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized alpha-synuclein-C4 molecules. Alpha-synuclein-C4 offers the means for directly probing amyloid formation and interactions of alpha-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease. 相似文献
10.
Modular broad-host-range expression vectors for single-protein and protein complex purification 总被引:1,自引:0,他引:1
Fodor BD Kovács AT Csáki R Hunyadi-Gulyás E Klement E Maróti G Mészáros LS Medzihradszky KF Rákhely G Kovács KL 《Applied and environmental microbiology》2004,70(2):712-721
A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species. 相似文献