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941.
Sanz-González SM Melero-Fernández de Mera R Malek NP Andrés V 《Journal of cellular biochemistry》2006,97(4):735-743
Excessive cellular proliferation is thought to contribute to neointimal lesion development during atherosclerosis and restenosis after angioplasty. Inhibition of cyclin-dependent kinase (CDK) activity by p27 inhibits mammalian cell growth. Mounting evidence indicates that p27 negatively regulates neointimal thickening in animal models of restenosis and atherosclerosis, and its expression in human neointimal lesions is consistent with such a protective role. Cell cycle progression is facilitated by cyclinE/CDK2-dependent phosphorylation of p27 on threonine 187 (T187) during late G1. The purpose of this study was to assess whether this phosphorylation event plays a role during atherosclerosis. To this end, we generated apolipoprotein E-null mice with both p27 alleles replaced by a mutated form non-phosphorylatable at T187 (apoE-/-p27T187A mice) and investigated the kinetics of atheroma development in these animals compared to apoE-/- controls with an intact p27 gene. Fat feeding resulted in comparable level of hypercholesterolemia in both groups of mice. Surprisingly, aortic p27 expression was not increased in fat-fed apoE-/-p27T187A mice compared with apoE-/- controls. Moreover, atheroma size, lesion cellularity, proliferation, and apoptotic rates were undistinguishable in both groups of fat-fed mice. Thus, in contrast to previous studies that highlight the importance of p27 phosphorylation at T187 on the control of p27 expression and function in different tissues and pathophysiological scenarios, our findings demonstrate that this phosphorylation event is not implicated in the control of aortic p27 expression and atheroma progression in hypercholesterolemic mice. 相似文献
942.
Corina Krause Jochen Kirschbaum Günther Jung Hans Brückner 《Journal of peptide science》2006,12(5):321-327
From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C-terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence:Ac-Aib-Ala-Aib-Ala-Aib-Ala(6)-Gln-Aib-Lx(9)-Aib-Gly-Aib(12)-Aib-Pro-Vx(15)-Aib-Vx(17)-Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (alpha-aminoisobutyric acid); Iva (D-isovaline); Lx (L-leucine or L-isoleucine); Vx (L-valine or D-isovaline); Fol (L-phenylalaninol)). 相似文献
943.
A number of studies have examined the structural properties of late folding intermediates of (beta/alpha)8-barrel proteins involved in tryptophan biosynthesis, whereas there is little information available about the early folding events of these proteins. To identify the contiguous polypeptide segments important to the folding of the (beta/alpha)8-barrel protein Escherichia coli N-(5'-phosphoribosyl)anthranilate isomerase, we structurally characterized fragments and circularly permuted forms of the protein. We also simulated thermal unfolding of the protein using molecular dynamics. Our fragmentation experiments demonstrate that the isolated (beta/alpha)(1-4)beta5 fragment is almost as stable as the full-length protein. The far and near-UV CD spectra of this fragment are indicative of native-like secondary and tertiary structures. Structural analysis of the circularly permutated proteins shows that if the protein is cleaved within the two N-terminal betaalpha modules, the amount of secondary structure is unaffected, whereas, when cleaved within the central (beta/alpha)(3-4)beta5 segment, the protein simply cannot fold. An ensemble of the denatured structures produced by thermal unfolding simulations contains a persistent local structure comprised of beta3, beta4 and beta5. The presence of this three-stranded beta-barrel suggests that it may be an important early-stage folding intermediate. Interactions found in (beta/alpha)(3-4)beta5 may be essential for the early events of ePRAI folding if they provide a nucleation site that directs folding. 相似文献
944.
Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species. 相似文献
945.
Steiner JM Yusa F Pompe JA Löffelhardt W 《The Plant journal : for cell and molecular biology》2005,44(4):646-652
The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa. 相似文献
946.
Fei Li Milind Gangal Celina Juliano Elliot Gorfain Susan S Taylor David A Johnson 《Journal of molecular biology》2002,315(3):459-469
While there is no question that ligands can induce large-scale domain movements that narrow (close) the active-site cleft of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK), the results from small-angle X-ray scattering, protein footprinting, and thermostability studies are inconsistent with regard to which ligands induce these movements. This inconsistency suggests a greater complexity of cAPK conformational dynamics than is generally recognized. As an initial step to study this issue in relation to the catalysis, a new method to measure cAPK domain closure was developed, and the state of domain closure and the local segmental flexibility at major steps of the cAPK catalytic cycle were examined with site-directed labeling and fluorescence spectroscopy. To achieve this, a C subunit mutant (F239C/C199A) was engineered that allowed for fluorescein 5-maleimide (donor) conjugation of F239C in the large lobe and tetramethylrhodamine (acceptor) conjugation of C343 in the small lobe. Domain closure was assessed as an increase in the efficiency of energy transfer between donor and acceptor. The anisotropy decay of fluoroscein 5-maleimide, conjugated to a site of cysteine substitution (K81C) in the small lobe of the C subunit was used to assess the local backbone flexibility around the B helix. The effects of substrate/pseudosubstrate (ATP and PKI(5-24)), a fragment of protein kinase inhibitor) and products (ADP and phosphorylated PKS) on domain closure and B helix flexibility were measured. The results show that domain closure is not tightly coupled to the flexibility around K81C. Moreover, although substrates/pseudosubstrate and products independently close the active-site cleft, only the substrates substantially decreased the backbone flexibility around the B helix. Because this order-to-disorder transition coincides with the phosphoryl transfer transition, the results suggest the existence of an internal entropy contribution to catalysis. 相似文献
947.
Substrate inhibition is a common phenomenon in enzyme chemistry, which is observed only with a fast-reacting substrate enantiomer. We report here for the first time substrate inhibition of an enantioselective enzyme by both substrate enantiomers. The enantioselective substrate inhibition, i.e., different mode of inhibition by each substrate enantiomer, of (S)-specific omega-transaminase was found with various chiral amines. A kinetic model based on ping-pong bi-bi mechanism has been developed and kinetic parameters were measured. The kinetic model reveals that the inhibition by (R)-amine results from formation of Michaelis complex with enzyme-pyridoxal 5'-phosphate, whereas the inhibition by (S)-amine results from the formation of the complex with enzyme-pyridoxamine 5'-phosphate. Substrate inhibition constants (K(SI)) of each (S)-enantiomer of four chiral amines showed a linear correlation with those of cognate (R)-amines. Such a correlation was also found between the K(SI) values and Michaelis constants of (S)-amines. These correlations indicate that recognition mechanisms and active site structures of both enzyme-pyridoxal 5'-phosphate, enzyme-pyridoxamine 5'-phosphate are similar. Taken together with the results, high propensity for non-productive substrate binding strongly suggests that binding pockets of the omega-transaminase is loosely defined, which accounts for the enantioselective substrate inhibition. 相似文献
948.
Pilloff RK Devadas SK Enyedi A Raina R 《The Plant journal : for cell and molecular biology》2002,30(1):61-70
We describe the characterization of a novel gain-of-function Arabidopsis mutant, dll1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection. Despite the constitutive expression of defense-related responses, dll1 is unable to suppress the growth of virulent pathogens. However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses. The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants. This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease. Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death. Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process. 相似文献
949.
950.
Ichiro Tabuchi Sayaka Soramoto Miho Suzuki Koichi Nishigaki Naoto Nemoto Yuzuru Husimi 《Biological procedures online》2002,4(1):49-54
The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion(=viral particle)”(mRNA-peptide fusion),
is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands
ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared
the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification
of the in vitro virus with this “viral genome” was demonstrated.
Published: October 28, 2002 相似文献