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261.
Heine G Zucht HD Schuhmann MU Bürger K Jürgens M Zumkeller M Schneekloth CG Hampel H Schulz-Knappe P Selle H 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,782(1-2):353-361
Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy. 相似文献
262.
In addition to protein identification, characterization of post-translational modifications (PTMs) is an essential task in proteomics. PTMs represent the major reason for the variety of protein isoforms and they can influence protein structure and function. Upon matrix-assisted laser desorption/ionization (MALDI) most post-translationally modified peptides form a fraction of labile molecular ions, which lose PTM-specific residues only after acceleration. Compared to fully accelerated ions these fragment ions are defocused and show in reflector mass spectra reduced resolution. A short time Fourier transform using a Hanning window function now uses this difference in resolution to detect the metastable fragments. Its application over the whole mass range yields frequency distributions and amplitudes as a function of mass, where an increased low frequency proportion is highly indicative for metastable fragments. Applications on the detection of metastable losses originating from carboxamidomethylated cysteines, oxidized methionines, phosphorylated and glycosylated amino acid residues are presented. The metastable loss of mercaptoacetamide detected with this procedure represents a new feature and its integration in search algorithms will improve the specificity of MALDI peptide mass fingerprinting. 相似文献
263.
Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The structure of the empty capsid has been determined to a resolution of better than 15 A by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 A diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA. 相似文献
264.
Gökbuget N Hoelzer D 《Reviews in clinical and experimental hematology》2002,6(2):114-41; discussion 200-2
In the last decades outcome of adult acute lymphoblastic leukemia (ALL) has improved considerably. In large multicenter studies remission rates range from 75% to 89%, and long-term leukemia-free survival (LFS) from 28% to 39%. Major progress has also been made regarding better characterization of subtypes of ALL. Complete diagnostic procedures are essential to identify these subtypes which have significant differences in clinical and laboratory features and prognosis. LFS of > 50% can be expected in favorable subtypes such as T-ALL or mature B-ALL, while LFS of < 20% is expected in Ph/BCR-ABL positive ALL. Prognostic factors can be used for risk stratification and selection of treatment strategies can be adapted to the subtype and relapse risk. This includes measurement of minimal residual disease (MRD) to evaluate individualized treatment strategies adapted to the molecular response. Several new approaches for improvement in chemotherapy and stem cell transplantation (SCT) are under investigation. They include the use of intensified anthracyclines, asparaginase, cyclophosphamide or high-dose cytarabine during induction and intensive rotational chemotherapy during consolidation. Also SCT - mainly from sibling donors - is now part of standard treatment of de novo ALL, although it remains open whether indications should be based on prognostic factors or whether SCT should be offered to all patients with sibling donor. However, substantial progress can only be achieved by new, experimental strategies. These include new approaches for SCT, such as nonmyeloablative SCT, measurement of MRD, causal treatment with molecular targeting, e.g. with kinase inhibitors, and antibody therapy. 相似文献
265.
Matthias Huber Ralph Hahn Dieter Hess 《Molecular breeding : new strategies in plant improvement》2002,10(1-2):19-30
Improved gene transfer techniques are necessary to obtain adequatenumbers of stable transgenic wheat plants needed for practical purposes.Considering that wheat transformation is genotype-dependent, we used cv. Combiin all experiments, which had been selected from agronomically important Germanspring wheat cultivars because of its high transformation ability. In mostwheatgene transfer attempts, immature embryos or embryogenic scutellar calli weremicrobombarded. We compared both methods under optimised conditions, usingbar, uidA, andgfp as markers in co-transformation attempts. Integrationof the genes mentioned above was proven by Southern blotting, expression levelswere measured by assays on phosphinothricin acetyltransferase and-glucuronidase activities, and by monitoring for green fluorescentproteinin most developmental stages. Following bombardment of scutellar calli, anaverage transformation frequency of 0.13% was attained. Using immature embryos,mean transformation frequency (1.06%) was 8-fold higher. In addition, embryotechniques were over 2 weeks faster than scutellar callus procedures.Introducing gfp as a vital marker led to an improvement ofembryo-based techniques. In a first screening, transientgfp-expressing embryos were transferred tophosphinothricincontaining callus medium. Only gfp-expressing calli whichdeveloped on it were cultured further on phosphinothricin containingregeneration medium. Shoots obtained from gfp-expressingcalli were rooted on phosphinothricin-free medium, and cultured exvitro. Average transformation frequency (4.93%) was 38-fold higherthan with scutellar callus techniques. Differences between the transformationstrategies used were of high statistical significance. Combining greenfluorescent protein screening with phosphinothricin selection in embryo-basedtechniques offers a promising system to obtain high wheat transformationfrequencies. 相似文献
266.
Feng L Stathopoulos C Ahel I Mitra A Tumbula-Hansen D Hartsch T Söll D 《Extremophiles : life under extreme conditions》2002,6(2):167-174
Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive. Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization. We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine. We show that the T. thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS. The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes. The heterotrimeric T. thermophilus aspartyl-tRNA(Asn) amidotransferase can form Gln-tRNA in addition to Asn-tRNA: however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates. A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles. 相似文献
267.
Glutamyl-tRNA reductase (GluTR) catalyzes the first step of tetrapyrrole biosynthesis in plants, archaea and most bacteria.
The catalytic mechanism of the enzyme was elucidated both by biochemical data and the determination of the high-resolution
crystal structure of the enzyme from the archaeon Methanopyrus kandleri in complex with a competitive inhibitor. The dimeric enzyme has an unusual V-shaped architecture where each monomer consists
of three domains linked by a long `spinal' α-helix. The central catalytic domain specifically recognizes the glutamate moiety
of the substrate. It bears a conserved cysteine poised to nucleophilically attack the activated aminoacyl bond of glutamyl-tRNA.
Subsequently, the thioester intermediate is reduced to the product glutamate-1-semialdehyde via hydride transfer from NADPH
supplied by the second domain. A structure-based sequence alignment indicates that catalytically essential amino acids are
conserved throughout all GluTRs. Thus the catalytic mechanism derived for M. kandleri is common to all including plant GluTRs. Mutations described to influence the catalytic efficiency of the barley enzyme can
therefore be explained.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
268.
269.
ZK 156718, a low calcemic, antiproliferative, and prodifferentiating vitamin D analog. 总被引:1,自引:0,他引:1
Tanja Gaschott Andreas Steinmeyer Dieter Steinhilber Jürgen Stein 《Biochemical and biophysical research communications》2002,290(1):504-509
The physiologically active form of vitamin D, 1,25-dihydroxyvitamin D(3), plays an important role not only in the establishment and maintenance of calcium metabolism, but also in regulating cell growth and differentiation. Because the clinical usefulness of 1,25-dihydroxyvitamin D(3) is limited by its tendency to cause hypercalcemia, new analogs with a better therapeutic profile have been synthesized, including ZK 156718. We compared the effects of 1,25-dihydroxyvitamin D(3) and ZK 156718 on growth, differentiation, and on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Whereas ZK 156718 at the concentration [10(-8) M] was as potent as 10(-6) M 1,25-dihydroxyvitamin D(3) in inducing differentiation and p21(Waf1/Cip1) expression, it was even more effective in inhibiting cell growth and stimulating p27(Kip1) expression than 1,25-dihydroxyvitamin D(3) itself. In summary, our study presents a new and potent vitamin D analog with a decreased metabolic stability, making it useful for the treatment of a diversity of clinical disorders. 相似文献
270.
The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649-653), the model that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state. 相似文献