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AIMS: We aimed to reclassify a population-based cohort of 529 adult glioma patients to evaluate the prognostic impact of the 2016 World Health Organization (WHO) central nervous system tumour classification. Moreover, we evaluated the feasibility of gene panel next-generation sequencing (NGS) in daily diagnostics of 225 prospective glioma patients. METHODS: The retrospective cohort was reclassified according to WHO 2016 criteria by immunohistochemistry for IDH-R132H, fluorescence in situ hybridization for 1p/19q-codeletion and gene panel NGS. All tumours of the prospective cohort were subjected to NGS analysis up-front. RESULTS: The entire population-based cohort was successfully reclassified according to WHO 2016 criteria. NGS results were obtained for 98% of the prospective patients. Survival analyses in the population-based cohort confirmed three major prognostic subgroups, that is, isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas, IDH-mutant astrocytomas and IDH-wildtype glioblastomas. The distinction between WHO grade II and III was prognostic in patients with IDH-mutant astrocytoma. The survival of patients with IDH-wildtype diffuse astrocytomas carrying TERT promoter mutation and/or EGFR amplification overlapped with the poor survival of IDH-wildtype glioblastoma patients. CONCLUSIONS: Gene panel NGS proved feasible in daily diagnostics. In addition, our study confirms the prognostic role of glioma classification according to WHO 2016 in a large population-based cohort. Molecular features of glioblastoma in IDH-wildtype diffuse glioma were linked to poor survival corresponding to IDH-wildtype glioblastoma patients. The distinction between WHO grade II and III retained prognostic significance in patients with IDH-mutant diffuse astrocytic gliomas.
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Biomarcadores Tumorais/genética , Neoplasias Encefálicas/diagnóstico , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrocitoma/diagnóstico , Astrocitoma/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioma/diagnóstico , Glioma/genética , Humanos , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Prognóstico , Telomerase/genética , Adulto JovemRESUMO
AIMS: Glioblastomas are highly aggressive and treatment resistant. Increasing evidence suggests that tumour-associated macrophages/microglia (TAMs) facilitate tumour progression by acquiring a M2-like phenotype. Our objective was to investigate the prognostic value of TAMs in gliomas using automated quantitative double immunofluorescence. METHODS: Samples from 240 patients with primary glioma were stained with antibodies against ionized calcium-binding adaptor molecule-1 (IBA-1) and cluster of differentiation 204 (CD204) to detect TAMs and M2-like TAMs. The expression levels were quantified by software-based classifiers. The associations between TAMs, gemistocytic cells and glioblastoma subtype were examined with immuno- and haematoxylin-eosin stainings. Three tissue arrays containing glioblastoma specimens were included to study IBA-1/CD204 levels in central tumour and tumour periphery and to characterize CD204+ cells. RESULTS: Our data revealed that the amount of especially CD204+ TAMs increases with malignancy grade. In grade III-IV, high CD204 expression was associated with shorter survival, while high IBA-1 intensity correlated with a longer survival. In grade IV, CD204 showed independent prognostic value when adjusting for clinical data and the methylation status of O6-methylguanine-DNA methyltransferase. Our findings were confirmed in two bioinformatics databases. TAMs were more abundant in central tumour tissue, mesenchymal glioblastomas and gliomas with many gemistocytic cells. CD204+ TAMs co-expressed proteins related to tumour aggressiveness including matrix metallopeptidase-14 and hypoxia-inducible factor-1α. CONCLUSIONS: This is the first study to use automated quantitative immunofluorescence to determine the prognostic impact of TAMs. Our results suggest that M2-like TAMs hold an unfavourable prognostic value in high-grade gliomas and may contribute to a pro-tumourigenic microenvironment.
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Neoplasias Encefálicas/patologia , Glioma/patologia , Macrófagos/patologia , Microglia/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Metilação de DNA , Feminino , Glioma/metabolismo , Glioma/mortalidade , Humanos , Macrófagos/metabolismo , Masculino , Microglia/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Prognóstico , Taxa de Sobrevida , Microambiente Tumoral/fisiologiaRESUMO
AIMS: It is important to predict response to treatment with temozolomide (TMZ) in glioblastoma (GBM) patients. Both MGMT protein expression and MGMT promoter methylation status have been reported to predict the response to TMZ. We investigated the prognostic value of quantified MGMT protein levels in tumour cells and the prognostic importance of combining information of MGMT protein level and MGMT promoter methylation status. METHODS: MGMT protein expression was quantified in tumour cells in 171 GBMs from the population-based Region of Southern Denmark (RSD)-cohort using a double immunofluorescence approach. Pyrosequencing was performed in 157 patients. For validation we used GBM-patients from a Nordic Study (NS) investigating the effect of radiotherapy and different TMZ schedules. RESULTS: When divided at the median, patients with low expression of MGMT protein (AF-low) had the best prognosis (HR = 1.5, P = 0.01). Similar results were observed in the subgroup of patients receiving the Stupp regimen (HR = 2.0, P = 0.001). In the NS-cohort a trend towards superior survival (HR = 1.6, P = 0.08) was seen in patients with AF-low. Including MGMT promoter methylation status, we found for both cohorts that patients with methylated MGMT promoter and AF-low had the best outcome; median OS 23.1 and 20.0 months, respectively. CONCLUSION: Our data indicate that MGMT protein expression in tumour cells has an independent prognostic significance. Exclusion of nontumour cells contributed to a more exact analysis of tumour-specific MGMT protein expression. This should be incorporated in future studies evaluating MGMT status before potential integration into clinical practice.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/genética , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , O(6)-Metilguanina-DNA Metiltransferase/genética , Prognóstico , Taxa de SobrevidaRESUMO
Non-invasive kidney stone treatments such as shock wave lithotripsy (SWL) and burst wave lithotripsy (BWL) rely on the delivery of pressure waves through tissue to the stone. In both SWL and BWL, the potential to hinder comminution by exciting cavitation proximal to the stone has been reported. To elucidate how different stones alter prefocal cavitation in BWL, different natural and synthetic stones were treated in vitro using a therapy transducer operating at 350 kHz (peak negative pressure 7 MPa, pulse length 20 cycles, pulse repetition frequency 10 Hz). Stones were held in a confined volume of water designed to mimic the geometry of a kidney calyx, with the water filtered and degassed to maintain conditions for which the cavitation threshold (in the absence of a stone) matches that from in vivo observations. Stone targeting and cavitation monitoring were performed via ultrasound imaging using a diagnostic probe aligned coaxially with the therapy transducer. Quantitative differences in the extent and location of cavitation activity were observed for different stone types-e.g., "softer" stones (natural and synthetic) that disintegrate into "dusty" fragments produced larger prefocal cavitation clouds. Future work will focus on correlation of such cavitation metrics with stone fragmentation.
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Our goal is an office-based, handheld ultrasound system to target, detach, break, and/or expel stones and stone fragments from the urinary collecting system to facilitate natural clearance. Repositioning of stones in humans (maximum 2.5 MPa, and 3-second bursts) and breaking of stones in a porcine model (maximum 50 cycles, 20 Hz repetition, 30 minutes, and 7 MPa peak negative pressure) have been demonstrated using the same 350-kHz probe. Repositioning in humans was conducted during surgery with a ureteroscope in the kidney to film stone movement. Independent video review confirmed stone movements (≥ 3 mm) in 15 of 16 kidneys (94%). No serious or unanticipated adverse events were reported. Experiments of burst wave lithotripsy (BWL) effectiveness on breaking human stones implanted in the porcine bladder and kidney demonstrated fragmentation of 8 of 8 stones on post mortem dissection. A 1-week survival study with the BWL exposures and 10 specific-pathogen-free pigs, showed all findings were within normal limits on clinical pathology, hematology, and urinalysis. These results demonstrate that repositioning of stones with ultrasonic propulsion and breaking of stones with BWL are safe and effective.
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PURPOSE: Persistent unexplained vaginal discharge or bleeding in the pediatric population may be the only manifestation of a serious underlying medical or social problem. Therefore, these symptoms require careful and complete evaluation to identify the primary pathology accurately. We retrospectively reviewed charts of patients who presented for evaluation of persistent vaginal discharge or bleeding to determine if noninvasive imaging was a sensitive means of screening for gynecological pathology. MATERIALS AND METHODS: The records of 24 girls younger than 6 years who presented with vaginal discharge or bleeding were reviewed retrospectively. All patients were evaluated with noninvasive imaging, a pelvic examination while under anesthesia, vaginoscopy and cystoscopy. RESULTS: Noninvasive imaging was useful in identifying 5 of 7 vaginal foreign bodies. However, noninvasive imaging identified only 2 of 6 malignancies. These malignancies consisted of rhabdomyosarcoma (3 patients) and endodermal sinus tumor (3). Two girls also had benign vaginal mullerian papillomas that were not identified by noninvasive imaging. Noninvasive imaging did not aid in the diagnosis of sexual abuse. CONCLUSIONS: Based on these data, we recommend that all girls younger than 6 years who present with persistent vaginal discharge or bleeding be evaluated with pelvic examination while under anesthesia, to be followed by vaginoscopy and cystoscopy if no readily identifiable pathology is found by simple genital examination alone, regardless of the results of noninvasive imaging studies.
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Corpos Estranhos/diagnóstico , Descarga Vaginal/diagnóstico , Doenças Vaginais/diagnóstico , Pré-Escolar , Feminino , Corpos Estranhos/complicações , Humanos , Lactente , Rabdomiossarcoma/complicações , Rabdomiossarcoma/diagnóstico , Descarga Vaginal/etiologia , Doenças Vaginais/etiologia , Neoplasias Vaginais/complicações , Neoplasias Vaginais/diagnósticoRESUMO
Specific cleavage of RNA is catalysed by short oligodeoxynucleotides termed DNAzymes. DNAzymes consist of two binding arms that hybridize to a predetermined RNA sequence and a catalytic core that cleaves a phosphodiester bond held between the binding arms. DNAzymes are exemplified by the well-studied 10-23 DNAzyme, which compared with protein ribonucleases is highly specific, albeit slow. Here we report a significant improvement in cleavage kinetics, while maintaining specificity, by incorporation of LNA (locked nucleic acid) and alpha-L-LNA nucleotides into the binding arms of 10-23 DNAzyme. DNAzymes modified in this way (LNAzymes) enhance cleavage of a phosphodiester bond presented in a short RNA substrate as well as in longer and highly structured substrates, and efficient cleavage is maintained from single- to multiple-turnover conditions. Analysis of the cleavage reaction indicates that substrate hybridization is boosted by the presence of the locked residues within the LNAzymes, while no apparent change occurs in the catalytic strand-scission step.
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DNA Catalítico/química , DNA Catalítico/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , RNA/metabolismo , Sequência de Bases , Células/metabolismo , DNA Catalítico/genética , Desenho de Fármacos , Estrutura Molecular , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , RNA/química , RNA/genéticaRESUMO
The remarkable binding properties of LNA (Locked Nucleic Acid) and alpha-L-LNA (the alpha-L-ribo configured diastereoisomer of LNA) are summarized, and hybridization results for LNA/2'-O-Me-RNA chimera and LNAs with a "dangling" nucleotide are introduced. In addition, results from NMR investigations on the furanose conformations of the individual nucleotide monomers in different duplexes are presented. All these data are discussed with focus on the importance of conformational steering of unmodified nucleotides in partly modified LNA and alpha-L-LNA sequences in relation to the unprecedented binding properties of LNA and alpha-L-LNA.
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DNA/química , Oligonucleotídeos/química , RNA/química , DNA/metabolismo , Furanos/química , Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , RNA/metabolismo , Ribose/química , EstereoisomerismoRESUMO
Novel fusidic acid type antibiotics having flexible side chains are described. Saturation of the double bond between C-17 and C-20 of fusidic acid produces four stereoisomers differing in the configuration at C-17 and C-20. The structure-activity relationship of the stereoisomers was studied using computer-assisted analyses of low-energy conformations and crystallographic data. Only one of the four stereoisomers showed potent antibiotic activity comparable with that of fusidic acid, whereas the other three stereoisomers retained little or no activity. The orientation of the side chain is crucial, and there is only a limited space for bioactive side chain conformations. This investigation demonstrates the essential role of the side chain conformations in relation to antibacterial activity and contradicts earlier assumptions that the Delta17(20) bond is an essential feature in the molecule.
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Antibacterianos/síntese química , Ácido Fusídico/análogos & derivados , Ácido Fusídico/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Contagem de Colônia Microbiana , Ácido Fusídico/química , Ácido Fusídico/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação MolecularRESUMO
The phosphoramidite approach has been used for the automated synthesis of alpha-L-LNA, alpha-L-RNA, and oligomers composed of mixtures of alpha-L-LNA, alpha-L-RNA and DNA monomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. alpha-L-LNAs were shown to be significantly stabilized towards 3'-exonucleolytic degradation. Duplexes formed between RNA and alpha-L-LNA induced E. coli RNase H-mediated RNA cleavage, albeit very slow, at high enzyme concentration.
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Ácidos Nucleicos/química , RNA/química , DNA/química , Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Exonucleases/metabolismo , Hibridização de Ácido Nucleico , Ácidos Nucleicos/metabolismo , RNA/metabolismo , EstereoisomerismoRESUMO
Various Y-shaped branched oligonucleotides containing a 2'-0,3'-C-ethylene linked or 2'-0,4'-C-methylene linked bicyclic nucleotide as branching point were synthesized on an automated DNA synthesizer. Thermal denaturation experiments at 260 and 284 nm showed increased thermal stabilities of complexes formed between these Y-shaped oligonucleotides and complementary DNA compared with those formed with the corresponding linear reference. The most significant effect was observed when LNA (locked nucleic acid) monomers were used in the triplex forming branch.
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Pareamento de Bases , Compostos Bicíclicos com Pontes/química , DNA/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/metabolismo , DNA/metabolismo , Estrutura Molecular , Hibridização de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismoRESUMO
The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.
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Caseínas/química , Caseínas/metabolismo , Membrana Celular/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Dimiristoilfosfatidilcolina/metabolismo , Ligação de Hidrogênio , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Metanol/metabolismo , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilgliceróis/metabolismo , Estrutura Secundária de Proteína , Dodecilsulfato de Sódio/metabolismo , TermodinâmicaRESUMO
NMR spectroscopic analysis of the C-terminal Kunitz domain fragment (alpha3(VI)) from the human alpha3-chain of type VI collagen has revealed that the side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X-ray crystallographic structure, while the minor conformation (m) cannot structurally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conformations of the Trp21 side chain obtained by energy minimization identified two energy minima: One corresponding to the conformation of Trp21 observed in the X-ray crystallographic structure and solution structure of alpha3(VI) (the M conformation) and the second corresponding to the m conformation predicted by NMR spectroscopy. A transition pathway between the M and m conformation is suggested. The free-energy difference between the two conformers obtained by the thermodynamic integration method is calculated to 1.77+/-0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformers of the alpha3(VI) molecule were investigated by molecular dynamic. Essential dynamics analysis of the two resulting 800 ps trajectories reveals that when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are observed in the mobility of the binding loop (residues Thr13-Ile18), which is more flexible in the m conformation than in the M conformation. This suggests that the reorientation of Trp2 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding loop and Trp21.
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Colágeno/química , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Termodinâmica , Triptofano/químicaRESUMO
It is demonstrated that the spin-state-selective pulse sequence elements, S3E and S3CT, previously introduced for measurement of J coupling constants in 15N-labeled proteins can be applied for work with peptides and proteins with 13C at the natural abundance level. In addition, a method is described for suppression of crosstalk caused by passive spin flips and pulse imperfections, which otherwise results in systematically underestimated J coupling constants and thereby inaccurate structural constraints. This method is also applicable for crosstalk suppression in applications of S3E and S3CT to 13C- or 15N-labeled samples. Experimental confirmation is obtained using a 10 mM BPTI sample focusing on 13C in the alpha position. The measured J coupling constants include 3J(HN-Halpha) and 3J(Halpha-Hbeta) related to the phi and chi1 angles, respectively.
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Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Proteínas/química , Isótopos de Carbono/análise , Isótopos de Nitrogênio/análise , Peptídeos/química , Prótons , Marcadores de SpinRESUMO
The solution structure and backbone dynamics of the 58-residue C-terminal Kunitz domain fragment [alpha3(VI)] of human alpha3-chain type VI collagen has been studied by two-dimensional 1H-1H and 1H-15N nuclear magnetic resonance spectroscopy at 303 K. The solution structure is represented by an ensemble of 20 structures calculated with X-PLOR using 612 distance and 47 dihedral angle restraints. The distance restraints were obtained by a complete relaxation matrix analysis using MARDIGRAS. The root mean squared (rms) deviation is 0.91 A for the backbone atoms of the residues Thr2(8)-Gly12(18), Arg15(21)-Tyr35(41), and Gly40(46)-Pro57(63). The central beta-sheet [residues Ile18(24)-Tyr35(41)] and the C-terminal alpha-helix [residues Gln48(54)-Cys55(61)] are better defined with a backbone rms deviation of 0.46 A. The solution structure of alpha3(VI) is virtually identical to the crystal structure of alpha3(VI) and to the solution structure of bovine pancreatic trypsin inhibitor (BPTI). The 15N spin-lattice and spin-spin relaxation rates and the 1H-15N heteronuclear nuclear Overhauser enhancement (NOE) were analyzed using both the "model-free" formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping procedure [Farrow, N. A., Szabo, A., Torchia, D. A., & Kay, L. E. (1995) J. Biomol.NMR 6, 153-162]. The results obtained from the "model-free" analysis include an overall correlation time tauc of 3. 00 ns and backbone order parameters S2 in the range from 0.28 to 0. 93. The necessity of including an exchange term in the analysis of the relaxation data from 14 residues indicated that these residues are involved in motions on the micro- to millisecond time scale. The majority of the 14 residues are located in the vicinity of the Cys14(20)-Cys38(44) disulfide bond, suggesting the presence of a disulfide bond isomerization similar to the one observed in BPTI [Otting, G., Liepinsh, E., & Wüthrich, K. (1993) Biochemistry 32, 3571-3582]. It is suggested that this disulfide bond isomerization is the main reason for the surprisingly small effect on trypsin inhibition observed when Thr13(19) of alpha3(VI) is substituted with Pro.
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Colágeno/química , Sequência de Aminoácidos , Animais , Aprotinina/química , Bovinos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Alinhamento de Sequência , Software , Tripsina/metabolismoRESUMO
The human alpha 3-chain type VI collagen C-terminal Kunitz domain fragment (alpha 3(VI)) has been studied by two dimensional 1H-1H and 1H-13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and a number of unusual H alpha chemical shifts, which are highly conserved in Kunitz-domain proteins, are also observed for a alpha 3(VI). Furthermore, a series of exchange cross peaks observed in 1H-1H spectra shows that a large number of protons in the central beta-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser47(53) H alpha, Arg32(28) H(gamma 1) and H(gamma 2), and GLN48(54) H(beta 2), all located in the vicinity of the Trp21(27) ring in the crystal structure of alpha 3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609-617], have very different chemical shifts in the two conformations, the most affected being Gln48(54) H(beta 2) (delta sigma = 3 ppm), which is placed directly above the Trp21(27) ring in the crystal structure of alpha 3(VI). It should be concluded that the origin of the multiple conformations of the central beta-sheet is a reorientation of the Trp21(27) ring. From the intensities of corresponding signals in the two conformations, the populations, the population of the minor conformation was found to be 6.4 +/- 0.2% of that of the major conformation, while a rate constant kM = 1.01 +/- 0.05 s-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys14(20)-Cys38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3571-3582], is also likely to occur in alpha 3(VI).
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Colágeno/química , Sequência de Aminoácidos , Animais , Aprotinina/química , Bovinos , Colágeno/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Estrutura Secundária de Proteína , Triptofano/químicaRESUMO
A 2D NMR experiment for assignment of backbone carbon resonances in small and medium-sized (15)N-labelled proteins with (13)C at natural abundance is presented. The experiment is a two-dimensional variant of the HNCO triple-resonance experiment and is demonstrated by application to a 6 kDa protein at relatively low concentration (2 mM) and temperature (30°C). The experiment is particularly suitable for assignment of carbonyl resonances.
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A titration study of the dimeric Asp(B9) mutant of human insulin was performed using two-dimensional NMR spectroscopy. Based on 10 NOESY spectra recorded in the pH range 1.73-3.93, the pKa values of the seven carboxyl groups in the mutant were determined, and the titration shifts of 46 pH-dependent protons in non-ionizable groups were investigated. Further, the pKa values of the two histidine imidazole rings were determined from a series of 1D spectra recorded in the pH range 6.65-10.0. The titration shifts of all pH-dependent protons were analyzed by a nonlinear least-squares fitting procedure, using an equation that describes a one-step titration. Also the pH dependence of the exchange rate of the amide proton of Phe(B24) was determined in the applied pH range. On the basis of the experimental results, it is concluded that the Asp(B9) residue forms an N-cap of the B-chain alpha-helix through an interaction between the side-chain carboxyl group of the residue and the dipole of the helix. Further, the titration data show that salt bridges are established between Glu(B13) and His(B10) and between Asn(A21) and Arg(B22) at pH values, where the interacting groups are ionized, and that a hydrogen bond exists between the amide proton of Val(A3) and the C-terminal carboxyl group of Thr(B30). Most surprisingly, the data analysis shows that the Asp(B9) insulin exists as a dimer throughout the investigated pH range, that is, also at pH values where there is a substantial negative charge repulsion in the monomer-monomer interface of the dimer.