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BACKGROUND: Borna disease virus 1 (BoDV-1) causes rare but severe zoonotic infections in humans, presenting as severe encephalitis. The case-fatality risk is very high and no effective countermeasures have been established so far. An immunopathology is presumed, while data on immune responses in humans are limited. Evidence of a role of the complement system in various neurological disorders and central nervous viral infections is increasing and specific inhibitors are available as therapeutic options. METHODS: In this study, we investigated factors of the complement system in the cerebrospinal fluid (CSF) of patients with BoDV-1 infections (n = 17) in comparison to non-inflammatory control CSF samples (n = 11), using a bead-based multiplex assay. In addition, immunohistochemistry was performed using post-mortem brain tissue samples. RESULTS: We found an intrathecal elevation of complement factors of all complement pathways and an active cascade during human BoDV-1 infections. The increase of certain complement factors such as C1q was persistent and C3 complement deposits were detected in post-mortem brain sections. Intrathecal complement levels were negatively correlated with survival. CONCLUSION: Further investigations are warranted to clarify, whether targeting the complement cascade by specific inhibitors might be beneficial for patients suffering from severe BoDV-1 encephalitis.
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BACKGROUND: Within endemic regions in southern and eastern Germany, Borna disease virus 1 (BoDV-1) causes rare zoonotic spill-over infections in humans, leading to encephalitis with a high case-fatality risk. So far, intra-vitam diagnosis has mainly been based on RT-qPCR from cerebrospinal fluid (CSF) and serology, both being associated with diagnostic challenges. Whilst low RNA copy numbers in CSF limit the sensitivity of RT-qPCR from this material, seroconversion often occurs late during the course of the disease. CASE PRESENTATION: Here, we report the new case of a 40 - 50 year-old patient in whom the detection of virus-specific T cells via ELISpot corroborated the diagnosis of BoDV-1 infection. The patient showed a typical course of the disease with prodromal symptoms like fever and headaches 2.5 weeks prior to hospital admission, required mechanical ventilation from day three after hospitalisation and remained in deep coma until death ten days after admission. RESULTS: Infection was first detected by positive RT-qPCR from a CSF sample drawn four days after admission (viral load 890 copies/mL). A positive ELISpot result was obtained from peripheral blood collected on day seven, when virus-specific IgG antibodies were not detectable in serum, possibly due to previous immune adsorption for suspected autoimmune-mediated encephalitis. CONCLUSION: This case demonstrates that BoDV-1 ELISpot serves as additional diagnostic tool even in the first week after hospitalisation of patients with BoDV-1 encephalitis.
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Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable ß-sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two ß-strands connected by varying loop lengths and composition (an example sequence: RWITVTI - loop - KKIRVWE). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: RWITVTI-(GGGGKK)2 GGGG-KKIRVWE) remaining folded (ΔGU = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: RWITVRIW-(GGGGKK)2 GGGG-WKTIRVWE) supports a remarkably stable ß-sheet (ΔGU = 6.3 kJ/mol at 295 K, Tm = â¼55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.
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Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peptídeos/síntese química , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
We have extended our studies of Trp/Trp to other Aryl/Aryl through-space interactions that stabilize hairpins and other small polypeptide folds. Herein we detail the NMR and CD spectroscopic features of these types of interactions. NMR data remains the best diagnostic for characterizing the common T-shape orientation. Designated as an edge-to-face (EtF or FtE) interaction, large ring current shifts are produced at the edge aryl ring hydrogens and, in most cases, large exciton couplets appear in the far UV circular dichroic (CD) spectrum. The preference for the face aryl in FtE clusters is W â« Y ≥ F (there are some exceptions in the Y/F order); this sequence corresponds to the order of fold stability enhancement and always predicts the amplitude of the lower energy feature of the exciton couplet in the CD spectrum. The CD spectra for FtE W/W, W/Y, Y/W, and Y/Y pairs all include an intense feature at 225-232 nm. An additional couplet feature seen for W/Y, W/F, Y/Y, and F/Y clusters, is a negative feature at 197-200 nm. Tyr/Tyr (as well as F/Y and F/F) interactions produce much smaller exciton couplet amplitudes. The Trp-cage fold was employed to search for the CD effects of other Trp/Trp and Trp/Tyr cluster geometries: several were identified. In this account, we provide additional examples of the application of cross-strand aryl/aryl clusters for the design of stable ß-sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 337-356, 2016.
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BACKGROUND: Zoonotic Borna disease virus 1 (BoDV-1) causes fatal encephalitis in humans and animals. Subsequent to the detection of two paediatric cases in a Bavarian municipality in Germany within three years, we conducted an interdisciplinary One Health investigation. We aimed to explore seroprevalence in a local human population with a risk for BoDV-1 exposure as well as viral presence in environmental samples from local sites and BoDV-1 prevalence within the local small mammal population and its natural reservoir, the bicoloured white-toothed shrew (Crocidura leucodon). METHODS: The municipality's adult residents participated in an anonymised sero-epidemiological study. Potential risk factors and clinical symptoms were assessed by an electronic questionnaire. Small mammals, environmental samples and ticks from the municipality were tested for BoDV-1-RNA. Shrew-derived BoDV-1-sequences together with sequences of the two human cases were phylogenetically analysed. RESULTS: In total, 679 citizens participated (response: 41 %), of whom 38 % reported shrews in their living environment and 19 % direct shrew contact. No anti-BoDV-1 antibodies were detected in human samples. BoDV-1-RNA was also undetectable in 38 environmental samples and 336 ticks. Of 220 collected shrews, twelve of 40 C. leucodon (30%) tested BoDV-1-RNA-positive. BoDV-1-sequences from the previously diagnosed two paediatric patients belonged to two different subclades, that were also present in shrews from the municipality. INTERPRETATION: Our data support the interpretation that human BoDV-1 infections are rare even in endemic areas and primarily manifest as severe encephalitis. Sequence analysis linked both previous paediatric human infections to the local shrew population, but indicated independent infection sources. FUNDING: The project was partly financed by funds of the German Federal Ministry of Education and Research (grant numbers: 01KI2005A, 01KI2005C, 01KI1722A, 01KI1722C, 01KI2002 to MaBe, DR, RGU, DT, BS) as well as by the ReForM-A programme of the University Hospital Regensburg (to MaBa) and by funds of the Bavarian State Ministry of Health, Care and Prevention, project "Zoonotic Bornavirus Focal Point Bavaria - ZooBoFo" (to MaBa, MaBe, BS, MMB, DR, PS, RGU).
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Doença de Borna , Vírus da Doença de Borna , Encefalite , Saúde Única , Animais , Humanos , Criança , Vírus da Doença de Borna/genética , Doença de Borna/epidemiologia , Musaranhos/genética , Estudos Soroepidemiológicos , RNA Viral/genética , Alemanha/epidemiologiaRESUMO
Although much has been learned about the design of models of beta-sheets during the last decade, modest fold stabilities in water and terminal fraying remain a feature of most beta-hairpin peptides. In the case of hairpin capping, nature did not provide guidance for solving the problem. Some observations from prior turn capping designs, with further optimization, have provided a generally applicable, "unnatural" beta cap motif (alkanoyl-Trp at the N terminus and Trp-Thr-Gly at the C terminus) that provides a net contribution of 6 + kJ/mol to beta-hairpin stability, surpassing all other interactions that stabilize beta-hairpins including the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel beta-strands and reduces fraying at the ends of hairpins and other beta-sheet models. Utilizing this motif, 10- to 22-residue peptide scaffolds of defined stereochemistry that are greater than 98% folded in water have been prepared. The beta-cap can also be used to staple together short antiparallel beta-strands connected by a long flexible loop.
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Proteínas/química , Acetilação , Sequência de Aminoácidos , Dicroísmo Circular , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas/metabolismoRESUMO
More than 40 human infections with the zoonotic Borna disease virus 1 (BoDV-1) have been reported to German health authorities from endemic regions in southern and eastern Germany. Diagnosis of a confirmed case is based on the detection of BoDV-1 RNA or BoDV-1 antigen. In parallel, serological assays such as ELISA, immunoblots, and indirect immunofluorescence are in use to detect the seroconversion of Borna virus-reactive IgG in serum or cerebrospinal fluid (CSF). As immunopathogenesis in BoDV-1 encephalitis appears to be driven by T cells, we addressed the question of whether an IFN-γ-based ELISpot may further corroborate the diagnosis. For three of seven BoDV-1-infected patients, peripheral blood mononuclear cells (PBMC) with sufficient quantity and viability were retrieved. For all three patients, counts in the range from 12 to 20 spot forming units (SFU) per 250,000 cells were detected upon the stimulation of PBMC with a peptide pool covering the nucleocapsid protein of BoDV-1. Additionally, individual patients had elevated SFU upon stimulation with a peptide pool covering X or phosphoprotein. Healthy blood donors (n = 30) and transplant recipients (n = 27) were used as a control and validation cohort, respectively. In this pilot study, the BoDV-1 ELISpot detected cellular immune responses in human patients with BoDV-1 infection. Its role as a helpful diagnostic tool needs further investigation in patients with BoDV-1 encephalitis.
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Doença de Borna , Vírus da Doença de Borna , Encefalite , Animais , Humanos , Vírus da Doença de Borna/genética , Projetos Piloto , Leucócitos Mononucleares/metabolismo , Doença de Borna/epidemiologia , Doença de Borna/patologia , Interferon gamaRESUMO
More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.
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Doença de Borna , Vírus da Doença de Borna , Encefalite Transmitida por Carrapatos , Encefalite Viral , Encefalite , Infecções por Flavivirus , Animais , Humanos , Vírus da Doença de Borna/genética , Doença de Borna/epidemiologia , Doença de Borna/genética , Encefalite Viral/epidemiologia , Encefalite Transmitida por Carrapatos/diagnóstico , Encefalite Transmitida por Carrapatos/epidemiologia , Alemanha/epidemiologiaRESUMO
Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage.
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Anticorpos/imunologia , Doença de Borna/diagnóstico , Doença de Borna/imunologia , Vírus da Doença de Borna/fisiologia , Nucleoproteínas/imunologia , Fosfoproteínas/imunologia , Proteínas Virais/imunologia , Idoso , Animais , Chlorocebus aethiops , Humanos , Proteínas Recombinantes/imunologia , Células VeroRESUMO
By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i-2 Trp ring, a particularly stable (DeltaG(U) > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH(2), results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed "edge-to-face" (EtF) Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater beta-propensity of Trp. The pi-cation, and Trp/Gly-H(N) interactions have been defined. The latter can give rise to >3 ppm upfield shifts for the Gly-H(N) in -WX(n)G- units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred "face" residue in aryl/aryl pairings, presumably because of its greater pi basicity.
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Amidas/metabolismo , Cátions/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Triptofano/metabolismo , Amidas/química , Cátions/química , Ligação de Hidrogênio , Indóis/química , Indóis/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Peptídeos/genética , Mutação Puntual , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Termodinâmica , Temperatura de Transição , Triptofano/químicaRESUMO
An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.
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Citometria de Fluxo/métodos , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/terapia , Avaliação de Resultados em Cuidados de Saúde/métodos , Humanos , Agências Internacionais , Síndromes Mielodisplásicas/classificação , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sociedades CientíficasRESUMO
BACKGROUND: Detection of basal levels of CD34 progenitor cells is a rare event analysis enumerating cells down to 1 cell/µl. A reproducible analytic approach was used in three independent clinical trials in which multiple sequential assays were obtained from the same individual. METHODS: A 4 color panel combining, HLA-DR, CD34, CD45, and CD11b was used in a dual platform analysis to quantify CD34 progenitor cells in peripheral blood, with quality control focused at the lowest measurements (i.e., basal levels), where assay error is greatest. RESULTS: Repeat testing of individuals every 4 h over the course of 6 days provided a unique opportunity to assess the precision of the analytic technique and identified basal differences between individuals. In a second study, the basal levels were stable for 10 weeks while in a third study the individual differences were maintained for 18 months. This approach was then used to monitor the kinetics of mobilization of CD34 cells following G-CSF stimulation every 4 h. CONCLUSIONS: The differences between individuals in basal levels of CD34 were shown to be a biologic constant, stable for 18 months and not a result of the variability of the assay, shown by low coefficients of variation for each individual. These results can be used to augment a quality control program by monitoring individuals over time to establish intra and inter-laboratory assay precision. In addition, the response of six individuals to G-CSF demonstrated differences in absolute numbers of mobilized CD34 progenitor cells but showed identical kinetics, peaking at 80-110 h.
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Antígenos CD34/metabolismo , Preservação de Sangue , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/citologia , Adolescente , Adulto , Contagem de Células Sanguíneas , Contagem de Células , Feminino , Variação Genética , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: In patients with unexplained cytopenias, abnormal karyotyping studies can be found with inconclusive light microscopic findings. Multidimensional flow cytometry (FCM) can identify myelomonocytic cells with aberrant phenotypes often not seen by standard morphology. METHODS: In 431 patients presenting with unexplained cytopenia(s) FCM results were compared to abnormal karyotyping and FISH results recognized as associated with myelodysplastic syndrome (MDS) in the 2008 WHO classification, to assess the degree of and types of phenotypic abnormalities observed using a previously reported flow cytometric scoring system (FCSS). Fluorescence activated cell sorting was also used to identify subpopulations of abnormal maturing myelomonocytic cells that carry the genotypic abnormality. RESULTS: For marrows with complex (three or more karyotypic abnormalities), two abnormalities, isolated chromosome seven anomalies, del(5q) or del(13q), 100% of cases were positive when using a FCSS cutoff of ≥ 2. Trisomy 8, del(20 q), and minus Y had flow scores ≥ 2 in 72, 60, and 18%, respectively, but in some cases the flow score was high, indicating myeloid dysplasia. Most patients (16/22) with high myeloid progenitor cells (MyPC) (> 20%) also exhibited maturing myeloid cell abnormalities by FCM. Morphology was negative in the maturing myeloid cells in many cases with phenotypically abnormal myeloid cells. CONCLUSIONS: The high correlation between genotypic and phenotypic abnormalities suggests a possible increased utility of flow cytometry in the diagnosis of patients with unexplained cytopenias and may be useful in future clinical studies and in the classification by the WHO, using the FCSS rather than simple counting of flow cytometric abnormalities.
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Genótipo , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Células Progenitoras Mieloides/patologia , Fenótipo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Adulto JovemRESUMO
While end capping in alpha-helices is well understood, the concept of capping a beta-hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a beta-hairpin. While cross-strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed beta-hairpin a conservative, hydrophobic, V --> I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H-bonded site and the sidechain at the C-terminal non-H-bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C-terminal site, for example an E --> A mutation (DeltaDeltaG(U) = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster.
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Peptídeos/química , Estabilidade Proteica , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Estrutura Secundária de ProteínaRESUMO
The similarity of an intranasal salmon calcitonin (sCT) employing chlorobutanol as preservative (Calcitonin Salmon Nasal Spray) was compared to the reference listed drug (RLD) employing benzalkonium chloride as preservative (Miacalcin Nasal Spray). Various orthogonal methods assessed peptide structuring, dynamics, and aggregation state. Mass spectrometry, amino acid analysis, and N-terminal sequencing all demonstrated similarity in primary structure. Near- and far-UV circular dichroism (CD) data supported similarity in secondary and tertiary sCT structure. Nuclear magnetic resonance studies further supported similarity of three-dimensional structure and molecular dynamics of the peptide. Other methods, such as sedimentation velocity and size exclusion chromatography, demonstrated similarity in peptide aggregation state. These latter methods, in addition to reversed phase chromatography, were also employed for monitoring stability under forced degradation, and at the end of recommended shelf storage and patient use conditions. In all cases and for all methodologies employed, similarity to the RLD was observed with respect to extent of aggregation and other degradation processes. Finally, ELISA and bioassay data demonstrated similarity in biological properties. These investigations comprehensively demonstrate physicochemical similarity of Calcitonin Salmon Nasal Spray and the RLD, and should prove a useful illustration to pharmaceutical scientists developing alternative and/or generic peptide or protein products.
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Antiasmáticos/administração & dosagem , Compostos de Benzalcônio/química , Calcitonina/administração & dosagem , Clorobutanol/química , Peptídeos/química , Conservantes Farmacêuticos/química , Administração Intranasal , Aerossóis , Aminoácidos/análise , Antiasmáticos/química , Bioensaio , Calcitonina/química , Varredura Diferencial de Calorimetria , Química Farmacêutica , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Armazenamento de Medicamentos , Ensaio de Imunoadsorção Enzimática , Fracionamento por Campo e Fluxo , Espectroscopia de Ressonância Magnética , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , UltracentrifugaçãoRESUMO
Minimized beta hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, DeltaG(U)(298) >or= +7 kJ/mol) and 12-residue (KTWNPATGKWTE, DeltaG(U)(298) = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-pi interaction between Lys(1) and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hepsilon3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the -TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small DeltaC(p) for unfolding (+420 J K(-)(1)/mol). In the case of Asx-Pro-Xaa-Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH(2)) with the fold stability reduced to DeltaG(U)(278) = -0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also form the same hairpin-stabilizing W/W interaction.