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1.
Scand J Trauma Resusc Emerg Med ; 29(1): 128, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34461967

RESUMO

BACKGROUND: Metabolic and electrolyte imbalances are some of the reversible causes of cardiac arrest and can be diagnosed even in the pre-hospital setting with a mobile analyser for point-of-care testing (POCT). METHODS: We conducted a retrospective observational study, which included analysing all pre-hospital resuscitations in the study region between October 2015 and December 2016. A mobile POCT analyser (Alere epoc®) was available at the scene of each resuscitation. We analysed the frequency of use of POCT, the incidence of pathological findings, the specific interventions based on POCT as well as every patient's eventual outcome. RESULTS: N = 263 pre-hospital resuscitations were included and in n = 98 of them, the POCT analyser was used. Of these measurements, 64% were performed using venous blood and 36% using arterial blood. The results of POCT showed that 63% of tested patients had severe metabolic acidosis (pH < 7.2 + BE < - 5 mmol/l). Of these patients, 82% received buffering treatment with sodium bicarbonate. Potassium levels were markedly divergent normal (> 6.0 mmol/l/ < 2.5 mmol/l) in 17% of tested patients and 14% of them received a potassium infusion. On average, the pre-hospital treatment time between arrival of the first emergency medical responders and the beginning of transport was 54 (± 20) min without POCT and 60 (± 17) min with POCT (p = 0.07). Overall, 21% of patients survived to hospital discharge (POCT 30% vs no POCT 16%, p = 0.01, Φ = 0.16). CONCLUSIONS: Using a POCT analyser in pre-hospital resuscitation allows rapid detection of pathological acid-base imbalances and potassium concentrations and often leads to specific interventions on scene and could improve the probability of survival.


Assuntos
Reanimação Cardiopulmonar , Parada Cardíaca Extra-Hospitalar , Serviço Hospitalar de Emergência , Humanos , Parada Cardíaca Extra-Hospitalar/diagnóstico , Parada Cardíaca Extra-Hospitalar/terapia , Testes Imediatos , Estudos Retrospectivos
2.
Notf Rett Med ; : 1-6, 2021 Aug 24.
Artigo em Alemão | MEDLINE | ID: mdl-34456622

RESUMO

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. Consequences of infection prevention measures during such contagion events can cause disadvantages especially for patients in out-of-hospital cardiac arrest (OHCA). Methods: Retrospective analysis of OHCAs in one county from January-May in 2018, 2019 and 2020, with the first appearance of the SARS-CoV­2 pandemic in 2020 and a high incidence of the influenza virus in 2018. Results: A total of 497 OHCAs were investigated (2018 n = 173; 2019 n = 149; 2020 n = 175). In this study, a constant resuscitation incidence (85-99 resuscitations/100,000 population/year) and locally typical patients (mean 70 years, 66% male; median PES 3) were found. There were no statistically significant differences in the initial situation of the patients (number of observed OHCAs, frequency of lay resuscitations, suspected causes of OHCAs, initial ECG rhythm) and the treatment course (frequency of return of spontaneous circulation [ROSC]/hospital admission/survival to hospital discharge, neurological outcome). None of the OHCA patients in 2020 tested positive for SARS-CoV­2 and 3 patients in 2018 tested positive for the influenza virus. Discussion: The lockdown during the first wave of SARS-CoV­2 pandemic does not seem to have affected the outcome of OHCA patients without coronavirus disease 2019 (COVID-19) in the end.

3.
Resuscitation ; 157: 219-224, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33022311

RESUMO

AIM OF THE STUDY: For out-of-hospital-cardiac-arrest (OHCA) due to ventricular fibrillation (VF) guidelines recommend early defibrillation followed by chest compressions for two minutes before analyzing shock success. If rhythm analysis reveals VF again, it is obscure whether VF persisted or reoccurred within the two-minutes-cycle of chest compressions after successful defibrillation. We investigated the time of VF-recurrence in OHCA. METHODS: We examined all cases of OHCA presenting with initial VF rhythm at arrival of ALS-ambulance (Marburg-Biedenkopf-County, 246.648 inhabitants) from January 2014 to March 2018. Three independent investigators analyzed corpuls3® ECG-recordings. We included ECG-data from CPR-beginning until four minutes after the third shock. VF termination was defined as the absence of a VF-waveform within 5 s of shock delivery. VF recurrence was defined as the presence of a VF-waveform in the interval 5 s post shock delivery. RESULTS: We included 185 shocks in 82 patients. 74.1% (n = 137) of all shocks terminated VF, but VF recurred in 81% (n = 111). The median (IQR) time of VF-recurrences was 27 s (13.5 s/80.5 s) after shock. 51.4% (n = 57) of VF-recurrence occurred 5-30 s after shock, 13.5% (n = 15) VF-recurrence occurred 31-60 s after shock, 21.6% (n = 24) of VF-recurrence occurred 61-120 s after shock, 13.5% (n = 15) of VF-recurrence occurred 121-240 s after shock. CONCLUSIONS: Although VF was terminated by defibrillation in 74.1%, VF recurred in 81% subsequent to the chest compression interval. Thus, VF reappears frequently and early. It is unclear to which extend chest compressions influence VF-relapse. Further studies need to re-evaluate the algorithm, timing of antiarrhythmic therapy or novel defibrillation strategies to minimize refibrillation during shockable OHCA.


Assuntos
Reanimação Cardiopulmonar , Parada Cardíaca Extra-Hospitalar , Ambulâncias , Cardioversão Elétrica , Humanos , Parada Cardíaca Extra-Hospitalar/terapia , Recidiva , Fibrilação Ventricular/terapia
4.
Eur J Radiol ; 131: 109244, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32905956

RESUMO

PURPOSE: To ensure that patients survive cardiac arrest, cardiopulmonary resuscitation (CPR) is needed. However, the procedure itself can lead to severe injuries. This study aims to examine both possibilities of resuscitation - mechanical or manual - with regard to their risk of injury. To this end, we compare the injuries patterns in both groups of patients after successful resuscitation based on computer tomography (CT). METHODS: This single-centre retrospective study included 32 patients (female: 21.87 %, male: 78.12 %, Mean age: 60.22 ± 13.93 years) with cardiac arrest followed by successful mechanical CPR, who underwent an early whole-body CT. A control group of 32 patients (female: 21.87 %, male: 78.12 %, mean age: 60.75 ± 13.34 years) that had been resuscitated successfully with manual CPR was matched according to gender and age for a better statistical comparison. Patients with cardiac arrest due to trauma were excluded from the study population. RESULTS: Mechanically resuscitated patients showed significantly more CPR-related injuries than those who were resuscitated manually (100 % vs. 84.37 %; p = 0.02). In particular, dislocated rib fractures (40.47 vs. 23.80 mean rank, p < 0.01), sternal fractures (74.19 % vs. 25 %; p < 0,01), bleeding complications (29.03 % vs. 3.12 %; p = 0.01), pneumothorax (38.71 % vs. 9.37 %; p = 0.01), mediastinal haematomas (58.01 % vs. 25 %, p = 0.01) and liver lacerations (29.03 % vs. 0 %, p = 0.04) were observed significantly more in patients after mechanical CPR compared to those with manual resuscitation. CONCLUSIONS: The guideline-based use of mechanical CPR results in a significant increase of internal and musculoskeletal injuries compared to manual CPR.


Assuntos
Reanimação Cardiopulmonar/efeitos adversos , Reanimação Cardiopulmonar/métodos , Parada Cardíaca/terapia , Traumatismos Torácicos/diagnóstico por imagem , Traumatismos Torácicos/etiologia , Tomografia Computadorizada por Raios X/métodos , Reanimação Cardiopulmonar/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fraturas das Costelas/diagnóstico por imagem , Fraturas das Costelas/etiologia , Sobreviventes
5.
Med Klin Intensivmed Notfmed ; 115(2): 88-93, 2020 Mar.
Artigo em Alemão | MEDLINE | ID: mdl-30014263

RESUMO

Loss of consciousness is a frequent cause for an emergency call to the emergency medical services (EMS). It can be associated with life-threatening conditions. A distinction must be made between transient loss of consciousness (TLOC) and syncope, which is of cardiovascular origin by definition. Initial assessment in prehospital emergency care should follow the ABCDE algorithm including a 12-lead ECG. The presence of important risk factors such as occurrence in supine position, physical stress, palpitations, history of heart diseases, and any abnormalities in the ECG warrants hospital admission. Initial treatment without admission to an emergency department may only be acceptable for healthy patients without any risk factors and injuries, when vital signs are normal and an orthostatic etiology seems most likely.


Assuntos
Serviços Médicos de Emergência , Medicina de Emergência , Serviço Hospitalar de Emergência , Humanos , Síncope/diagnóstico , Inconsciência
6.
Emerg Radiol ; 27(1): 23-29, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31468207

RESUMO

PURPOSE: Non-traumatic cardiac arrest (CA) and return of spontaneous circulation (ROSC) after cardiopulmonary resuscitation (CPR) are often associated with multiple pathologies. Expecting a high prevalence of important findings, a whole-body CT (WBCT) could be of relevance for therapy. The aim of this study is to investigate the feasibility and diagnostic yield of an early WBCT in this setting. METHODS: This single-center retrospective study included 100 consecutive patients (27 female; 73 male; mean age 68.5± 12.57 years) with non-traumatic, in- and out-of-hospital CA and ROSC following CPR, who underwent a contrast-enhanced WBCT within 6 h after ROSC over 12 months. CT findings were determined corresponding to anatomical region. RESULTS: Early WBCT was successfully carried out in 100% of the patients with CA and ROSC after CPR. Acute pathologies were found not only in the chest but also in the head (15%) and the abdomen (6%). Early global brain edema (n = 12), acute stroke (n = 3), pulmonary embolism (n = 10), pneumothorax (26%), acute abdominal pathologies (n = 6), iatrogenic bleeding (4%), and CPR-related injuries (93%) were detected by CT right from the beginning of the post-cardiac arrest care. CONCLUSIONS: An early WBCT is feasible and provides added diagnostic value for patients with ROSC after non-traumatic CA.


Assuntos
Reanimação Cardiopulmonar , Parada Cardíaca/complicações , Parada Cardíaca/terapia , Tomografia Computadorizada por Raios X/métodos , Imagem Corporal Total , Idoso , Meios de Contraste , Feminino , Humanos , Masculino , Estudos Retrospectivos
7.
Scand J Immunol ; 77(3): 213-20, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23298344

RESUMO

Dendritic cells (DCs) are the most potent antigen-presenting cells and are the key link between the innate and adaptive immune response. Only a few reports with study populations of up to 50 individuals have been published with age-based reference values for DC subpopulations in healthy children. Therefore, we aimed to establish reference ranges in a larger study population of 100 healthy children, which allowed age-matched subgroups. Most previous studies were performed using a dual-platform approach. In this study, a single-platform approach in a lyse no-wash procedure was used. DC subpopulations were defined as follows: CD45(+) CD85k(+) HLA-DR(+) CD14(-) CD16(-) CD33(+) cells as myeloid DCs (mDCs) and CD45(+) CD85k(+) HLA-DR(+) CD14(-) CD16(-) CD123(+) cells as plasmacytoid DCs (pDCs). Reference ranges were established using a semi-parametric regression of age-matched absolute and relative DC counts. We found a significant decline with increasing age in the medians of mDCs (P = 0.0003) and pDCs per µl peripheral blood (PB) (P = 0.004) and in the 50%, 90% and 95% reference ranges. We also identified significantly lower absolute cell counts of mDCs per µl PB in girls than in boys for all age groups (P = 0.0015). Due to the larger paediatric study population and single-platform approach, this study may give a more precise overview of the normal age-matched development of DC subpopulations and may provide a basis for analyzing abnormal DC counts in different illnesses or therapies such as post stem cell transplantation.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Adolescente , Fatores Etários , Antígenos CD/imunologia , Antígenos CD/metabolismo , Contagem de Células , Criança , Pré-Escolar , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Lactente , Recém-Nascido , Subunidade alfa de Receptor de Interleucina-3/imunologia , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Células Mieloides/citologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Análise de Regressão , Fatores Sexuais
8.
Eur Surg Res ; 47(4): 189-95, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21986340

RESUMO

UNLABELLED: The exothermal reaction of polymethylmethacrylate leads to an extensive interaction between bone cement and the synthetic material of the application system. This chemical reaction changes the structure of the cement and might generate air inclusions. METHODS AND MATERIALS: Two application systems for bone cement made of polycarbonate (PC) and polypropylene (PP) were evaluated. The application systems were mounted in a testing unit. The testing device injects a defined amount of bone cement with a certain pressure. After the injection procedure a microscopic examination was carried out. RESULTS: There were no differences in the size and the design of the used syringes. Forty procedures were carried out. The time frame for application of the cement was 5 min in the PC group and 9 min in the PP group. There was a remarkable interaction between the plastics and the cement with the appearance of numerous air inclusions in the PC group. Barely any interaction was found in the PP group. CONCLUSION: Application systems made of PP enable a prolonged application time and a reduced number of air inclusions. Further research, especially on a molecular level as well as material tests on the quality of the applied bone cement, should be carried out.


Assuntos
Cimentos Ósseos/química , Cimento de Policarboxilato/química , Polipropilenos/química , Seringas , Cimentos Ósseos/uso terapêutico
9.
Transgenic Res ; 10(5): 409-22, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11708651

RESUMO

Anther-targeted expression of E. coli DNA (Adenosine-N6-)-Methyltransferase (DAM) in maize was tested as a means to produce male-sterile plants. A high frequency of male-sterile plants with reduced anther size was observed when DAM was regulated by the maize anther-specific promoter 5126 (5126:DAM) and placed upstream of the herbicide resistance gene, pat, regulated by the cauliflower mosaic virus (CaMV) 35S promoter (35S:PAT). In contrast, placement of 5126:DAM upstream of a pat gene regulated by either the maize ubiquitin (UBI:PAT) or rice actin (rACTIN:PAT) promoters resulted in male-fertile plants. Based on these observed differences, DAM-mediated sterility was used as a phenotypic marker to assess the contribution of factors affecting gene expression such as orientation of the transcription units, choice of regulatory sequences mediating expression of adjacent genes, and effects of varying the anther-specific promoter regulating DAM. Constructs that place a portion of the CaMV 35S promoter, including the native AS-1 sequences, between 5126:DAM and UBI:PAT yielded a high frequency of male-sterile plants with reduced anther size. Significant differences in the frequency of male-sterile events and the associated anther size were also observed when the position of 35S:PAT was changed relative to 5126:DAM. These data provide evidence that gene expression in transformed maize plants can be impacted by simply altering the order, orientation or regulatory sequences of adjacent genes.


Assuntos
Acetiltransferases/genética , Regulação da Expressão Gênica de Plantas , Reprodução/fisiologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Zea mays/genética , Zea mays/fisiologia , Actinas/genética , Biomarcadores , Caulimovirus/genética , Escherichia coli/genética , Ordem dos Genes , Genes de Plantas , Biologia Molecular/métodos , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas , Seleção Genética , Esporos/genética , Transformação Genética , Ubiquitina/genética
10.
J Mol Biol ; 305(2): 361-73, 2001 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-11124911

RESUMO

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins.


Assuntos
Engenharia de Proteínas , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Substituição de Aminoácidos/genética , Dicroísmo Circular , Guanidina/farmacologia , Microscopia de Fluorescência , Modelos Moleculares , Peso Molecular , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Maleabilidade , Desnaturação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas/genética , Solventes , Temperatura , Termodinâmica , Ultracentrifugação
11.
Biochemistry ; 39(36): 11024-33, 2000 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-10998239

RESUMO

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.


Assuntos
Apoptose , Micelas , Fosforilcolina/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/química , Sequência de Aminoácidos , Apoptose/fisiologia , Sítios de Ligação , Dicroísmo Circular , Detergentes/química , Endopeptidases/química , Humanos , Hidrólise , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Éteres Fosfolipídicos/química , Fosforilcolina/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Dodecilsulfato de Sódio/química , Soluções , Relação Estrutura-Atividade , Ultracentrifugação , Água/química , Proteína bcl-X
12.
Nature ; 408(6815): 1004-8, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11140637

RESUMO

The inhibitor-of-apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes. Recently, a mammalian protein called Smac (also named DIABLO) was identified that binds to the IAPs and promotes caspase activation. Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function. To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac. The peptide binds across the third beta-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein. The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions. This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Mitocondriais , Proteínas/metabolismo , Sequência de Aminoácidos , Antineoplásicos/química , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Proteínas de Transporte/química , Caspase 9 , Inibidores de Caspase , Clonagem Molecular , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Escherichia coli , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X
13.
Int J Androl ; 22(4): 275-81, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10442301

RESUMO

The aim of this work was to determine whether it is possible to distinguish between fertile (control group, already fathers) and infertile men (suspected infertility), by comparing the fluorescence intensity of the sperm-DNA after incubation with appropriate dyes. First we examined two different DNA-specific dyes (DAPI and YOYO-1) using bull spermatozoa. Based on good results in immunohistochemical applications, YOYO-1 was chosen for further work. The fluorescence-intensity of 200 single, morphologically normal spermatozoa in each semen sample were measured in a cytophotometer, means + SD determined and histograms delineated. Of 20 samples from the control group, 17 had markedly higher fluorescence-intensity than did 7/15 of the suspected infertile men. It is concluded that the DNA of the latter seven samples was less accessible to the dye than was the DNA of the control group. There are cases of infertility known in which there is loss of one or more of the DNA-binding proteins, which in spermatozoa are mainly (85%) protamines. The relationship between the stainability of the sperm-DNA and the packaging with DNA-binding proteins is discussed. Two of the histograms showed abnormalities in the distribution of the fluorescence-intensities, one sample was extremely fragile and most of the sperm lysed during the staining-procedure. Five samples showed normal histograms in comparison with the control group.


Assuntos
DNA/análise , Fertilidade , Infertilidade Masculina/patologia , Espermatozoides/patologia , Coloração e Rotulagem , Animais , Benzoxazóis , Bovinos , Estudos de Avaliação como Assunto , Corantes Fluorescentes , Humanos , Indóis , Masculino , Compostos de Quinolínio
14.
Proc Natl Acad Sci U S A ; 95(14): 7909-14, 1998 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-9653114

RESUMO

Cytohesin-1 (B2-1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the beta2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein's guanine nucleotide exchange factor function and beta2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 alpha-helices that form a unique tertiary fold. The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining 1H-15N and 1H-13C chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-1 occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the beta2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase.


Assuntos
Moléculas de Adesão Celular/química , Proteínas de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Antígenos CD18/metabolismo , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Fatores de Troca do Nucleotídeo Guanina , Humanos , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína
15.
Biochemistry ; 36(9): 2450-8, 1997 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-9054549

RESUMO

The de novo design and characterization of a series of 51-residue helix-turn-helix peptides intended to dimerize into antiparallel four-stranded coiled coils is described. The sequence is based on a coiled coil heptad repeat Ncap-(Aa Zb Zc Ld Ze Zf Zg)3-turn- (Xa Zb Zc Ld Ze Zf Zg)3-Ccap-CONH2, where X is either Val or Ala. The overall topology was intended to be similar to that found in the Escherichia coli protein ROP. The design strategy included consideration of geometric complementarity of the packing of side chains within the hydrophobic core as well as the use of specific interfacial interactions, both of which were intended to favor the desired ROP-like topology. Additionally, the sequence was designed to destabilize potential alternative structures that might compete with the desired topology. The peptides (RLP-1, RLP-2, and RLP-3) assemble into stable alpha-helical dimers and exhibit the hallmarks of a native protein as judged by its spectroscopic properties, and the lack of binding to hydrophobic dyes. Also, the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence of the CD spectra and confirmed by differential scanning calorimetry (DSC). The values determined by the two methods are in excellent agreement and are in the range of those of naturally occurring proteins of this size. These results suggest that it is now possible to design native-like helical proteins that should serve as templates for the further design of functional proteins.


Assuntos
Proteínas de Bactérias/química , Sequências Hélice-Alça-Hélice , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Centrifugação com Gradiente de Concentração , Escherichia coli , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Desnaturação Proteica , Estrutura Secundária de Proteína , Termodinâmica
16.
Acta Chem Scand ; 50(8): 688-96, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8756357

RESUMO

The successful design of proteins requires careful consideration of the multiplicity of forces that stabilize their three-dimensional structures including hydrophobic interactions, hydrogen-bonding, electrostatics and weakly polar interactions. Early attempts to design proteins relied too heavily on hydrophobic interactions to provide stability, resulting in structures with dynamic properties. Addition of more specific interactions to these initial designs gives rise to proteins with more native-like properties. This manuscript describes the design of native-like three- and four-helix bundles, and their cloning and expression of these proteins.


Assuntos
Engenharia de Proteínas , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica/genética , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Biossíntese de Proteínas/genética , Conformação Proteica
17.
Biochemistry ; 35(23): 7422-8, 1996 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-8652519

RESUMO

The global and local stabilities of a eukaryotic ferricytochrome c variant with an engineered disulfide are examined. The disulfide connects position 20, which is usually a valine, to position 102, which is usually a threonine. The cross-linked variant is approximately 1.2 kcal mol-1 less stable than the wild-type protein at 298 K, pH 4.6, in H2O and D2O. Circular dichroism studies show that the decreased stability results from structure-induced stabilization of the denatured state [Betz, S. F., & Pielak, G. J. (1992) Biochemistry 31, 12337-12344]. Here, we use proton chemical shift, paramagnetic shift, and amide proton exchange data to obtain atomic level structural and energetic information. Chemical and paramagnetic shift data indicate only minor native state structural changes. Local stability is obtained from amide proton-deuterium exchange data, using model peptide intrinsic exchange rates. As expected, the exchange data indicate that cross-link incorporation decreases the majority of local stabilities. Near the cross-link, however, local stability seems to increase despite the overall global stability decrease. Furthermore, local stability changes for hydrophobic core residues seem to be greater than the global stability change. We interpret these observations as cross-link-induced changes in exchange competent states and relate them to changes in the denatured state.


Assuntos
Grupo dos Citocromos c/química , Citocromos c , Conformação Proteica , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Calorimetria , Dicroísmo Circular , Grupo dos Citocromos c/biossíntese , Dissulfetos , Estabilidade de Medicamentos , Variação Genética , Espectroscopia de Ressonância Magnética , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Termodinâmica , Treonina
18.
Biochemistry ; 35(21): 6955-62, 1996 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-8639647

RESUMO

The de novo design of peptides and proteins has emerged as an attractive approach for investigating protein structure and function. Here, the design, synthesis, and characterization of a new series of alpha-helical peptides intended to form antiparallel four-stranded coiled coils is described. Computer models were generated without the use of extant protein structures and were used to refine the sequence. The peptides are of the general formula Ncap-(XaZbZcLdZeZfZg)3-Ccap, where X is either Ala, Val, Thr, or Leu, and Ncap and Ccap are sequences designed to satisfy the helices unpaired amide nitrogens and carbonyl oxygens, respectively. The hydrophobic residues (at positions a and d) were chosen so that geometric packing of large and small hydrophobes would favor an antiparallel arrangement. Special attention was also given to residues at the helix--helix interfaces. These residues were chosen to balance potential attractive and repulsive electrostatic forces so that the desired topology was favored while other possible folds were destabilized. Two of the four peptides associate under neutral conditions into the desired tetramers. One of the complexes (a = Val) behaves like a native-like protein as judged by NMR, thermodynamics, and apolar dye (ANS) binding. The other tetrameric complex (a = Leu) exhibits broader NMR resonances, diminished values of delta H and delta Cp, and tight binding of the hydrophobic dye ANS, similar to early designed proteins. These results reinforce the importance of optimizing van der Waals packing interactions in protein design but demonstrate that hydrophobic packing must be balanced with hydrogen-bonding and electrostatic interactions to produce novel native-like proteins.


Assuntos
Peptídeos/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Calorimetria , Simulação por Computador , Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Matemática , Modelos Estruturais , Dados de Sequência Molecular , Peptídeos/síntese química , Espectrometria de Fluorescência , Termodinâmica
19.
Fold Des ; 1(1): 57-64, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-9079364

RESUMO

BACKGROUND: The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles. RESULTS & CONCLUSIONS: Crystals diffracting to 3.5 A resolution have been grown of a 16-residue peptide (alpha 1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha 1, has previously been shown to yield crystals that diffract to 1.2 A resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.


Assuntos
Peptídeos/química , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Dicroísmo Circular , Cristalização , Desenho de Fármacos , Estabilidade de Medicamentos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/síntese química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Soluções , Espectrometria de Fluorescência
20.
Science ; 270(5238): 935-41, 1995 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-7481798

RESUMO

The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of alpha-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.


Assuntos
Conformação Proteica , Engenharia de Proteínas , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica , Dedos de Zinco
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