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INTRODUCTION: In order for young children to be able to undergo a Magnetic Resonance Imaging (MRI) examination, general anesthesia is often required. The aim of this study was to compare the image quality, times, and costs of the examinations of infant brains performed with MRI either during sedation with dexmedetomidine administered by radiographers or anesthesia with propofol administered by anesthesia staff. METHODS: This study was a quantitative retrospective study of 27 consecutive standard brain examinations performed under sedation or anesthesia, involving 15 children under sedation and 12 under anesthesia. The age of the children was from 0.5 to five years old. The image quality was evaluated by three radiologists experienced in pediatric MRI examinations. Information such as examination time and the expense of the examination was also collected. RESULTS: There was no statistically significant difference in the general image quality, but one image series was assessed to have significantly better image quality under sedation than under anesthesia, but all images had very high quality. However, it emerged that children under anesthesia were at the hospital on average 55 min longer and the scanner room was occupied 20 min longer on average. The anesthesia examinations were three times more expensive. CONCLUSION: This study demonstrated equivalent image quality between sedation and anesthesia. In addition, sedation was less time-consuming and had a lower price, partly because no extra anesthetic staff were required. The use of intranasal sedation offers a possibility to expand the competence area for radiographers. IMPLICATIONS FOR PRACTICE: If radiographers learn to perform intranasal sedation, examinations can be performed in less time, at a third of the staff costs while maintaining image quality.
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Dexmedetomidina , Hipnóticos y Sedantes , Lactante , Niño , Humanos , Preescolar , Estudios Retrospectivos , Anestesia General , Imagen por Resonancia Magnética/métodosRESUMEN
INTRODUCTION: Adverse reactions to iodinated contrast media, which is used during computed tomography (CT) examinations, are rare. As a result, radiographers have limited experience handling those situations and may feel uncertainty and a lack of confidence. The aim of this study was to investigate radiographers' confidence in handling hypersensitivity reactions to contrast media during CT examinations. METHODS: A survey in the form of a questionnaire was conducted to gather both quantitative and qualitative data. There were 31 clinics that participated in this study, of which four were university hospitals, 17 were medium-sized hospitals and 10 were small hospitals. In total, the questionnaires were distributed to 700 radiographers. The questionnaire contained 12 questions and was distributed via email with a link to the questionnaire. RESULTS: Two hundred-ninety radiographers participated in the survey. 72% of the respondents answered in the middle of the four-point scale (2-3) in response to the statement "I feel confident in handling hypersensitivity reactions". 65% answered that they did not have routines for training regularly regarding hypersensitivity reactions. Qualitative data showed that many of the respondents wished to receive education and training regularly. CONCLUSIONS: The confidence of radiographers regarding the management of hypersensitivity reactions was deficient and most of the respondents wished they felt more confident. IMPLICATION FOR PRACTICE: To increase radiographers' confidence in handling hypersensitivity reactions, it is recommended that the radiology clinics review their routines and the possibility to implement regular training.
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Yodo , Radiología , Humanos , Medios de Contraste/efectos adversos , Yodo/efectos adversos , Radiografía , Radiología/educación , Técnicos Medios en SaludRESUMEN
INTRODUCTION: Guidelines concerning intravenous iodinated contrast media (CM) during computed tomography (CT) examinations are important to follow to minimize the risk for post-contrast acute kidney injury (PC-AKI). The purpose of this study was to investigate the radiology departmental policy compliance with Swedish guidelines concerning PC-AKI. METHODS: In February 2020, an electronic survey was distributed to the responsible radiographer at 41 radiology departments in all university hospitals and medium-sized hospitals in Sweden. The questions focused on routines around renal functional tests, individualized contrast administration and handling of patients with diabetes mellitus taking metformin. RESULTS: The response rate was 83%. Seventy-six percent (n = 26) of radiology departments calculated estimated glomerular filtration rate (eGFR) from serum creatinine prior to CM administration, but only 24% (n = 8) followed the recommendation to calculate eGFR from both serum creatinine and cystatin C. For acute/inpatients, 55% (n = 18) followed the recommendation that renal functional tests should be performed within 12 h before CM administration. For elective patients, 97% (n = 33) followed the recommendation to have eGFR newer than three months which is acceptable for patients with no history of disease that may have affected renal function. Approximately 80% of the radiology departments followed the recommendation that CM dose always should be individually adjusted to patient eGFR. Seventy-six percent (n = 26) followed the recommendation to continue with metformin at eGFR ≥ 45 ml/min. CONCLUSION: Compliance with the national guidelines was high regarding routines around renal functional tests, dose adjustment of CM and metformin discontinuation. Improvements can be made in using both cystatin C and serum creatinine for eGFR calculations as well as ensuring renal function tests within 12 h for acute/inpatients with acute disease that may affect renal function. IMPLICATIONS FOR PRACTICE: This study raises awareness of the importance of adhering to guidelines in healthcare. To have knowledge about the current level of compliance regarding PCI-AKI is important to maintain and develop effective clinical implementation of guidelines. The variation in practice seen in this study emphasizes the need of more effective implementation strategies to ensure adherence with best practice.
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Lesión Renal Aguda , Intervención Coronaria Percutánea , Radiología , Lesión Renal Aguda/inducido químicamente , Medios de Contraste/efectos adversos , Adhesión a Directriz , Humanos , Factores de Riesgo , SueciaRESUMEN
OBJECTIVES: To develop and evaluate a procedure for quantifying the hepatocyte-specific uptake of Gd-BOPTA and Gd-EOB-DTPA using dynamic contrast-enhanced (DCE) MRI. METHODS: Ten healthy volunteers were prospectively recruited and 21 patients with suspected hepatobiliary disease were retrospectively evaluated. All subjects were examined with DCE-MRI using 0.025 mmol/kg of Gd-EOB-DTPA. The healthy volunteers underwent an additional examination using 0.05 mmol/kg of Gd-BOPTA. The signal intensities (SI) of liver and spleen parenchyma were obtained from unenhanced and enhanced acquisitions. Using pharmacokinetic models of the liver and spleen, and an SI rescaling procedure, a hepatic uptake rate, K (Hep), estimate was derived. The K (Hep) values for Gd-EOB-DTPA were then studied in relation to those for Gd-BOPTA and to a clinical classification of the patient's hepatobiliary dysfunction. RESULTS: K (Hep) estimated using Gd-EOB-DTPA showed a significant Pearson correlation with K (Hep) estimated using Gd-BOPTA (r = 0.64; P < 0.05) in healthy subjects. Patients with impaired hepatobiliary function had significantly lower K (Hep) than patients with normal hepatobiliary function (K (Hep) = 0.09 ± 0.05 min(-1) versus K (Hep) = 0.24 ± 0.10 min(-1); P < 0.01). CONCLUSIONS: A new procedure for quantifying the hepatocyte-specific uptake of T (1)-enhancing contrast agent was demonstrated and used to show that impaired hepatobiliary function severely influences the hepatic uptake of Gd-EOB-DTPA. KEY POINTS: ⢠The liver uptake of contrast agents may be measured with standard clinical MRI. ⢠Calculation of liver contrast agent uptake is improved by considering splenic uptake. ⢠Liver function affects the uptake of the liver-specific contrast agent Gd-EOB-DTPA. ⢠Hepatic uptake of two contrast agents (Gd-EOB-DTPA, Gd-BOPTA) is correlated in healthy individuals. ⢠This method can be useful for determining liver function, e.g. before hepatic surgery.
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Medios de Contraste/farmacocinética , Gadolinio DTPA/farmacocinética , Hepatopatías/diagnóstico , Hepatopatías/metabolismo , Hígado/metabolismo , Imagen por Resonancia Magnética/métodos , Meglumina/análogos & derivados , Compuestos Organometálicos/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Femenino , Humanos , Interpretación de Imagen Asistida por Computador , Masculino , Meglumina/farmacocinética , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Estudios Retrospectivos , Bazo/metabolismo , Resultado del TratamientoRESUMEN
The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.
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Antígenos de Carbohidratos Asociados a Tumores/química , Glicopéptidos/síntesis química , Mucina-1/química , N-Acetilglucosaminiltransferasas/química , Ácido N-Acetilneuramínico/química , Péptidos/química , Sialiltransferasas/química , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Epítopos de Linfocito T/química , Humanos , Insectos , Ratones , Datos de Secuencia Molecular , Mucinas/química , N-Acetilglucosaminiltransferasas/biosíntesis , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Péptidos/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sialiltransferasas/biosíntesis , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Linfocitos T/inmunologíaRESUMEN
[structure: see text] A simple and efficient method for monitoring and optimizing carbohydrate synthesis on polymeric support by using (19)F NMR spectroscopy is described. The method relies on the use of fluorinated variants of protective groups that are in common use in oligosaccharide synthesis.
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Técnicas Químicas Combinatorias , Glicósidos/química , Resonancia Magnética Nuclear Biomolecular/métodos , Flúor , Glicósidos/síntesis química , Oligosacáridos/síntesis química , Oligosacáridos/químicaRESUMEN
Fluorobenzoyl groups have been investigated as alternatives to acetyl and benzoyl protective groups in carbohydrate and glycopeptide synthesis. D-Glucose and lactose were protected with different fluorobenzoyl groups and then converted into glycosyl bromides in high yields (>80% over two steps). Glycosylation of protected derivatives of serine with these donors gave 1,2-trans glycosides in good yields (approximately 60--70%) and excellent stereoselectivity without formation of ortho esters. The resulting glycosylated amino acid building blocks were then used in solid-phase synthesis of two model O-linked glycopeptides known to be unusually sensitive to beta-elimination on base-catalyzed deacylation. When either a 3-fluoro- or a 2,5-difluorobenzoyl group was used for protection of each of the two model glycopeptides the extent of beta-elimination decreased from 80% to 10% and from 50% to 0%, respectively, as compared to when using the ordinary benzoyl group. Fluorobenzoyl groups thus combine the advantages of the benzoyl group in formation of glycosidic bonds (i.e., high stereoselectivity and low levels of ortho ester formation) with the ease of removal characteristic of the acetyl group.
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Carbohidratos/química , Glicopéptidos/síntesis química , Hidrocarburos Fluorados/química , Catálisis , Cromatografía en Capa Delgada , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría UltravioletaAsunto(s)
Antibacterianos/química , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Proteínas Fimbrias , Fimbrias Bacterianas/efectos de los fármacos , Péptidos/química , Proteínas Periplasmáticas , Aminoácidos/síntesis química , Aminoácidos/farmacología , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/química , Diseño de Fármacos , Modelos Moleculares , Chaperonas Moleculares/química , Péptidos/síntesis química , Péptidos/farmacología , Piridonas/síntesis química , Piridonas/farmacología , Resonancia por Plasmón de SuperficieRESUMEN
Three fluorinated linkers which are analogues of linkers commonly used in solid-phase peptide synthesis have been prepared. One of the linkers was used in combination with gel-phase 19F NMR spectroscopy to develop conditions for solid-phase synthesis of two libraries of pilicides, i.e. compounds designed to inhibit assembly of adhesive pili in uropathogenic Escherichia coli. Attachment to and cleavage from the linker could be monitored based on the chemical shift of the fluorine atom of the linker. In addition, use of the linker as internal standard allowed quantification and optimization of reactions occurring further away from the linker when fluorinated building blocks were employed. Importantly, high-quality 19F NMR spectra were obtained for compounds linked to a TentaGel resin in a standard NMR tube using an ordinary NMR instrument.
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Biblioteca de Péptidos , Péptidos/síntesis química , Técnicas Químicas Combinatorias , Espectrometría de Masas , Resonancia Magnética Nuclear BiomolecularRESUMEN
The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-gamma secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.
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Artritis/inmunología , Cartílago/inmunología , Colágeno/inmunología , Linfocitos T/inmunología , Animales , Artritis/sangre , Células Cultivadas , Glicosilación , Inmunoglobulina G/sangre , Ganglios Linfáticos/inmunología , Ratones , Ratones Transgénicos , Péptidos/inmunologíaRESUMEN
A C-linked isostere of beta-D-galactosylated hydroxynorvaline has been prepared in eight steps from per-O-benzylated galactopyranolactone. Addition of a homoallylic Grignard reagent to the lactone, reduction of the resulting hemiacetal with triethylsilane, and a Wittig reaction with Garner's aldehyde were key steps in this synthesis. The C-linked building block was then incorporated at position 264 into the fragment CII(256--270) from typeII collagen by solid-phase synthesis using a combination of the tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) protective group strategies. Deprotection of the benzylated C-linked galactosyl moiety was achieved simultaneously with cleavage of the glycopeptide from the solid phase by using triethylsilyl trifluoromethanesulfonate in TFA. Helper T-cell hybridomas obtained in a mouse model for rheumatoid arthritis responded to the C-linked glycopeptide when presented by classII MHC molecules. However, 10- to 20-fold higher concentrations were required as compared to when O-linked beta-D-galactosylated hydroxynorvaline or hydroxylysine (Hyl) were present at position 264 of CII(256--270). Thus, replacement of a single oxygen atom by a methylene group in the carbohydrate moiety of a glycopeptide antigen had a substantial influence on the T-cell response. This reveals that T cells are able to recognize the carbohydrate moiety of glycopeptide antigens with high specificity. Finally, the results suggest that structural modifications of beta-D-Gal-Hyl(264) in CII(256--270) may give altered peptide ligands that can be used for induction of tolerance in autoimmune rheumatoid arthritis.
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Colágeno Tipo II/inmunología , Glicopéptidos/síntesis química , Linfocitos T/inmunología , Animales , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Conformación de Carbohidratos , Colágeno Tipo II/química , Galactosa/análogos & derivados , Galactosa/química , Glicopéptidos/metabolismo , Glicosilación , Antígenos HLA/inmunología , Hibridomas/inmunología , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/metabolismo , Interleucina-2/metabolismo , Ligandos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Estructura Molecular , Ratas , Valina/análogos & derivados , Valina/químicaRESUMEN
We examined the antigenic specificity of two T cell hybridomas elicited against the disaccharide galabiose attached to the fifth residue of the I-Ak binding peptide 52-61 of lysozyme. By making changes in the saccharide molecule and in the peptide, we conclude that the outer galactose residue of the galabiose moiety is directly recognized by the T cells together with the exposed side chains of the peptide. The overall spatial display of this galactose moiety on the 52-61 peptide is likewise important.
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Aminoácidos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Disacáridos/inmunología , Epítopos de Linfocito T/metabolismo , Glicopéptidos/inmunología , Glicopéptidos/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Aminoácidos/metabolismo , Animales , Sitios de Unión/inmunología , Linfocitos T CD4-Positivos/inmunología , Disacáridos/metabolismo , Glicina/inmunología , Glicina/metabolismo , Glicopéptidos/química , Hibridomas , Ratones , Ratones Endogámicos CBA , Muramidasa/inmunología , Muramidasa/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Serina/inmunología , Serina/metabolismoRESUMEN
The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.
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Fimbrias Bacterianas/química , Chaperonas Moleculares/química , Periplasma/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli , Modelos Moleculares , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Unión Proteica/genética , Pliegue de ProteínaRESUMEN
Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256-270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Valpha2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256-270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256-270) glycosylated with a monosaccharide (beta-D-galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.
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Artritis/inmunología , Colágeno/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Artritis/etiología , Artritis/genética , Artritis Reumatoide/etiología , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Secuencia de Bases , Colágeno/química , ADN/genética , Modelos Animales de Enfermedad , Glicosilación , Hibridomas/inmunología , Inmunización , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Estructura Molecular , Ratas , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
The 2-bromoethyl beta-glycosides of the disaccharide galabiose [Gal(alpha1-4)Gal] and the trisaccharides globotriose [Gal(alpha1-4)Gal(beta1-4)Glc] and 3'-sialyllactose [Neu5Ac(alpha2-3)Gal(beta1-4)Glc] have been prepared by improved routes. The 2-bromoethyl glycosides were then used in cesium carbonate promoted alkylations of the sulfhydryl groups of cysteine and homocysteine residues in T cell stimulating peptides. This convergent and general approach was used to prepare 16 neoglycopeptides which were obtained in 52-95% yields after purification by HPLC. 1H NMR spectroscopy revealed that beta-elimination and epimerization of neoglycopeptide stereocentres did not occur during the synthesis.
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Glicopéptidos/síntesis química , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Cisteína/química , Glicopéptidos/química , Glicopéptidos/inmunología , Glicósidos/química , Homocisteína/química , Humanos , Epítopos Inmunodominantes/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Linfocitos T/inmunologíaRESUMEN
In an attempt to raise anti-Tn antibodies, an alpha-N-acetyl-D-galactosamine glycosylated peptide based on the tandem repeat of the intestinal mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMVTPTPTPTC) was glycosylated in vitro using concentrated alpha-N-acetylgalactosaminyltransferases activity from porcine submaxillary glands which resulted in the incorporation of 8-9 mol of Ga/NAc. Rabbits and mice developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not show reactivity with the unglycosylated MUC2 peptide or with other GalNAc glycosylated peptides. A mouse monoclonal antibody (PMH1) representative of the observed immune response was generated and its immunohistological reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the staining was not restricted to the supranuclear area but extended to the entire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glycoforms of MUC2. The present work presents a useful approach for development of anti-mucin antibodies directed to different glycoforms of individual mucins.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Glicopéptidos/inmunología , Mucinas/inmunología , Animales , Western Blotting , Mapeo Epitopo , Glicopéptidos/síntesis química , Glicosilación , Humanos , Inmunohistoquímica , Ratones , Peso Molecular , Mucina 2 , Conejos , Distribución TisularRESUMEN
The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.
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Artritis/metabolismo , Colágeno/metabolismo , Glicopéptidos/metabolismo , Antígenos H-2/química , Antígenos H-2/fisiología , Epítopos Inmunodominantes/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Presentación de Antígeno , Artritis/etiología , Artritis/inmunología , Colágeno/efectos adversos , Glicopéptidos/química , Glicopéptidos/inmunología , Antígenos H-2/biosíntesis , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/inmunología , Procesamiento Proteico-Postraduccional , Ratas , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Relación Estructura-ActividadRESUMEN
Desmopressin (1-desamino-[DArg8]vasopressin, is a synthetic analogue of the neurohypophyseal peptide hormone vasopressin which has high antidiuretic and antibleeding potency. The structure of desmopressin has been determined in aqueous solution by two-dimensional NMR techniques and molecular dynamics simulations. Both standard and time-averaged distance restraints were used in structure calculations because of the inherent flexibility in small peptides. 21 models calculated with standard restraints were compared with structures refined with time-averaged distance restraints and were found to be good representatives of the conformational ensemble of desmopressin. The macrocyclic ring forms an inverse gamma-turn centered around Gln4. Residues 1 and 2, the disulphide bridge and the three-residue acyclic tail were found to be flexible in solution. Residues 4-6 in the ensemble of calculated structures contain essentially the same backbone conformation as in the crystal structure of pressinoic acid, the cyclic moiety of vasopressin, whereas residues 2-6 superimpose on the NMR-derived conformation of oxytocin bound to neurophysin. The results presented in this work suggest that, in addition to the differences in sequence between desmopressin and vasopressin, differences in conformational and dynamic properties between the two compounds explain their pharmacological differences.
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Desamino Arginina Vasopresina/química , Conformación Proteica , Amidas , Secuencia de Aminoácidos , Simulación por Computador , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Programas InformáticosRESUMEN
PapD is the prototype member of a family of periplasmic chaperones which are required for assembly of virulence associated pili in pathogenic, gram-negative bacteria. In the present investigation, an ELISA has been developed for evaluation of compounds as inhibitors of PapD. Synthetic peptides, including an octamer, derived from the C-terminus of the pilus adhesin PapG were able to inhibit PapD in the ELISA. Evaluation of a panel of octapeptides in the ELISA, in combination with NMR studies, showed that the peptides were bound as extended beta-strands by PapD in aqueous solution. The PapD-peptide complex was stabilized by backbone to backbone hydrogen bonds and interactions involving three hydrophobic peptide side chains. This structural information, together with previous crystal structure data, provides a starting point in efforts to design and synthesize compounds which bind to chaperones and interfere with pilus assembly in pathogenic bacteria.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Proteínas Periplasmáticas , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ensayo de Inmunoadsorción Enzimática , Indicadores y Reactivos , Cinética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Soluciones , Relación Estructura-ActividadRESUMEN
Interaction of the Escherichia coli PapD chaperone with the synthetic peptide PapG308-314 (Thr-Met-Val-Leu-Ser-Phe-Pro), corresponding to the seven C-terminal residues of the PapG pilus subunit, was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The observation of cross-peaks corresponding to either intraresidue or sequential C(alpha)H/NH and C(beta)H/NH TRNOEs and the absence of sequential NH(i)/NH(i+1) TRNOEs indicate that the peptide binds to PapD in an extended conformation. In addition, line-broadening effects gave information of the peptide's mode of interaction with PapD. These observations were in excellent agreement with a recent crystal structure of a PapG peptide complexed with PapD.