RESUMEN

We have recently developed a range of synthetic retinoid analogues which include the compounds EC23 and EC19. They are stable on exposure to light and are predicted to be resistant to the normal metabolic processes involved in the inactivation of retinoids in vivo. Based on the position of the terminal carboxylic acid groups in the compounds we suggest that EC23 is a structural analogue of all-trans retinoic acid (ATRA), and EC19 is an analogue of 13-cis retinoic acid. Their effects on the differentiation of pluripotent stem cells has been previously described in vitro and are consistent with this hypothesis. We present herein the first description of the effects of these molecules in vivo. Retinoids were applied to the anterior limb buds of chicken embryos in ovo via ion-exchange beads. We found that retinoid EC23 produces effects on the wing digits similar to ATRA, but does so at two orders of magnitude lower concentration. When larger quantities of EC23 are applied, a novel phenotype is obtained involving production of multiple digit 1s on the anterior limb. This corresponds to differential effects of ATRA and EC23 on sonic hedgehog (shh) expression in the developing limb bud. With EC23 application we also find digit 1 phenotypes similar to thumb duplications described in the clinical literature. EC23 and ATRA are shown to have effects on the entire proximal-distal axis of the limb, including hitherto undescribed effects on the scapula. This includes suppression of expression of the scapula marker Pax1. EC23 also produces effects similar to those of ATRA on the developing face, producing reductions of the upper beak at concentrations two orders of magnitude lower than ATRA. In contrast, EC19, which is structurally very similar to EC23, has novel, less severe effects on the face and rarely alters limb development. EC19 and ATRA are effective at similar concentrations. These results further demonstrate the ability of retinoids to influence embryonic development. Moreover, EC23 represents a useful new tool to investigate developmental processes and probe the mechanisms underlying congenital abnormalities in vertebrates including man.

Asunto(s)

Desarrollo Embrionario/efectos de los fármacos , Extremidades/embriología , Cara/embriología , Esbozos de los Miembros/efectos de los fármacos , Retinoides/farmacología , Animales , Benzoatos , Embrión de Pollo/metabolismo , Proteínas Hedgehog/metabolismo , Reacción en Cadena de la Polimerasa , Tetrahidronaftalenos

RESUMEN

Nucleostemin (NS), a nucleolar GTPase, is highly expressed in stem/progenitor cells and in most cancer cells. However, little is known about the regulation of its expression. Here, we identify the NS gene as a novel direct transcriptional target of the c-Myc oncoprotein. We show that Myc overexpression enhances NS transcription in cultured cells and in pre-neoplastic B cells from Eµ-myc transgenic mice. Consistent with NS being downstream of Myc, NS expression parallels that of Myc in a large panel of human cancer cell lines. Using chromatin immunoprecipitation we show that c-Myc binds to a well-conserved E-box in the NS promoter. Critically, we show NS haploinsufficiency profoundly delays Myc-induced cancer formation in vivo. NS+/-Eµ-myc transgenic mice have much slower rates of B-cell lymphoma development, with life spans twice that of their wild-type littermates. Moreover, we demonstrate that NS is essential for the proliferation of Myc-overexpressing cells in cultured cells and in vivo: impaired lymphoma development was associated with a drastic decrease of c-Myc-induced proliferation of pre-tumoural B cells. Finally, we provide evidence that in cell culture NS controls cell proliferation independently of p53 and that NS haploinsufficiency significantly delays lymphomagenesis in p53-deficient mice. Together these data indicate that NS functions downstream of Myc as a rate-limiting regulator of cell proliferation and transformation, independently from its putative role within the p53 pathway. Targeting NS is therefore expected to compromise early tumour development irrespectively of the p53 status.

Asunto(s)

Proteínas de Unión al GTP/genética , Haploinsuficiencia , Proteínas Nucleares/genética , Oncogenes/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Humanos , Linfoma/genética , Linfoma/patología , Ratones , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a chronic autoimmune arthropathy. Beta 2-adrenergic receptors are a link between the sympathetic nervous system and the immune system. Associations between variants in the gene encoding the beta 2-adrenergic receptor (ADRB2) and autoimmune disorders such as rheumatoid arthritis (RA) have been demonstrated. We aimed to investigate ADRB2 variants for association with JIA. METHODS: Genotypes and haplotypes of two ADRB2 variants (G16R and Q27E) were determined in 348 children with JIA and 448 healthy controls by direct molecular haplotyping using melting-curve analysis of a fluorescently labelled loci-spanning probe. Case-control analysis was performed to investigate whether ADRB2 variants were associated with JIA. RESULTS: No association was found between JIA and alleles, genotypes, or haplotypes of ADRB2. Specifically, the haplotype that demonstrated a strong association with RA (R16/Q27) was not associated with JIA. None of the variants demonstrated association after stratification by JIA subtypes or gender. CONCLUSIONS: Our results indicate that ADRB2 variants are not associated with JIA or any of the major JIA subtypes. These observations suggest that, although they share several clinical and pathological features, JIA and RA have unique genetic associations.

Asunto(s)

Artritis Juvenil/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Receptores Adrenérgicos beta 2/genética , Niño , Femenino , Haplotipos , Humanos , Masculino

RESUMEN

The mode of action of retinoids in relation to their activity in the adult central nervous system and the potential of synthetic retinoid analogues is reviewed. Investigation into the activity of such molecules will further our understanding of the retinoid pathway during nervous system development and in various neurological disease states.

Asunto(s)

Modelos Biológicos , Sistema Nervioso/efectos de los fármacos , Retinoides/farmacología , Retinoides/fisiología , Adulto , Animales , Humanos , Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/tratamiento farmacológico

RESUMEN

Twenty isolates of group B streptococcus (GBS) were recovered from the milk of cows with bovine mastitis on three farms located in the south and south-east of Brazil between 1987 and 1988. These isolates were characterised by molecular methods and compared with a collection of 103 human GBS isolates from colonised and infected patients in the same region between 1980 and 2003. Some of the bovine isolates shared identical or similar pulsed-field gel electrophoresis (PFGE) patterns with a PFGE clone of human GBS type V. In addition, these bovine and human isolates also possessed the same ribotype. Multilocus sequence typing (MLST) of representative isolates confirmed the genetic relationship between the human and bovine GBS isolates with identical PFGE patterns, which clustered in the same ST-26 clonal complex. These data support the hypothesis that some bovine GBS strains are related closely to human isolates and may infect humans, or vice versa. Further comparative genomic analyses of GBS isolates from bovine and human origins are required to investigate this hypothesis further.

Asunto(s)

Mastitis Bovina/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/clasificación , Streptococcus agalactiae/genética , Animales , Técnicas de Tipificación Bacteriana , Brasil , Bovinos , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Ribotipificación , Análisis de Secuencia de ADN , Serotipificación , Infecciones Estreptocócicas/veterinaria

RESUMEN

Juvenile rheumatoid arthritis (JRA) is mediated by Th1-immune responses. In children with JRA, synovial T cells express high levels of the Th1-chemokine receptor CC chemokine receptor 5 (CCR5), which has been implicated in susceptibility to rheumatoid arthritis. To test the hypothesis that genetic variation in CCR5 is associated with susceptibility to JRA, we analyzed patterns of variation in the 5'cis-regulatory region of CCR5 in 124 multiplex families from a JRA-affected sibpair registry. After sequencing the upstream region of CCR5, variants were tested for association with JRA by transmission disequilibrium testing. A single nucleotide polymorphism, C-1835T, was significantly undertransmitted to children with early-onset JRA (P<0.01). C-1835T was genotyped in 424 additional simplex and multiplex families. CCR5-1835T allele was undertransmitted in the cohort of all probands with JRA (P<0.02), as well as in those with early-onset (P<0.01) or pauciarticular JRA (P<0.05). Another variant, a 32-bp deletion in the open reading frame of CCR5 (CCR5-Delta32) was also tested in approximately 700 simplex and multiplex families. CCR5-Delta32 was also significantly undertransmitted to probands with early-onset JRA (P<0.05). Both variants are in regions under natural selection, and result in functional consequences. Our results suggest these CCR5 variants are protective against early-onset JRA.

Asunto(s)

Artritis Juvenil/genética , Polimorfismo Genético , Receptores CCR5/genética , Secuencia de Bases , Niño , Preescolar , Estudios de Cohortes , Femenino , Eliminación de Gen , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido Simple

RESUMEN

From an initial cohort of 15 children, between the ages of 5 and 10 years, who had expressive oral language problems, 4 were shown in an earlier study to be both motor and language impaired. Two explanatory hypotheses were proposed to account for this communality: (a) cerebellar deficit; (b) inter- and/or intrahemispheric deficit. In order to explore the validity of the latter explanation, the same group of children, together with a matched control group, were required to carry out two sensory matching tests designed to tap inter- and intrahemispheric information processing abilities: hand-hand and foot-hand. The results, discussed in the light of Liederman's shielding model, provided more support for the hypothesis of an interhemispheric information processing problem from left to right rather than an intrahemispheric problem.

Asunto(s)

Corteza Cerebral/anomalías , Corteza Cerebral/fisiopatología , Lateralidad Funcional/fisiología , Trastornos del Desarrollo del Lenguaje/etiología , Trastornos de la Destreza Motora/etiología , Vías Nerviosas/anomalías , Vías Nerviosas/fisiopatología , Niño , Preescolar , Cognición/fisiología , Femenino , Humanos , Trastornos del Desarrollo del Lenguaje/patología , Trastornos del Desarrollo del Lenguaje/fisiopatología , Masculino , Modelos Neurológicos , Destreza Motora/fisiología , Trastornos de la Destreza Motora/patología , Trastornos de la Destreza Motora/fisiopatología , Pruebas Neuropsicológicas , Reproducibilidad de los Resultados , Conducta Verbal/fisiología

RESUMEN

Previous work divided serotype III group B streptococci (GBS) into 3 major phylogenetic lineages (III-1, III-2, and III-3) on the basis of bacterial DNA restriction digest patterns (RDPs). Most neonatal invasive disease was caused by III-3 strains, which implies that III-3 strains are more virulent than III-2 or III-1 strains. In the current studies, all RDP III-3 and III-1 strains expressed hyaluronate lysase activity; however, all III-2 strains lack hyaluronate lysase activity, because the gene that encodes hyaluronate lysase, hylB, is inactivated by IS1548. Subtractive hybridization was used to identify 9 short DNA sequences that are present in all the III-3 strains but not in any of the III-2 or III-1 strains. With 1 exception, these III-3-specific sequences were not detected in nonserotype III GBS. These data further validate the RDP-based subclassification of GBS and suggest that lineage-specific genes will be identified, which account for the differences in virulence among the lineages.

Asunto(s)

Polisacárido Liasas/genética , Streptococcus/genética , Southern Blotting , Enzimas de Restricción del ADN , Elementos Transponibles de ADN , ADN Bacteriano/análisis , ADN Bacteriano/genética , Humanos , Hibridación de Ácido Nucleico , Filogenia , Polisacárido Liasas/química , Análisis de Secuencia de ADN , Streptococcus/clasificación , Streptococcus/enzimología , Virulencia/genética

RESUMEN

Ruthenium(II) complexes can be used to oxidise N-Boc hydroxylamine the the presence of tert-butylhydroperoxide to the corresponding nitroso dienophile, which is trapped using cyclohexa-1,3-diene as the hetero-Diels-Alder adduct; direct evidence has been obtained for the intervention of a triphenylphosphine oxide-stabilised ruthenium(IV) oxocomplex as the catalytically active species.

RESUMEN

TfdA is a non-heme iron enzyme which catalyzes the first step in the oxidative degradation of the widely used herbicide (2, 4-dichlorophenoxy)acetate (2,4-D). Like other alpha-keto acid-dependent enzymes, TfdA utilizes a mononuclear Fe(II) center to activate O(2) and oxidize substrate concomitant with the oxidative decarboxylation of alpha-ketoglutarate (alpha-KG). Spectroscopic analyses of various Cu(II)-substituted and Fe(II)-reconstituted TfdA complexes via electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), and UV-vis spectroscopies have greatly expanded our knowledge of the enzyme's active site. The metal center is coordinated to two histidine residues as indicated by the presence of a five-line pattern in the Cu(II) EPR signal, for which superhyperfine splitting is attributed to two equivalent nitrogen donor atoms from two imidazoles. Furthermore, a comparison of the ESEEM spectra obtained in H(2)O and D(2)O demonstrates that the metal maintains several solvent-accessible sites, a conclusion corroborated by the increase in multiplicity in the EPR superhyperfine splitting observed in the presence of imidazole. Addition of alpha-KG to the Cu-containing enzyme leads to displacement of an equatorial water on copper, as determined by ESEEM analysis. Subsequent addition of 2,4-D leads to the loss of a second water molecule, with retention of a third, axially bound water. In contrast to these results, in Fe(II)-reconstituted TfdA, the cosubstrate alpha-KG chelates to the metal via a C-1 carboxylate oxygen and the alpha-keto oxygen as revealed by characteristic absorption features in the optical spectrum of Fe-TfdA. This binding mode is maintained in the presence of substrate, although the addition of 2,4-D does alter the metal coordination environment, perhaps by creating an O(2)-binding site via solvent displacement. Indeed, loss of solvent to generate an open binding site upon the addition of substrate has also been suggested for the alpha-keto acid-dependent enzyme clavaminate synthase 2 [Zhou et al. (1998) J. Am. Chem. Soc. 120, 13539-13540]. Nitrosyl adducts of various Fe-TfdA complexes have also been investigated by optical and EPR spectroscopy. Of special interest is the tightly bound NO complex of Fe-TfdA.(alpha-KG).(2,4-D), which may represent an accurate model of the initial oxygen-bound species.

Asunto(s)

Ácido 2,4-Diclorofenoxiacético/química , Metales/química , Oxigenasas de Función Mixta/química , Ácido 2,4-Diclorofenoxiacético/metabolismo , Alcaligenes , Sitios de Unión , Biodegradación Ambiental , Cobre/química , Deuterio , Espectroscopía de Resonancia por Spin del Electrón , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Hierro/química , Hierro/metabolismo , Ligandos , Metales/metabolismo , Oxigenasas de Función Mixta/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxígeno/metabolismo

RESUMEN

A series of novel 6-substituted 5,6-dihydro-5-hydroxy-alpha-pyrone esters, 1 approximately 3, isolated from fermentations of a Phomopsis sp. (Xenova culture collection no. X22502) have been identified as inhibitors of lipopolysaccharide (LPS)-induced cytokine production. These include the (6S)-4,6-dimethyldodecadien-2E,4E-dienoyl ester of phomalactone, 1, and two analogues bearing a prop-2E-enoic acid moiety at the 6-position of the alpha-pyrone ring. (6S)-4,6-Dimethyl-2E,4E-dienoic acid, 4, and a hydroxylated analogue, 5, were also isolated and characterised. The most potent cytokine production inhibitor was 1, which inhibited LPS-induced tumour necrosis factor alpha (TNFalpha) production by U937 cells and LPS-induced interleukin 1beta (IL-1beta) production by peripheral blood mononuclear cells (PBMC) with IC50 values of 80 nM and 190 nM respectively. The effect of 1 in PBMC was selective for IL-1beta relative to TNFalpha. The inhibition of IL-1beta production by 1 involved a post-translational mechanism of action at the level of IL-1beta secretion as demonstrated by the lack of an effect on cell-associated IL-1beta production. 1 showed no effect on the activity of caspase 1 in cytosolic extracts from the THP1 monocytic cell line.

Asunto(s)

Interleucina-1/biosíntesis , Lactonas/aislamiento & purificación , Lactonas/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Pironas/aislamiento & purificación , Factor de Necrosis Tumoral alfa/biosíntesis , Células U937/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ésteres/química , Ésteres/aislamiento & purificación , Ésteres/farmacología , Fermentación , Humanos , Lactonas/química , Estructura Molecular , Pironas/química , Pironas/farmacología , Relación Estructura-Actividad , Células U937/metabolismo

RESUMEN

Cytomegalovirus (CMV) is a significant pathogen among immunocompromised patients. We compared supernatant and sediment fractions of centrifuged urine for the optimal recovery of CMV by shell vial culture and polymerase chain reaction (PCR). Of 336 urine specimens, 31 (9.23%) were positive by shell vial culture; of these 29 (93.5%) were identified using the sediment fraction and 17 (54.8%) using the supernatant fraction (p = 0.001, chi2). Of the 29 positive sediment fraction specimens, 24 (82.8%) were identified as CMV positive at 24 h and 5 (17.2%) were identified as positive at 48 h. Two (0.064%) of the total 31 positive specimens were lost to microbial contamination in the sediment inoculated cultures. Of the 17 supernatant fraction specimens, 9 (53.9%) were identified as CMV positive at 24 h and 8 (47.1%) were identified as positive at 48 h. Fourteen (45.2%) of the total 31 positive specimens were lost to either toxicity or microbial contamination in the sediment-inoculated cultures. Thirty-four CMV culture-positive specimens were tested by PCR; 5 of these specimens (14.7%) were PCR negative for both sediment and supernatant fractions; 26 (76.5%) were found to be positive using the sediment fraction and negative using the supernatant; 3 (8.8%) were PCR positive for both the sediment and the supernatant. None of the 34 was identified as positive using the supernatant fraction only (p = 0.001, chi2). These findings demonstrate that the method of specimen preparation can significantly affect the outcome of diagnostic testing for CMV from urine specimens.

Asunto(s)

Infecciones por Citomegalovirus/orina , Citomegalovirus/aislamiento & purificación , Antígenos Virales/genética , Línea Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , ADN Viral , Humanos , Proteínas Inmediatas-Precoces/genética , Reacción en Cadena de la Polimerasa

RESUMEN

This study investigated the degree to which speed of stereoscopic translational motion (i.e. moving binocular disparity information) can be discriminated in a display that minimizes position information. Observers viewed dynamic random-element stereograms depicting arrays of randomly positioned stereoscopic dots that moved bidirectionally. Two tasks were performed: a speed discrimination task and a displacement discrimination task. Across a range of conditions, speed could be discriminated under conditions in which displacement could not. Thus, speed of stereoscopic motion can be discriminated when position information is minimal. This result indicates that stereoscopic motion is sensed in a way that cannot be explained by feature tracking or by inferring the motion from memory of position and time.

Asunto(s)

Percepción de Profundidad/fisiología , Percepción de Movimiento/fisiología , Umbral Diferencial , Femenino , Humanos , Masculino , Reconocimiento Visual de Modelos/fisiología , Factores de Tiempo , Disparidad Visual/fisiología

RESUMEN

Whereas all other members of the extradiol-cleaving catechol dioxygenase family are iron-dependent, the 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is dependent on manganese for catalytic activity. Recently, the endogenous iron ligands of one family member, the 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), were identified crystallographically as two histidines and a glutamic acid [Sugiyama, K., et al. (1995) Proc. Jpn. Acad., Ser. B 71, 32-35; Han, et al. (1995) Science 270, 976-980; Senda, T., et al. (1996) J. Mol. Biol. 255, 735-752]. Though BphC and MndD have low overall sequence identity (23%), the three BphC metal ligands are all conserved in MndD (H155, H214, and E266). In order to determine whether these residues also act as ligands to manganese in MndD, site-directed mutants of each were constructed, purified, and analyzed for activity and metal content. Mutations H155A, H214A, and E266Q yielded purified enzymes with specific activities of <0.1% of that of the wild-type dioxygenase and bound 0.4, 1.8, and 33% of the wild-type level of manganese, respectively. The relatively high level of manganese [with a Mn(II) EPR signal distinctly different from that of the wild-type enzyme] observed for E266Q suggests that the glutamine may act as a weak ligand to the metal. Mutant E266D, which retains the potential metal binding capability of a carboxylate group, exhibited 12% of the wild-type activity in crude extracts, suggesting that Mn remains bound; however, this mutant protein was too unstable to be purified and analyzed for metal content. On the basis of the low activity and metal content of mutant proteins, we propose that the conserved residues H155, H214, and E266 ligate manganese in MndD. As is the case with the superoxide dismutases, the extradiol-cleaving catechol dioxygenases appear to utilize identical coordinating residues for their iron- and manganese-dependent enzymes.

Asunto(s)

Arthrobacter/enzimología , Dioxigenasas , Manganeso/metabolismo , Oxigenasas/metabolismo , Secuencia de Aminoácidos , Arthrobacter/genética , Sitios de Unión , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oxigenasas/genética

RESUMEN

A manganese-dependent 3,4-dihydroxyphenylactate 2,3-dioxygenase from Arthrobacter globiformis strain CM-2 (MndD) cloned in Escherichia coli has been purified to homogeneity. Sedimentation equilibrium analysis indicates an alpha 4 homotetrameric holoenzyme structure (4 x 38,861 Da). Steady-state kinetic analysis of MndD with a variety of substrates and inhibitors yields very similar relative rates to the known Fe(II)- and Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenases from Pseudomonas ovalis and Bacillus brevis, respectively. Yet, unlike the Fe(II)-dependent enzyme, MndD retains almost all activity in the presence of H2O2 and CN- and is inactivated by Fe(II). ICP emission analysis confirms the presence of 3.0 +/- 0.2 g-atoms Mn (and only 0.7 +/- 0.2 g-atoms Fe) per tetrameric holoenzyme molecule. Comparison of MndD samples with varying metal content, including an apo and partial-apo enzyme preparation, shows a strong positive correlation between specific activity and Mn content. EPR spectra of MndD as isolated exhibit a nearly isotropic g = 2.0 signal having 6-fold hyperfine splitting (A = 95 G) typical of octahedrally coordinated Mn(II) in a protein. Quantitation of the EPR spin yields 3.4 +/- 0.3 g-atoms of Mn(II) per holoenzyme. When exposed anaerobically to its natural substrate, 3,4-dihydroxyphenylacetate (3,4-DHPA), the EPR spectrum undergoes a dramatic change characterized by the attenuation of the g = 2 signal and the appearance of new signals at g = 1.2, 2.9, 4.3, and 16. The g = 4.3 signal displays 6-fold hyperfine splitting (A = 95 G) that unambiguously assigns it to the Mn(II) center. The appearance of these new signals indicates a large increase in zero-field splitting suggestive of a change in ligand coordination to the Mn(II) center. Similarly perturbed signals are seen in the EPR spectra of MndD complexed with the comparably active substrate analog, D,L-3,4-dihydroxymandelate, or the tight-binding inhibitor, p-nitrocatechol, but not in the complexes with weaker binding substrates and inhibitors. The fact that only strong-binding substrates and inhibitors significantly perturb the Mn(II) EPR signal strongly suggests that the substrate coordinates to the Mn(II) center in the catalytic pathway.

Asunto(s)

Arthrobacter/enzimología , Dioxigenasas , Manganeso/metabolismo , Oxigenasas/metabolismo , Catecol 2,3-Dioxigenasa , Cromatografía por Intercambio Iónico , Clonación Molecular , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Cinética , Peso Molecular , Oxigenasas/química , Oxigenasas/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato

RESUMEN

The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.

RESUMEN

The present study describes the development of an enzyme-linked immunosorbent assay capable of quantifying serum antibody of all four canine IgG subclasses. A panel of subclass-restricted and subclass-specific monoclonal antibodies was used to measure IgG subclasses in the serum of healthy dogs, as well as in dogs with a range of clinical diseases. The subclasses have been redefined as IgG1, IgG2, IgG3 and IgG4 based on a comparison with the relative concentration and electrophoretic mobilities of human IgG subclasses. In serum samples from healthy dogs, the concentration of IgG1 (mean, 8.17 +/- 0.95 mg ml-1) and IgG2 (mean, 8.15 +/- 3.16 mg ml-1) were very similar and considerably higher than the levels of IgG3 (mean, 0.36 +/- 0.43 mg ml-1) and IgG4 (mean, 0.95 +/- 0.45 mg ml-1). There was no apparent difference in the level of subclasses between the different breeds comprising this normal population. Sera from dogs with a range of immune-mediated or inflammatory diseases all had markedly elevated levels of IgG2 (more than 13 mg ml-1), but IgG1 decreased (less than 4 mg ml-1) to levels below the normal range.

Asunto(s)

Enfermedades de los Perros/inmunología , Perros/inmunología , Inmunoglobulina G/sangre , Anemia Hemolítica Autoinmune/sangre , Anemia Hemolítica Autoinmune/inmunología , Anemia Hemolítica Autoinmune/veterinaria , Animales , Enfermedades del Ano/sangre , Enfermedades del Ano/inmunología , Enfermedades del Ano/veterinaria , Proteínas Sanguíneas/análisis , Enfermedades de los Perros/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Forunculosis/sangre , Forunculosis/veterinaria , Hipotiroidismo/sangre , Hipotiroidismo/inmunología , Hipotiroidismo/veterinaria , Inmunoglobulina G/clasificación , Masculino , Mieloma Múltiple/sangre , Mieloma Múltiple/inmunología , Mieloma Múltiple/veterinaria , Valores de Referencia , Albúmina Sérica/análisis , Seroglobulinas/análisis

RESUMEN

Canine IgG is composed of four subclasses, which are defined as IgG1, IgG2, IgG3 and IgG4 on the basis of data from fast protein liquid chromatography, and their electrophoretic mobilities and relative concentrations in serum. This paper describes the preparation of mAbs specific for determinants on canine IgG2, IgG3 and IgG4. The mAb specific for IgG2 resulted from a conventional immunisation protocol. The mAb specific for IgG3 was a result of immunisation with IgG3 combined with the suppression of the immune response to IgG1 by passively administered anti-IgG1 antibody. The mAb specific for IgG4 resulted from immunisation with Fab or Fc fragments which were obtained by the cleavage of the IgG4 molecule with papain. The specificity of each mAb was established by using an enzyme-linked immunosorbent assay which showed that all three specific clones recognised a determinant in the Fd region of the canine immunoglobulin molecule.

Asunto(s)

Anticuerpos Monoclonales , Perros/inmunología , Inmunoglobulina G/sangre , Animales , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Epítopos/análisis , Inmunización , Fragmentos Fab de Inmunoglobulinas , Fragmentos Fc de Inmunoglobulinas , Inmunoglobulina G/clasificación , Ratones , Ratones Endogámicos BALB C/inmunología , Valores de Referencia

RESUMEN

Raman, absorbance, and kinetic measurements were used to determine how the serine protease active site feature known as the oxyanion hole interacts with an acyl-enzyme intermediate. The substrate, p-(dimethylamino)benzoylimidazolide (DAB-Im), was synthesized and used to prepare DAB-acyl-enzymes of wild-type (WT) and N155G subtilisin-BPN' (the N155G mutant lacks a fully functioning oxyanion hole), alpha-chymotrypsin (CHT), and bovine trypsin (TRY). DAB-acyl-enzyme deacylation rate constants, k3, were found to span a 720-fold range at pH 7.8 (DAB-WT > DAB-TRY > DAB-N155G > DAB-CHT). DAB-N155G was found to deacylate 80-fold slower than DAB-WT, indicating a 2.6 kcal/mol loss of transition-state binding energy due to this mutation. Absorbance spectra revealed strongly red-shifted absorbance lambda max values for all of the DAB-acyl-enzymes. The red shift was found to be 2.0 nm less in DAB-N155G, indicating that the oxyanion hole is partially responsible for this electronic perturbation of the DAB chromophore at the active site. Raman difference spectra of the DAB-acyl-enzymes measured at pH 5.0 and 8.6, with 18O-labeling of the carbonyl, show that the molecular motions most perturbed by the active site are three associated with the scissile acyl bond. Most interesting is the carbonyl stretching vibration, v(C = O), whose motion extends into the hydrolytic reaction coordinate. Comparison of the v(C = O) of DAB-WT and DAB-N155G reveals that the oxyanion hole does indeed form a hydrogen-bonding interaction with the carbonyl oxygen, the strength of which increases at pH 8.6. Interestingly, the DAB-TRY carbonyl forms very strong hydrogen bonds, even at pH 5.0, but DAB-CHT does not, even at pH 8.6. The low-frequency (1661 cm-1) v(C = O)'s of pH 5.0 DAB-TRY and pH 8.6 DAB-WT are proposed to correspond to a tetrahedrally distorted carbonyl center like that observed in the crystal structure of guanidinobenzoyl-TRY (Mangel et al., 1990). The strength of hydrogen bonding between the DAB-acyl-enzyme's carbonyl and the oxyanion hole, as gauged by the v(C = O) frequency, was found to correlate positively with an increased deacylation rate. This correlation, as well as calculated acyl-enzyme carbonyl bond lengths, which indicate a 0.015-A lengthening due to the oxyanion hole interaction, was found to be in good agreement with previously published resonance Raman data of alpha, beta-unsaturated arylacryloyl-acyl-enzymes (Tonge & Carey, 1990b, 1992).

Asunto(s)

Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Acilación , Aniones , Sitios de Unión , Catálisis , Quimotripsina/metabolismo , Cristalización , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Espectrofotometría , Espectrometría Raman , Termodinámica , Tripsina/metabolismo , p-Dimetilaminoazobenceno/metabolismo