Defective calcium binding to fibrillin-1: consequence of an N2144S change for fibrillin-1 structure and function

Fibrillin-1 is a major structural component of 10-12 nm connective tissue microfibrils and has a modular organisation that includes 43 calcium binding epidermal growth factor-like (cbEGF) domains and seven transforming growth factor beta-binding protein-like (TB) domains. Mutations in the fibrillin-1 (FBN1) gene cause the Marfan syndrome (MFS) and related connective tissue disorders. We have previously investigated an N2144S change, identified in a MFS patient, which removes one of the key calcium binding ligands within cbEGF domain 32. In this study the structural consequences of the N2144S amino acid change for the folding and calcium binding properties of mutant and wild-type TB6-cbEGF32 and cbEGF32-33 domain pairs have been analysed by nuclear magnetic resonance. The presence of an N2144S substitution does not alter the native fold of either the TB6 domain, or cbEGF domains 32 and 33. Comparison of calcium dissociation constants measured for the wild-type and mutant pairs shows that: (i) the affinity of cbEGF32 is weakly enhanced by N-terminal linkage of TB6 relative to cbEGF32 in isolation; (ii) the affinity of cbEGF32 is approximately ninefold decreased by the N2144S substitution in the TB-cbEGF pair; and (iii) reduced affinity of cbEGF32 does not result in lower affinity of cbEGF33 for calcium. Together, these data suggest that the TB6-cbEGF32 linkage is flexible and the structural effect of the mutation is localised to the interdomain linkage. We have also investigated the effect of defective calcium binding to cbEGF32 on fibrillin-1 produced by N2144S MFS fibroblasts. 35S-pulse-chase analysis shows that the N2144S substitution does not detectably affect fibrillin-1 biosynthesis, rate of secretion or processing. Deposition of reducible fibrillin-1 into the extracellular matrix was also unaffected. The implications of these results for the assembly and properties of the microfibril are discussed.     

Cracking the auditory genetic code: nonsyndromic hereditary hearing impairment

Vascular anomalies of the head and neck are common lesions of childhood. The vascular anomalies can be divided into hemangiomas and vascular malformations. Each of these lesions has a characteristic imaging appearance. Correctly classifying the anomaly is essential in directing the treatment of these lesions.     

Paramagnetic liposomes as MRI contrast agents: influence of liposomal physicochemical properties on the in vitro relaxivity

Because of the possible application of tea in the prevention of oral and esophageal cancers, the salivary levels of tea catechins were determined in six human volunteers after drinking tea. Saliva samples were collected after thoroughly rinsing the mouth with water. After drinking green tea preparations equivalent to two to three cups of tea, peak saliva levels of (-)-epigallocatechin (EGC; 11.7-43.9 microg/ml), EGC-3-gallate (EGCG; 4.8-22 microg/ml), and (-)-epicatechin (EC; 1.8-7.5 microg/ml) were observed after a few minutes. These levels were 2 orders of magnitude higher than those in the plasma. The elimination half-life (t(1/2)) of the salivary catechins was 10-20 min, much shorter than that of the plasma. Holding a tea solution in the mouth for a few minutes without swallowing produced even higher salivary catechin levels, but taking tea solids in capsules resulted in no detectable salivary catechin level. Holding an EGCG solution in the mouth resulted in EGCG and EGC in the saliva and, subsequently, EGC in the urine. The results suggest that EGCG was converted to EGC in the oral cavity, and both catechins were absorbed through the oral mucosa. A catechin esterase activity that converts EGCG to EGC was found in the saliva. The enzyme was likely of human origin, but the activity was not inhibited by common human esterase inhibitor. The present results suggest that slowly drinking tea is a very effective way of delivering rather high concentrations of catechins to the oral cavity and then the esophagus.     

Blood protein adsorption onto a polymeric biomaterial of polyethylene glycol and poly[(2‐hydroxyethyl methacrylate)‐co‐acrylonitrile] and evaluation of in vitro blood compatibility

A semi‐interpenetrating polymer network (semi‐IPN) of polyethylene glycol (PEG) and crosslinked poly[(2‐hydroxyethyl methacrylate)‐co‐acrylonitrile] was prepared and adsorption of bovine serum albumin (BSA) on the IPN surfaces was investigated. The dynamic nature of the adsorption process was studied, and the effects of various experimental factors such as pH and ionic strength on the adsorption isotherms of BSA were investigated. Various kinetic parameters, such as the adsorption coefficient, rate constant for adsorption and penetration rate constants, were calculated. For assessment of in vitro blood compatibility of the IPN surfaces, water sorption, blood clot formation tests and percent hemolysis measurements were performed. Copyright © 2004 Society of Chemical Industry     

Carotid restenosis: operative and endovascular management

PURPOSE: Surgical management of carotid restenosis (CR) after carotid endarterectomy (CEA) has been associated with a higher perioperative complication rate than that of primary CEA. We recently used carotid angioplasty-stenting (CAS) as an alternative to operative management in patients who had undergone CEA within three years, and we retrospectively compared these results with those of operative management of CR and the overall results of CEA. METHODS: CEA was performed on 1065 adult patients (58% symptomatic, 42% asymptomatic), 62% of whom were men (n = 660) and 38% of whom were women (n = 405), from 1989 to 1997. Before our initiation of a program of CAS, 16 operative procedures (1.9% of CEAs) were performed for CR in 14 adult patients (7 women and 7 men). During the last 20 months, CAS was used in the management of 17 CRs (16 patients; 9 women and 7 men). RESULTS: The 30-day stroke morbidity-death rate for all CEAs (n = 1065) was 1.4%; 11 strokes (1. 0%) occurred (4 major strokes with disability and 7 strokes with minor or no disability), and 4 deaths (0.4%) occurred (2 deaths caused by myocardial infarction, 1 caused by intracranial hemorrhage, and 1 caused by stroke). Operative management of CR (n = 16) included patch angioplasty in 12 cases (autologous vein patches in 10 cases and synthetic patches in 2 cases), whereas interposition grafting was used in 4 cases (saphenous vein in 3 instances and synthetic [polytetrafluoroethylene] in one case). No strokes or deaths were observed. One recurrent laryngeal nerve palsy occurred (6.2%). Among the 16 patients undergoing 17 CAS procedures, the technical procedures were accomplished in all patients. No strokes or deaths occurred. No recurrent restenoses (50% or greater) have been identified within or adjacent to the CAS procedures. CONCLUSION: CR caused by myointimal hyperplasia can be managed by operative techniques or CAS with comparable periprocedural complications. Although long-term follow-up will be required to determine the incidence of recurrent restenosis, CAS may become the preferred procedure in these cases. A randomized clinical trial ultimately will be necessary to determine the role of CAS, as compared with that of operative management.     

Biliary excretion in persons with low blood glutathione levels

To examine their possible predictive value for the development of asthma, the serum concentration of eosinophil cationic protein (ECP) and the total eosinophil count were measured at admission in 25 children aged 1-17 months hospitalized for their first episode of bronchiolitis. After an average of three years the parents of 23 index patients answered a questionnaire to determine development of asthma. Eight children were defined as having asthma at follow-up based on at least three episodes of wheezing. The remaining 15 children had experienced only one or two episodes of wheezing, and all of these children had been wheeze free for the last year. The serum concentrations of ECP were similar in children who subsequently developed asthma (8.0 microg/l; 3.6 to 14.2 (median; quartiles)) and in those who did not (12 microg/l; 4.5 to 16.8). Moreover, the total eosinophil counts were similar in asthmatic (0.10 x 10(9)/l; 0.04 to 0.20) and non-asthmatic patients (0.09 x 10(9)/l; 0.02 to 0.13). In conclusion, our study suggest that neither the serum concentration of ECP nor the total eosinophil count can predict the development of asthma when measured in children admitted for their first episode of bronchiolitis, but larger studies need to be carried out to confirm these results.     

Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state

The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degreesC. In contrast, no burst phase change in fluorescence occurs during unfolding at 10 degreesC. At 22 and 34 degreesC, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, IU1, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, IU2, has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22 degreesC. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of IU1 and IU2 decrease steadily with an increase in temperature from 10 to 34 degreesC, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions.     

PCR-ELISA for the detection of Brugia malayi infection using finger-prick blood

Stone model casts of a patient's maxillary and mandibular arches were used to fabricate a clear, soft, vacuum-formed custom mouth guard that was scalloped to end 1.0 mm supragingivally. The patient was given prophylaxis and oral hygiene home care instruction, and instructions regarding the placement of an at-home bleaching gel into the mouth guard and the mouth guard into the mouth. The patient was asked to wear the mouth guard for two hours daily before bedtime for one week and to return to the clinic for evaluation. This protocol was followed for three weeks for each arch. At the end of the three weeks, the stains on the most affected teeth were reduced dramatically.     

Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses

The addition of Ags to mononuclear leukocyte cultures typically elicits modest Ab responses, implying that cosignals beyond those provided by T cells and macrophages may be needed. Recently, we reported that Ab responses could be dramatically enhanced (10-1000-fold) by the addition of follicular dendritic cells (FDC), suggesting that FDC may provide an important costimulatory signal. This result prompted a study of molecules involved in FDC-mediated enhancement of Ab responses stimulated by specific Ag with memory T and B cells or nonspecifically by the addition of LPS. In this study, we report evidence supporting the concept that FDC bear a ligand that engages complement receptor II (CR2 or CD21) on B cells and provides a critical cosignal for both Ag-specific and polyclonal responses. A blockade of the CR2 ligand on FDC by the use of soluble CR2 or a blockade of CR2 on B cells by use of CR2 knockout mice (or B cells with CR2 blocked) reduced Ab responses from the microg/ml to the ng/ml range (10-1000-fold reductions). FDC from C3 knockout mice, which cannot generate the CR2-binding fragments (iC3b, C3d, and C3dg), were unable to provide costimulatory activity, suggesting the CR2 ligand on FDC consists of C3 fragments. FDC trap complement-activating Ag-Ab complexes, and it appears that FDC present B cells with both specific Ag to engage B cell receptors and a CR2 ligand to engage B cell-CR2. In short, optimal induction of specific Ab responses appears to require the combination of specific Ag and costimulatory molecules from both T cells and FDC.