Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

The molecular genetics of Marfan syndrome and related disorders.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

A G1127S change in calcium-binding epidermal growth factor-like domain 13 of human fibrillin-1 causes short range conformational effects.

Human fibrillin-1, an extracellular matrix glycoprotein, has a modular organization that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats. A missense mutation that changes a highly conserved glycine to serine (G1127S) has been identified in cbEGF13, which results in a variant of Marfan syndrome, a connective tissue disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair indicated that the G1127S mutation caused defective folding of cbEGF13 but not cbEGF14. We have used limited proteolysis methods and two-dimensional NMR spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair and a cbEGF12-14 triple domain construct. Protease digestion studies of the cbEGF12-13 G1127S mutant pair indicated that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure to the normal domain pair, because all identified cleavage sites showed calcium-dependent protection from proteolysis. However, small changes in the conformation of cbEGF13 G1127S, revealed by the presence of a new protease-sensitive site and comparative two-dimensional NOESY data, suggested that the fold of the mutant domain was not identical to the wild-type, but was native-like. Additional cleavage sites identified in cbEGF12-14 G1127S indicated further subtle changes within the mutant domain but not the flanking domains. We have concluded the following in this study. (i) Covalent linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and (ii) conformational effects introduced by G1127S are localized to cbEGF13. This study demonstrates that missense mutations in fibrillin-1 cbEGF domains can cause short range structural effects in addition to long range effects previously observed with a E1073K mutation in cbEGF12.

Alcoholism in the emergency department: an epidemiologic study.

OBJECTIVE: To describe the epidemiology of alcoholism in ED patients. METHODS: Over a two-month period, every adult patient brought by ambulance to the ED of a large municipal hospital was prospectively enrolled by questionnaire. Data collected included demographics, previous ED use, triage complaint-related diagnoses, hospital admission rates, and ethanol levels (if determined). The CAGE alcoholism questions were administered to all patients by trained assistants. The only exclusion criterion was the inability to communicate while in the ED. A chi-square analysis was used to compare categorical variables. RESULTS: A total of 2,658 patients were enrolled in the study; 226 were unable to respond to the CAGE questions. Five hundred eighty-eight of the remaining 2,432 patients (24%) were defined as being alcoholic by an affirmative response to at least two of the CAGE questions. All four questions were answered affirmatively by 17% of the total patients. Alcoholic patients were more likely to be male (88% vs 60%), unemployed (87% vs 71%), undomiciled (46% vs 20%), polysubstance users (52% vs 25%), and tobacco users (77% vs 41%), and to have had an ED visit in the previous six months (51% vs 35%) (p < 0.001 for all tests). Ethanol levels ranged from zero to 573 mg/dL. Whereas no positive response to a single CAGE question was predictive of a final diagnosis of alcoholism, a blood ethanol level more than 300 mg/dL predicted an affirmative response to at least two CAGE questions in 97% of cases. CONCLUSIONS: Alcoholism should be presumed to be present in a substantial number of patients who present to urban EDs by ambulance.

Bilateral temporomandibular joint dislocation in a 10-month-old infant after vomiting.

We describe a case of bilateral dislocation of the temporomandibular joints (TMJ) in a 10-month-old infant. TMJ dislocations can occur after opening the mouth widely with or without direct trauma.

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1.

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.

Physician bias during patient selection in the pediatric emergency department.

STUDY OBJECTIVES: To evaluate whether pediatric or emergency medicine residents exhibit a bias when they select patients from triage based on the chief complaint, ie, medical versus surgical in the pediatric emergency department (PED). DESIGN: A retrospective chart review of a convenience sample of consecutive patients, excluding those seen during times when both pediatric and emergency medicine residents were not simultaneously present. SETTING: Urban Municipal PED with 25,000 visits annually. TYPE OF PARTICIPANTS: Pediatric residents, emergency medicine residents, and medical students rotating through the PED and their supervising attending physicians. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Five hundred and ninety-nine charts were included in the study. On the basis of the triage complaint the initial diagnosis was classified as either surgical or medical. Surgical diagnoses were assigned to those patients who required a surgical procedure, involved a surgical subspecialty or were victims of trauma and represented 151 (25.2%) of the patients seen. Medical diagnoses were assigned to the nonsurgical patients and represented 448 (74.8%) of the patients seen. There are roughly three pediatric residents to each emergency resident working in our PED. Of the 367 patients seen by the pediatric residents, 73 (19.9%) had surgical diagnoses, and 294 (80.1%) had medical diagnoses. Of the 158 patients seen by the emergency residents, 59 (37.3%) had surgical diagnoses and 99 (62.7%) had medical diagnoses. chi2 analysis was used to compare categorical variables. The P value was considered significant at <0.05. DISCUSSION: Residents, both pediatric and emergency medicine, were instructed to see patients based upon the severity of the patient illness as judged by the triage nurse unless the patients' illnesses were of equal severity, in which case they were to be seen in the order in which they presented. The null hypothesis was that in the absence of physician bias, both pediatric and emergency medicine residents would see the same proportion of surgical and medical patients. The results showed that a bias exists. Emergency medicine residents saw a greater proportion of surgical patients, and pediatric residents saw a greater proportion of general medical patients. A limitation of this study may be the that the supervising attending physician selected residents to see certain patients to expedite PED flow. CONCLUSIONS: Recognizing that bias in the selection of patients seen exists is important in ensuring a balanced education experience.

A Gly --> Ser change causes defective folding in vitro of calcium-binding epidermal growth factor-like domains from factor IX and fibrillin-1.

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refolded in vitro adopt the native fold. In contrast, the Gly --> Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.

NMR analysis of cbEGF domains gives new insights into the structural consequences of a P1148A substitution in fibrillin-1.

Fibrillin-1 is a modular glycoprotein and a major component of the 10-12 nm microfibrils of the extracellular matrix. Mutations in the fibrillin-1 (FBN 1) gene result in the connective tissue disease the Marfan syndrome (MFS) and related disorders. The calcium binding EGF-like (cbEGF) domain is the predominant structural motif of the protein and >70% of mutations leading to MFS disrupt this domain. A missense mutation which changes a proline to alanine (P1148A) in cbEGF domain 13 has been associated with a number of fibrillin disorders including MFS and Shprintzen-Goldberg syndrome. However, it has also been described as a polymorphism. In this study comparative NMR analyses on wild-type and mutant forms of covalently-linked fibrillin cbEGF domain pairs have been performed to investigate the structural consequences of this substitution. A comparison of the two-dimensional NOESY spectra of the wild-type and mutant forms of cbEGF domains 12 & 13 and cbEGF domains 13 & 14 indicated that the proline to alanine amino acid change does not introduce a significant structural defect into cbEGF domain 13 or the adjacent domains and most likely represents a polymorphism. These results demonstrate how, in the case of a protein with a well defined domain organisation such as fibrillin-1, comparative NMR analyses can be used to substantiate genetic evidence for the polymorphic status of an amino acid.

Phenoxymethylpenicillin amidohydrolases from Penicillium chrysogenum.

A phenoxymethylpenicillin amidohydrolase which hydrolyses phenoxymethylpenicillin to 6-aminopenicillanic acid (6-APA) has been isolated from two species of Penicillium chrysogenum. The amidohydrolase had a molecular mass of approx. 42 kDa. Its activity with benzylpenicillin as substrate was only 1.5% of that with phenoxymethylpenicillin and it was inhibited by its products. No penicillin formation from 6-APA and phenoxyacetyl or phenylacetyl coenzyme A was observed. The enzyme is thus distinct from the phenylacetyl coenzyme A:6-APA acyltransferase, which also has amidohydrolase activity and is involved in the final stages of the biosynthesis of penicillins.

Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.

Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes.

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans.

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.

The disposition of antipyrine and its metabolites in young and elderly healthy volunteers.

1 Oxidative metabolism of antipyrine (AP) was compared in 11 elderly (greater than 65 years) and 12 young (less than 40 years) volunteers. All subjects were non-smokers, consumed little if any alcohol and were in good health. 2 After a single dose of AP 500 mg, its clearance from saliva and profiles of the parent drug and its major metabolites in urine were determined using high-performance liquid chromatography. 3 Mean total AP clearance from saliva was lower in the elderly (P less than 0.05). Mean weight-normalised volume of distribution was also smaller (P less than 0.01) so that elimination half-life in the elderly was not significantly different from that in the young. 4 The percentage dose excreted in 48 h urine as norantipyrine (NORA) and its clearance for production were lower in the elderly (P less than 0.001 and P less than 0.01 respectively). Urinary 3-hydroxymethylantipyrine (HMA) and free antipyrine were present in greater quantities in 48 h urine in the elderly (P less than 0.001 and P less than 0.05) while the amounts of 4-hydroxyantipyrine (OHA) were almost identical in the two age groups. 5 The findings suggest that there is a selective impairment of N-demethylation in the elderly which may have important implications for dosage of elderly patients with drugs metabolised by this route.

Postcesarean wound infection by Mycoplasma hominis in a patient with persistent postpartum fever.

Mycoplasma hominis was isolated in pure culture from a wound infection following delivery by cesarean section. The importance of recognizing this organism as a potential pathogen of the female genital tract is emphasized. Two commercially available isolation systems that allow the recovery of this organism are also described.

A515U: a prodrug of acyclovir with increased oral bioavailability.

The tolerance and pharmacokinetics of A515U, a xanthine oxidase-activated prodrug of acyclovir have been investigated in healthy volunteers and in two phase-I clinical studies in immunocompromised patients. In all cases the bioavailability of acyclovir following oral administration of A515U was substantially increased over that achieved in the same subjects with oral acyclovir itself. Plasma acyclovir levels were similar to those previously attainable only with intravenous acyclovir. This increase in bioavailability may permit reductions in the frequency of administration and extend the range of herpes virus infections amenable to oral therapy. A515U was very well tolerated, with no significant clinical adverse events being attributed to the drug.

Agranulocytosis associated with maloprim: review of cases.

Twenty-one cases of agranulocytosis following dosage with one tablet of Maloprim given once or twice weekly are reviewed. Of the 18 individuals for whom the dose is certain 12 were taking twice-weekly Maloprim. Time of onset was 7-9 weeks in 15 of 19 cases where duration of dosage is known. Nine patients died, 12 recovered. Of those who died six were taking Maloprim twice weekly, one once weekly and two at an uncertain dosage. The available data suggest that this is an idiosyncratic reaction to dapsone exacerbated by the concomitant administration of pyrimethamine.

Retrospective evaluation of the isolation and identification of herpes simplex virus with Cultureset and human fibroblasts.

A total of 442 specimens from various anatomic sites was cultured for herpes simplex virus during the past 2 years. Most specimens were obtained from the respiratory tracts and cutaneous lesions of immunocompromised hosts (232 specimens) or the female genital tract (138 specimens). Two tubes containing human newborn foreskin fibroblasts and two Ortho Cultureset tubes containing Vero cells were inoculated with each specimen. The 384 inoculated specimens were stained with Cultureset peroxidase-antiperoxidase reagents within 48 h and again at 3, 4, or 5 days if initially negative. Fibroblasts were inspected for cytopathic effect for 7 days. Of these 384 specimens, Cultureset detected 57 of 62 positive specimens within 48 h; fibroblasts detected 58 positive specimens by 7 days. The calculated sensitivity and specificity for Cultureset at 48 h were 91.9 and 100%, respectively. However, when all results were considered, including those that became positive in Cultureset after 48 h, the calculated sensitivity and specificity for Cultureset were 98.8 and 100%, respectively. We conclude that Cultureset is a reliable method for detection of herpes simplex virus when two tubes are inoculated and stained as described.

The clinical pharmacology of acyclovir and its prodrugs.

Acyclovir (Zovirax) is a highly specific antiherpes virus agent. Extensive investigations of the pharmacokinetics in man have shown it to have a useful half-life of about three hours and to be largely excreted unchanged in the urine. Crystaluria can be avoided provided the patient is well hydrated and attention is paid to the dosing instructions especially in patients with renal failure. In vitro ED50s (the drug concentration inhibiting virus replication by 50%) bear some general relevance to effective plasma levels in man. A new prodrug of acyclovir, 2-amino-9-[2-hydroxyethoxy methyl]-9H-purine (A515U), which is converted to acyclovir by xanthine oxidase is rapidly absorbed from the human gut and converted to acyclovir. This prodrug provides the opportunity to design regimes that are more convenient for the patient and may be more effective than acyclovir itself in the therapy of the less sensitive herpes viruses (e.g. Epstein-Barr virus and the Cytomegalovirus).

The disposition of bupropion and its metabolites in healthy male volunteers after single and multiple doses.

The pharmacokinetics of bupropion and 3 of its basic metabolites were determined in 8 young, healthy, male volunteers after single and multiple oral doses of bupropion. Plasma profiles were obtained: 1) after a single 100 mg oral dose of bupropion hydrochloride, 2) following administration of 100 mg 8-hourly for 14 days and 3) again after a single 100 mg dose 14 days later. Plasma concentrations of the parent drug and metabolites were determined by high-performance liquid chromatography. Saliva secretion and pupil diameters were measured, subjective assessments of sleep made using visual analogue scales and side effects, blood counts and biochemistry were monitored. After the first dose mean elimination half lives (t1/2) of bupropion, and metabolites I and II were 8, 19 and 19 h respectively. On repeated administration there was little accumulation of the parent drug and no evidence for induction of its own metabolism. Accumulation of I was consistent with its rate of elimination after single doses while that of II was greater than predicted with prolongation of t1/2 to 35 h. Metabolite III was barely detectable after single doses but its accumulation on multiple dosing was consistent with its long half life (35 h) determined on occasion 2. Saliva secretion was significantly reduced during the multiple dosing period but there were no complaints of dry mouth. Subjective assessments of sleep were not significantly altered though one subject reported vivid dreams. There were no other adverse reactions.