A fixed denture for a child with epidermolysis bullosa simplex.

AIM: To report the caries treatment and delivery of a fixed denture for a 3-year-old girl with epidermolysis bullosa simplex (EBS). CASE REPORT: EBS is manifested on the skin or mucous membranes where skin separation is easily induced by trauma. Full- mouth rehabilitation under in-patient general anaesthesia was performed to the patient in conjunction with proper pre- and postoperative care. A fixed denture was fabricated and installed to replace the extracted teeth without later causing irritation on the mucosa. The prosthesis restored aesthetics and provided comfort without imposing the burden of compliance on the patient. CONCLUSION: Aided by meticulous pre- and postoperative care and oral hygiene reinforcement, comprehensive dental treatment coupled with fixed denture delivery can greatly improve the life quality and aesthetics for children with EBS.

Identification of novel loci for bipolar I disorder in a multi-stage genome-wide association study.

OBJECTIVE: Identification of genetic variants that influence bipolar I disorder (BPD-I) through genome-wide association (GWA) studies is limited in Asian populations. The current study aimed to identify novel common variants for BPD-I in an ethnically homogeneous Taiwanese sample using a multi-stage GWA study design. METHOD: At the discovery stage, 200 BPD-I patients and 200 controls that combined to form 16 pools were genotyped with 1 million markers. Utilizing a newly developed rank-based method, top-ranked markers were selected. After validation with individual genotyping, a fine-mapping association study was conducted to identify associated loci using 240 patients and 240 controls. At the last stage, independent samples were collected (351 cases and 341 controls) for replication. RESULTS: Among the top-ranked markers from the discovery stage, eight genes and 15 individual SNPs were evaluated in the fine-mapping stage. At this stage, rs7619173, which is not in a gene coding region, showed the most significant association (P = 2 ∗ 10(-5)) with BPD-I. Four genes had empirical P-values<0.05, including KCNH7 (P = 0.0047), MYST4 (P = 0.0047), NRXN3 (P = 0.0095), and SEMA3D (P = 0.037). For markers genotyped in replication samples, rs7619173 exhibited a significant association (P(combined) = 2 ∗ 10(-4)) after multiple testing correction, while markers rs11001178 (MYST4) and rs2217887 (NRXN3) showed weak associations (P(combined) = 0.02) with BPD-I. CONCLUSION: A multi-stage GWA design has the potential to uncover the underlying pathogenesis of a complex trait. Findings in the present study highlight three loci that warrant further investigation for bipolar.

Regulation of nuclear transport and degradation of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1.

The regulation of the vertebrate cell cycle is controlled by the function of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. The Xenopus laevis kinase inhibitor, p27(Xic1) (Xic1) is a member of the p21(Cip1)/p27(Kip1)/p57(Kip2) CDK inhibitor family and inhibits CDK2-cyclin E in vitro as well as DNA replication in Xenopus egg extracts. Xic1 is targeted for degradation in interphase extracts in a manner dependent on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we show that ubiquitination of Xic1 occurs exclusively in the nucleus and that nuclear localization of Xic1 is necessary for its degradation. We find that Xic1 nuclear localization is independently mediated by binding to CDK2-cyclin E and by nuclear localization sequences within the C terminus of Xic1. Our results also indicate that binding of Xic1 to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation. Moreover, we show that amino acids 180-183 of Xic1 are critical determinants of Xic1 degradation. This region of Xic1 may define a motif of Xic1 essential for recognition by the ubiquitin conjugation machinery or for binding an alternate protein required for degradation.

Mesenchymal stem cells as vehicles for gene delivery.

Mesenchymal stem cells contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and marrow stroma. Transduction of mesenchymal stem cells from species other than humans is required for the development of disease models in which mesenchymal stem cells-based gene delivery is evaluated. Attempts to transduce mesenchymal stem cells from some species with amphotropic retroviral vectors were unsuccessful, leading to comparative mesenchymal stem cells transductions with xenotropic and gibbon-ape leukemia virus envelope-pseudotyped retroviral vectors. Human, baboon, canine, and rat mesenchymal stem cells were transduced optimally with amphotropic vector supernatants. In contrast, sheep, goat, and pig mesenchymal stem cells showed highest transduction levels with xenotropic retroviral vector supernatant, and rabbit mesenchymal stem cells were transduced optimally with gibbon-ape-enveloped vectors. Using a myeloablative canine transplantation model and gene-marked canine mesenchymal stem cells, the biodistribution of infused and ex vivo expanded mesenchymal stem cells were examined. The majority of transduced canine mesenchymal stem cells were found in the bone marrow samples. The current study shows the use of mesenchymal stem cells as a delivery vehicle for gene transfer studies, and validates the feasibility of delivering mesenchymal stem cells to the marrow compartment for stromal regeneration after cancer-associated cytotoxic therapies.

Protein engineering of venom toxins by synthetic approach and NMR dynamic simulation: status of basic amino acid residues in waglerin I.

The tertiary structure of waglerin I has been determined by NMR and dynamic simulated annealing [Chuang et al., Biochim. Biophys. Acta 1292, 145-155 (1996)]. It is believed that the peptide basicity of waglerin may play an important role for its activity due to its high content of basic amino acids. In order to investigate the active site of the toxin, seven analogues of waglerin, [Ala3]-waglerin, [Ala7]-waglerin, [Ala10]-waglerin, [Ala14]-waglerin, [Ala18]-waglerin, [Ala20]-waglerin and [Ala22]-waglerin have been synthesized chemically by single replacement of basic amino acid residues one by one with Ala. By correlation of structures for each analogue with LD50 toxicity bioassays, it is found that the [Ala10]-waglerin exhibits no toxicity and the active site of the native toxin seems to reside in the proximity of the disulfide loop, which is spatially close to His10. Furthermore, the closer is the disulfide loop to the basic amino acid in waglerin, the more influential is the basic amino acid on the toxicity of waglerin. Based on the tertiary structure of waglerin, the structures of all synthetic analogues were derived based on computer-simulated modelling. By the pair-wise structural comparison, the disulfide loop in [Ala10]-waglerin analogue is found to be twisted as compared to the native form, in agreement with the lack of toxicity for this synthetic analogue.

Structural analysis of a biologically active echistatin analogue des(46-49)-[Ala8,37]-echistatin gamma with three disulfide bonds by 2D-NMR and computer graphics.

An echistatin analogue, designated as des(46-49)-[Ala8,37]-echistatin gamma, was synthesized chemically by solid-phase peptide synthesis. The analogue was made by replacing Cys8 and Cys37 residues with two alanines and the deletion of C-terminal peptide 46-49 of echistatin gamma, resulting in an artificial polypeptide of 45 amino acids with three disulfide bonds. In the platelet aggregation assay, the analogue exhibits almost the same activity as echistatin gamma, indicating that the linear sequence of des(46-49)-[Ala8,37]-echistatin gamma contains all of the primary-structure information that is required for proper folding of this synthetic polypeptide. The tertiary structure of the analogue, as determined from high-resolution nuclear magnetic resonance (NMR) coupled with dynamic simulated annealing, is very similar to that of echistatin alpha1 which differs from echistatin gamma by 8 residues. In particular the two important sites of the Arg-Gly-Asp (RGD) loop and the C-terminal Lys45, both of which show some degree of disorder, are maintained in similar spatial orientation and proximity as those in echistatin alpha 1 even without the constraint provided by the disulfide bond of the (Cys8-Cys37) pair. These results provide new insights in further defining distinct structural features of echistatin gamma, which are involved in supporting the active polypeptide conformation to achieve biological activity in the absence of one pair of disulfide bonds.

Determination of three-dimensional solution structure of waglerin I, a toxin from Trimeresurus wagleri, using 2D-NMR and molecular dynamics simulation.

The solution conformation of a synthetic snake venom toxin waglerin I, has been determined by using proton nuclear magnetic resonance spectroscopy. By a combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of waglerin I was completely assigned. A set of 247 interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 2 dihedral angle restraints (from coupling constant measurements) and 7 omega torsion angel restraints for prolines, formed the basis of three-dimensional structure determined by molecular dynamics techniques. The 19 structures that were obtained satisfy the experimental restraints, and display small deviation from idealized covalent geometry. Analysis of converged structures indicates that the toxin has no special secondary structure. In the solution structure of waglerin I, the central ring region is well defined but the N- and C-termini possesses more disorder.

Impact of a clinical pharmacist on cost saving and cost avoidance in drug therapy in an intensive care unit.

The authors performed a study to document the impact of a clinical pharmacist on cost saving and cost avoidance in an intensive care unit, and to evaluate the cost saving and avoidance to justify additional clinical pharmacist positions. Over 13 consecutive 5-day weeks, a clinical pharmacist with 50% teaching responsibility documented time spent and all interventions that impacted the cost of drug therapy. Both cost avoidance and cost saving were documented on change in route, change in dosage, change to another drug, discontinuation of therapy, discontinuation of therapeutic duplication, discontinuation of inappropriate therapy, notification of pharmacy of discrepancy, and improper drug-level monitoring avoidance. Cost analysis was calculated based on acquisition costs. The final cost saving was derived from adding cost avoided and cost saved minus pharmacist salary for the time spent in conducting the study. A total of 310 interventions were documented during the 13 weeks (65 days) of the study. The final cost saving was $79,723, which would extrapolate to an annual savings of $318,891. Although 31.3% of interventions involved change of dosage, interventions involving change to another drug (13.9%) had the largest economic impact ($62,527). The majority (85.4%) of the savings involved costs of medications saved (actual dollars saved rather than avoided). The authors concluded that the clinical pharmacist had a significant impact on the cost of drug therapy in the intensive care unit and that the cost of additional clinical pharmacist positions should be justified.

Conformation of a cyclic opioid peptide analog by NMR and molecular dynamics simulation.

The conformation of a new Ty-c[Lys-Phe-Asp]-NH2 cyclic opioid peptide synthesized by solid phase method, has been determined from two-dimensional NMR and distance geometry followed by restrained molecular dynamics simulation. The conformation of the ring is well-defined, but the exocylic Tyr-1 and Phe-3 side-chain moiety possesses significant orientational freedom.

Possible case of procainamide-induced intrahepatic cholestatic jaundice.

OBJECTIVE: To describe a possible case of procainamide-induced intrahepatic cholestatic jaundice that was recognized six weeks after the initiation of procainamide therapy and to summarize the five previously reported cases. CASE SUMMARY: A 77-year-old woman with a history of hypertension, insulin-dependent diabetes mellitus, temporal arteritis, and Wolff-Parkinson-White syndrome who had taken procainamide for six weeks presented to the hospital with disorientation and acute renal and hepatic dysfunction. In addition to disorientation, scleral icterus, and diffuse maculopapular rash, her physical examination was generally normal. There was no evidence of fever, nausea, vomiting, lymphadenopathy, or eosinophilia. Her liver enzyme concentrations increased significantly from baseline (beginning of procainamide therapy). Her N-acetylprocainamide (NAPA) concentration was elevated to 52 mg/L upon admission. Procainamide was discontinued and her NAPA concentration returned to within normal limits in two days. Diagnostic tests were performed to rule out active hepatitis, vasculitis, and liver malignancies. After procainamide was discontinued and prednisone treatment was started, she became more oriented and her liver enzyme concentrations slowly improved. DISCUSSION: Only five cases of procainamide-induced liver abnormalities have been previously reported; these included granulomatous hepatitis and intrahepatic cholestasis. The mechanism for liver dysfunction is not known; however, it is proposed to be a delayed hypersensitivity reaction. Clinical hallmarks of hypersensitivity include fever, eosinophilia, rash, and lymphadenopathy; nausea and vomiting also may be present. Of the five reported cases, all experienced fever and only one experienced pruritus. No patients had eosinophilia or lymphadenopathy. Because of the temporal increase in liver enzyme concentrations after six weeks of procainamide therapy, we believe that this case represents another possible procainamide-induced hypersensitivity reaction. CONCLUSIONS: Procainamide-induced liver dysfunction can occur from one day to six weeks after initiation of the drug and may subside one day to several weeks after discontinuation of therapy. Symptoms may include nausea, vomiting, rash, and fever. Liver enzyme concentrations are abnormal. It is important to recognize the possibility of such a reaction early so that procainamide therapy can be discontinued promptly to avoid further liver damage.

Solution conformation of cobrotoxin: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study.

The solution conformation of cobrotoxin has been determined by using proton nuclear magnetic resonance spectroscopy. With the combination of various two-dimensional NMR techniques, the 1H-NMR spectrum of cobrotoxin was completely assigned (Yu et al., 1990). A set of 435 approximate interproton distance restraints was derived from nuclear Overhauser enhancement (NOE) measurements. These NOE constraints, in addition to the 29 dihedral angle constraints (from coupling constant measurements) and 26 hydrogen bonding restraints (from the pattern of short-range NOEs), form the basis of 3-D structure determination by the hybrid distance geometry-dynamical simulated annealing method. The 23 structures that were obtained satisfy the experimental restraints, display small deviation from idealized covalent geometry, and possess good nonbonded contacts. Analysis of converged structures indicated that there are two antiparallel beta sheets (double and triple stranded), duly confirming our earlier observations. These are well defined in terms of both atomic root mean square (RMS) differences and backbone torsional angles. The average backbone RMS deviation between the calculated structures and the mean structure, for the beta-sheet regions, is 0.92 A. The mean solution structure was compared with the X-ray crystal structure of erabutoxin b, the homologous protein. This yielded information that both structures resemble each other except at the exposed loop/surface regions, where the solution structure seems to possess more flexibility.

Conformational properties of oxytocin in dimethyl sulfoxide solution: NMR and restrained molecular dynamics studies.

The conformation of oxytocin, the neurohypophyseal nonapeptide hormone, in solution in deuterated dimethyl sulfoxide has been determined by 1H-nmr. The structural determination is based on the experimental data set of nuclear Overhauser effect restraints. Obtained after the restrained molecular dynamics simulation on an initial structure of extended conformation, five resultant structures satisfy the experimental restraints well. These structures resemble that of the crystal structure of deamino-oxytocin, an analogue of oxytocin, in terms of a close correlation observed both at two beta-turn regions of the 20-membered tocin ring and at the tripeptide tail end. Based on this comparison and analysis of restrained molecular dynamics trajectories, we found that, although the turns are stabilized by the formation of hydrogen bonds, the oxytocin molecule possesses a slight twist in DMSO solution relative to the orientation of deamino-oxytocin in the crystalline state. Analyses of oxytocin conformation indicate that the tripeptide tail is more flexible than the tocin ring.

Use of granulocyte colony-stimulating factor in the treatment of pancytopenia secondary to colchicine overdose.

OBJECTIVE: To report a case of pancytopenia following colchicine overdose and to discuss the use of granulocyte colony-stimulating factor (G-CSF) for treating this severe complication. CASE SUMMARY: A 19-year-old man developed pancytopenia four days after ingestion of approximately 50-60 0.6-mg colchicine tablets in a suicide attempt. His pancytopenia resolved after subcutaneous administration of one 300-micrograms dose of G-CSF. The patient recovered from his other multiorgan disturbances during his hospitalization and was discharged from the hospital with elevated liver enzyme concentrations. CONCLUSIONS: Colchicine overdose is rare, but can be fatal. The use of G-CSF appears to be beneficial in alleviating bone marrow depression in colchicine overdose situations.

Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution.

The 1H-NMR spectra of cobrotoxin, a neurotoxic protein isolated from Formosan cobra Naja naja atra, have been studied by two-dimensional NMR techniques. Of 62 amino acid residues in cobrotoxin, the complete assignments of 58 residues have been made. The resonances from several of the remaining residues have been identified but not yet specifically assigned. The secondary structure of an antiparallel triple- and double-stranded beta-sheet has also been determined by observing the NOE.

Molecular cloning and hybridization studies on bluetongue virus serotype 17.

The dsRNA of bluetongue virus (BTV) serotype 17 has been reverse transcribed and dsDNA copies of the viral RNA have been cloned into the plasmid vector pBR-322. Segments ranging from 3 kilobases to less than 500 bases have been cloned and at present 1 of the clones has been identified by hybridization to the genome segment of its origin (genome segment 7). Identification of further clones is proceeding. The techniques for northern blotting BTV dsRNA onto 2-aminophenylthioether (APT) paper and the detection of the transferred RNA by 32P labelled dsRNA or cDNA probes have been standardized. Cross-hybridization studies can be used to detect genetic relationships of different serotypes and isolates of BTV.