Imipramine metabolism in relation to the sparteine and mephenytoin oxidation polymorphisms--a population study

1. Sparteine and mephenytoin phenotyping tests were carried out in 327 healthy Danish subjects. Two weeks later each subject took 25 mg imipramine followed by urine collection for 24 h. The urinary content of imipramine, desipramine, 2-hydroxy-imipramine and 2-hydroxy-desipramine was assayed by h.p.l.c. 2. The medians of the hydroxylation ratios (i.e. 2-hydroxy-metabolite over parent compound) were 6 to 14 times higher in 300 extensive metabolizers of sparteine (EMs) as compared with 27 poor metabolizers (PMs), but none of the ratios separated the two phenotypes completely. 3. There were 324 EM of mephenytoin (EMM) and three PM (PMM) in the sample. The demethylation ratios between desipramine, 2-hydroxy-desipramine and their corresponding tertiary amines showed statistically significant correlations with the mephenytoin S/R isomer ratio (Spearman's rs: -0.20 and -0.27, P < 0.05). 4. The demethylation ratios were higher in 80 smokers than in 245 non-smokers. This indicates that CYP1A2, which is induced by cigarette smoking, also catalyzes the N-demethylation of imipramine. 5. CYP2D6 genotyping was carried out by PCR in 325 of the subjects, and the D6-wt allele was amplified in 298 EMs, meaning that they were genotyped correctly. One PMs was D6-wt/D6-B, another PMs had the genotype D6-wt/ and hence both were misclassified as EMs. The remaining 25 PMs were D6-A/D6-B (n = 5), D6-B/ (n = 18) or D6-D/D6-D (no PCR amplification, n = 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments

Recent studies have reported that mutant genomic cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) sequences can be corrected in transformed CF airway epithelial cell lines by targeted replacement with small fragments of DNA with wild-type sequence. To determine if the observed genotype modification following small fragment homologous replacement (SFHR) was limited to transformed CF cell lines, further studies were carried out in both transformed and non-transformed primary normal airway epithelial cells. The endogenous genotype of these normal cell lines was modified following liposome or dendrimer transfection using DNA fragments with DeltaF508 CFTR sequence (488 nt, complementary single strands) designed to also contain a unique restriction enzyme cleavage site (Xho I). Replacement at the appropriate genomic locus by exogenous DeltaF508 CFTR DNA and its expression as mRNA was demonstrated by PCR amplification of genomic DNA and mRNA-derived cDNA as well as Xho I digestion of the PCR products. These studies show that SFHR occurs in both transformed and non-transformed primary human airway epithelial cells and indicate that single base substitution (the silent mutation giving rise to the Xho I site) and deletion or insertion of at least three consecutive bases can be achieved in both normal and CF epithelial cells. Furthermore, these studies reiterate the potential of SFHR as a strategy for a number of gene targeting applications, such as site-specific mutagenesis, development of transgenic animals, development of isogenic cell lines and for gene therapy.     

Functional anatomy of the cartilage of the distal phalanx and digital cushion in the equine foot and a hemodynamic flow hypothesis of energy dissipation

OBJECTIVES: To examine macro- and microscopic characteristics of cartilage of the distal phalanx (ungual cartilage [UC]) and digital cushion in the equine foot and to relate them to the foot's function of energy dissipation. ANIMALS: 85 horses and 5 foals of various breeds and ages. PROCEDURE: Feet, obtained at necropsy, were perfused with India ink (n = 30), latex (5), or polymer plastic (10). Select feet were examined histologically for tissue architecture and to identify elastic fibers. Immunochemistry to identify substance P peptides in nerves (feet from foals) and gold chloride impregnation of axons (n = 10) were performed. Feet were sectioned transversely (n = 27) or coronally (62 feet in a matched-paired study). Ungual cartilage was measured at the navicular bone. Digital cushions were examined for relative tissue composition between forefeet and hind feet. RESULTS: Ungual cartilage formed an axial projection that extended towards the midline to overlie the bars, and dorsally along the semilunar line of the distal phalanx. Ungual cartilage of forefeet was significantly larger than that of hind feet. The digital cushion was composed of fat and elastic tissues in feet with thin UC, or fibrous and fibrocartilaginous tissue and elastic tissue in feet with thicker UC. Sensory nerves and an extensive network of venovenous anastomoses were found in the UC. CONCLUSIONS AND CLINICAL RELEVANCE: Ungual cartilage and the digital cushion provide the basis for a hemodynamic flow hypothesis of energy dissipation. Maximum energy dissipation depends on proper hoof preparation and shoeing.     

Ca2+-dependent structural changes in C-type mannose-binding proteins

C-type animal lectins are a diverse family of proteins which mediate cell-surface carbohydrate-recognition events through a conserved carbohydrate-recognition domain (CRD). Most members of this family possess a carbohydrate-binding activity that depends strictly on the binding of Ca2+ at two sites, designated 1 and 2, in the CRD. The structural transitions associated with Ca2+ binding in C-type lectins have been investigated by determining high-resolution crystal structures of rat serum mannose-binding protein (MBP) bound to one Ho3+ in place of Ca2+, and the apo form of rat liver MBP. The removal of Ca2+ does not affect the core structure of the CRD, but dramatic conformational changes occur in the loops. The most significant structural change in the absence of Ca2+ is the isomerization of a cis-peptide bond preceding a conserved proline residue in Ca2+ site 2. This bond adopts the cis conformation in all Ca2+-bound structures, whereas both cis and trans conformations are observed in the absence of Ca2+. The pattern of structural changes in the three loops that interact with Ca2+ is dictated in large part by the conformation of the prolyl peptide bond. The highly conserved nature of Ca2+ site 2 suggests that the transitions observed in MBPs are general features of Ca2+ binding in C-type lectins.     

Outcome of 259 patients with primary proliferative polycythaemia (PPP) and idiopathic thrombocythaemia (IT) treated in a regional nuclear medicine department with phosphorus-32--a 15 year review

259 patients with primary proliferative polycythaemia (PPP) and idiopathic thrombocythaemia (IT) have been treated with 32P over the last 15 years. Complete follow-up data were obtained in 238 patients. PPP was the diagnosis in 183 patients and 76 patients had IT. The sex ratio in PPP was male/female 1.1:1 and in IT 1:1.4. Patients' ages ranged from 28 to 95 years (median 72 years). The number of 32P administrations per patient ranged from 1 to 13 (median 2) and the total administered activity per patient ranged from 81.4 to 4162 MBq (median 496 MBq). The outcome showed a normalization of the full blood count in 50% of patients after a single administration of 32P and in 73% after two treatments. 13 patients (5.5%) developed myelofibrosis; 18 (7.6%) developed leukaemia while other cancers arose in 19 patients (8%). 32P therapy proved to be of particular value in the elderly. 32P is easy to administer and is cost effective, compared with the alternative of chemotherapy where good compliance and frequent hospital visits are required.     

Radon penetration of concrete slab cracks, joints, pipe penetrations, and sealants

Radon movement through 12 test slabs with different cracks, pipe penetrations, cold joints, masonry blocks, sealants, and tensile stresses characterized the importance of these anomalous structural domains. Diffusive and advective radon transport were measured with steady-state air pressure differences controlled throughout the deltaP = 0 to 60 Pa range. Diffusion coefficients (deltaP = 0) initially averaged 6.5 x 10(-8) m2 s(-1) among nine slabs with only 8% standard deviation, but increased due to drying by 0.16% per day over a 2-y period to an average of 2.0 x 10(-7) m2 s(-1). An asphalt coating reduced diffusion sixfold but an acrylic surface sealant had no effect. Diffusion was 42 times higher in solid masonry blocks than in concrete and was not affected by small cracks. Advective transport (deltaP < or = 60 Pa) was negligible for the slabs (10(-16) m2 permeability), pipe penetrations, and caulked gaps, but was significant for cracks, disturbed pipe penetrations, cold joints, masonry blocks, and concrete under tensile stress. Crack areas calculated to be as small as 10(-7) m2 significantly increased radon advection. Algebraic expressions predict air velocity and effective crack width from enhanced radon transport and air pressures. Masonry blocks, open cracks, and slab cold joints enhance radon penetration but stressed slabs, undisturbed pipe penetrations, and sealed cracks may not.     

Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis

The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis. The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components. Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes. To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated. We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex. The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region. In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins. Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters.     

N-cadherin-catenin interaction: necessary component of cardiac cell compartmentalization during early vertebrate heart development

During early heart development the expression pattern of N-cadherin, a calcium-dependent cell adhesion molecule, suggests its involvement in morphoregulation and the stabilization of cardiomyocyte differentiation. N-cadherin's adhesive activity is dependent upon its interaction with the intracellular catenins. An association with alpha-catenin and beta-catenin also is believed to be involved in cell signaling. This study details the expression patterns of alpha-catenin, beta-catenin, and gamma-catenin, during definition of the cardiac cell population as distinct compartments in the anterior regions of the chick embryo between stages 5 and 9. The restriction of N-cadherin/catenin localization at stage 5+ from a uniform pattern in vivo, to specific cell clusters that demarcate areas where mesoderm separation is initiated, suggests that the N-cadherin/catenin complex is involved in boundary formation and in the subsequent cell sorting. The latter two processes lead to the specification and formation of the somatic and cardiac splanchnic mesoderm. N-cadherin colocalized with alpha- and beta-catenin at the cell membrane before and during the time that its expression becomes restricted to the lateral mesoderm and continues cephalocaudad into stage 8. These proteins continue to colocalize in the myocardium of the tubular heart. Plakoglobin is not expressed in this region during stages 6-8, but is detected in the myocardium later at stage 13. The observed in vivo expression patterns of alpha-catenin, beta-catenin, and plakoglobin suggest that these proteins are directly linked with the developmental regulation of cell junctions, as cardiac cells become stably committed and phenotypically differentiated to eventually form a mature myocardium. The localization of N-CAM also was analyzed during these stages to determine whether the N-cadherin-catenin localization was unique or whether other cell adhesion molecules were expressed similarly. The results indicate that the unique pattern of N-cadherin expression is not shared with N-CAM. We also show that perturbation of N-cadherin using a function perturbing N-cadherin antibody (NCD-2) inhibits normal early heart development and myogenesis in a cephalocaudad, stage-dependent manner. We propose a model whereby myocardial cell compartmentalization also defines the endocardial population. The presence of beta-catenin suggests that a similar signaling pathway involving Wnt (wingless)-mediated events may function in myocardial cell compartmentalization during early vertebrate heart development, as in Drosophila contractile vessel development.     

Clostridium difficile-associated diarrhea: epidemiology, risk factors, and infection control

OBJECTIVES: To evaluate the effectiveness of specific infection control measures on the incidence of Clostridium difficile-associated diarrhea (CDAD) and to identify risk factors for its development. SETTING: 370-bed, tertiary-care teaching hospital with approximately 12,000 to 15,000 admissions per year. METHODS: Several infection control measures were implemented in 1991 and 1992, and the attack rates of CDAD were calculated quarterly. Antibiotic use for 1988 through 1993 was analyzed. A case-control study was conducted from January 1992 to December 1992 to identify risk factors for acquisition of CDAD. RESULTS: From 1989 to 1992, the attack rate of CDAD increased from 0.49% to 2.25%. An increase in antibiotic use preceded the rise in the incidence of CDAD in 1991. Despite implementation of various infection control measures, the attack rate decreased to 1.32% in 1993, but did not return to baseline. Ninety-two cases and 78 controls (patients with diarrhea but with negative toxin assay) were studied. By univariate analysis, history of prior respiratory tract infections (odds ratio [OR], 3.6; 95% confidence interval [CI95], 1.2-10.4), the number of antibiotics, and the duration of exposure to second-generation cephalosporins (OR, 3.55; CI95, 1.47-9.41) and to ciprofloxacin (OR, 7.27; CI95, 1.13-166.0) were related significantly to the development of CDAD. By stepwise logistic regression analysis, only exposure to antibiotics and prior respiratory tract infections (P = .0001 and .0203, respectively) were found to be significant. CONCLUSION: Antibiotic pressure might have contributed to failure of infection control measures to reduce the incidence of CDAD to baseline.     

Value of thoracic computed tomography in the first assessment of severely injured patients with blunt chest trauma: results of a prospective study

OBJECTIVE: The aim of this prospective study was to evaluate whether early thoracic computed tomography (TCT) is superior to routine chest x-ray (CXR) in the diagnostic work-up of blunt thoracic trauma and whether the additional information influences subsequent therapeutic decisions on the early management of severely injured patients. PATIENTS AND METHODS: In a prospective study of 103 consecutive patients with clinical or radiologic signs of chest trauma (94 multiple injured patients with chest trauma, nine patients with isolated chest trauma), an average Injury Severity Score of 30 and an average Abbreviated Injury Scale thorax score of 3, initial CXR and TCT were compared after initial assessment in our emergency department of a Level I trauma center. RESULTS: In 67 patients (65%) TCT detected major chest trauma complications that have been missed on CXR (lung contusion (n = 33), pneumothorax (n = 27), residual pneumothorax after chest tube placement (n = 7), hemothorax (n = 21), displaced chest tube (n = 5), diaphragmatic rupture (n = 2), myocardial rupture (n = 1)). In 11 patients only minor additional pathologic findings (dystelectasis, small pleural effusion) were visualized on TCT, and in 14 patients CXR and TCT showed the same pathologic results. Eleven patients underwent both CXR and TCT without pathologic fundings. The TCT scan was significantly more effective than routine CXR in detecting lung contusions (p < 0.001), pneumothorax (p < 0.005), and hemothorax (p < 0.05). In 42 patients (41%) the additional TCT findings resulted in a change of therapy: chest tube placement, chest tube correction of pneumothoraces or large hemothoraces (n = 31), change in mode of ventilation and respiratory care (n = 14), influence on the management of fracture stabilization (n = 12), laparotomy in cases of diaphragmatic lacerations (n = 2), bronchoscopy for atelectasis (n = 2), exclusion of aortic rupture (n = 2), endotracheal intubation (n = 1), and pericardiocentesis (n = 1). To evaluate the efficacy of all those therapeutic changes after TCT the rates of respiratory failure, adult respiratory distress syndrome, and mortality in the subgroup of patients with Abbreviated Injury Scale thorax score of > 2 were compared with a historical control group, consisting of 84 patients with multiple trauma and with blunt chest trauma Abbreviated Injury Scale thorax score of > 2, prospectively studied between 1986 and 1992. Age (38 vs. 39 years), average Injury Severity Score (33 vs. 38), and the rate of respiratory failure (36 vs. 56%) were not statistically different between the two groups, but the rates of adult respiratory distress syndrome (8 vs. 20%; p < 0.05) and mortality (10 vs. 21%; p < 0.05) were significantly reduced in the TCT group. CONCLUSIONS: TCT is highly sensitive in detecting thoracic injuries after blunt chest trauma and is superior to routine CXR in visualzing lung contusions, pneumothorax, and hemothorax. Early TCT influences therapeutic management in a significant number of patients. We therefore recommend TCT in the initial diagnostic work-up of patients with multiple injuries and with suspected chest trauma because early and exact diagnosis of all thoracic injuries along with sufficient therapeutic consequences may reduce complications and improve outcome of severely injured patients with blunt chest trauma.