Direct determination of substituted azepinoindole enantiomers in rat plasma using silica stationary phase and beta-cyclodextrin as a mobile phase additive

DU 124884 is a racemic serotonin receptor agonist in an early stage of drug development. DU 124884 and its potential N-desmethyl metabolite, KC 9048, both contain a single chiral center. A direct enantioselective HPLC assay was developed and validated to quantify DU 124884 and KC 9048 in rat plasma. The drug and metabolite enantiomers were extracted from plasma and separated using silica stationary phase with an aqueous mobile phase containing beta-cyclodextrin (beta-CD), triethylamine, and 2-methyl-2-propanol. A variable wavelength detector was used to monitor absorbance at 231 nm. The assay calibration range was from 100 to 5000 ng/mL. Quality control sample precision (< or = 9% RSD) and accuracy (+/-10% error) were satisfactory for all four analytes (n = 12). The method was used to assess drug exposure during a pilot toxicology study in rats. Toxicokinetic study animals were dosed subcutaneously for 15 days at 0, 2.5, 10, and 40 mg of DU 124884.HCl kg-1 day-1. Blood was collected on the last day of dosing between 22 min and 4 h and 13 min after the last dose. The samples showed (+/-)-DU 124884 isomer ratios ranging from 1.1 to 1.3. These data suggest that DU 124884 undergoes stereoselective metabolism in rats. Levels of the N-desmethyl metabolite enantiomers were < 100 ng/mL.     

Antitumor activity of 9(R)-dihydrotaxane analogs

A novel reduced taxane, 13-acetyl-9(R)-dihydrobaccatin III (1) has been isolated from Taxus canadensis. The selective C-13 deacetylation of this isolate has allowed for the preparation of a wide variety of 9(R)-dihydrotaxane analogs. In general, this series has shown greater stability and water solubility than the 9-carbonyl series while retaining antimicrotubule and tumor cell cytotoxicity activities relative to taxol. Placement of polar functionalities at the C-7 position results in loss of activity whereas alkylation or acylation of either C-7 or C-9 hydroxyl groups ameliorate the activity.     

Mechanical airway obstruction secondary to retropharyngeal haematoma

Mechanical airway obstruction secondary to retropharyngeal haematoma is a life-threatening emergency and should be anticipated in all cervical spine injure patients regardless of the severity of trauma. Most retropharyngeal haematomas described in the literature have involved complicating factors such as anticoagulant therapy, tumour, aneurysm, infection or major cervical spine injury. Several authors have however described haematomas causing airway compromise with minor hyperextension injuries. We describe the case of a 19-year-old male who was involved in a motorcycle accident. He sustained an atlanto-occipital fracture-dislocation and a very large retropharyngeal haematoma which resulted in airway obstruction and a subsequent difficult intubation. We outline the normal anatomy of the retropharyngeal space, the pathogenesis of retropharyngeal haematomas, and outline techniques of intubation available.     

Müllerian duct cyst masquerading as chronic prostatitis: diagnosis with magnetic resonance imaging using a phased array surface coil

A retrospective study was designed to verify how often an anatomic variation caused the radial sensory nerve entrapment at the forearm (Wartenberg's syndrome). This variation, in which the superficial branch of the radial nerve emerges from under the fascia between two slips of a split brachioradialis tendon, was mentioned once in a clinical textbook as a basis for nerve entrapment but was found to occur in 5 of 150 dissected arms in 4 of 75 cadavers (3.3% of the investigated arms) in a recent anatomic study. To evaluate the incidence of this variation, 143 operative reports from patients who had Wartenberg's syndrome were reviewed. The variation was observed in seven patients. We conclude that any operation for Wartenberg's syndrome should include a thorough investigation of the site where the radial sensory nerve emerges from under the fascia and, if the nerve emerges through a split brachioradialis tendon, the anomalous tendon slip should be divided.     

Phosphoenolpyruvate carboxykinase from Trypanosoma cruzi. Purification and physicochemical and kinetic properties

Phospho enolpyruvate carboxykinase (PEPCK) has been purified to homogeneity from epimastigotes of the Tul 0 strain of Trypanosoma cruzi. The physicochemical parameters determined allowed the calculation of an average molecular mass of 120 kDa; the subunit molecular mass, about 61 kDa, is in good agreement with the value of 58.6 kDa recently determined from the sequence by Sommer et al. (FEBS Lett. 359 (1994) 125-129). The PEPCK from T. cruzi presented, in addition to its molecular mass, typical properties of other ATP-linked PEPCKs, namely strict specificity for ADP in the carboxylation reaction and lower specificity in the decarboxylation and exchange reactions, and synergistic activation by CdCl2 or MgCl2 when added in addition to MnCl2. The enzyme presented hysteretic behaviour, shown by a lag period in the carboxylation reaction, which was affected by dilution and preincubation. The decarboxylation reaction catalyzed by the T. cruzi PEPCK was not inhibited by excess of ATP-Mn. The apparent Km values for the carboxylation reaction, including the low value for PEP (0.035 mM) are compatible with an important role of PEPCK, as suggested by previous NMR experiments, on the CO2 fixation in vivo which leads to succinate excretion during aerobic fermentation of glucose.     

Ribozyme-mediated RNA degradation in nuclei suspension

Ribozymes containing 2'-fluoro- and 2'-amino-modified pyrimidine nucleosides in combination with terminal phosphorothioate linkages were targeted against HTLV-I tax RNA. In order to examine the activity of such chemically modified ribozymes in the nuclear environment, they were incubated with nuclei of a Tax-transformed mouse fibroblast cell line. Ribozyme cleavage of tax RNA was analyzed by the RNase protection assay. Comparison of the cleavage of tax RNA isolated nuclei with that of tax RNA present in nuclei suspension revealed a 30 times more efficient cleavage of the latter one. Pre-treatment with proteinase K and SDS abolished the enhancement of the ribozyme-mediated RNA cleavage. Catalytically inactive ribozymes did not yield any cleavage products. These results demonstrate an augmenting effect of nuclear proteins on the ribozyme-mediated RNA cleavage.     

Evidence for alpha-helical conformation of an essential N-terminal region in the human Bcl2 protein

A region occupying approximately 24 amino acids near the N terminus of human Bcl2 is essential for this cytoplasmic membrane protein's ability to inhibit apoptosis. Systematic mutagenesis of this N-terminal region indicates that only five hydrophobic and aromatic residues within it are specifically required for function. Computerized secondary structure prediction, together with circular dichroism spectroscopy of synthetic peptides, indicates that the region encompassing these five residues has the propensity to take on an alpha-helical conformation in the presence of SDS micelles, which presumably mimic the hydrophobic surfaces of cellular membranes or polypeptides. The five critical residues are predicted to be clustered on one face of this putative helix, where they might serve to mediate protein-protein contacts involved in the multimerization of Bcl2 or in the interaction of Bcl2 with other, as yet unidentified components of the apoptotic pathway. Apparent structural homologues of this helical motif are also present in at least some other anti-apoptotic proteins from the Bcl2 family but not in those family members that tend to potentiate, rather than inhibit, apoptosis.