Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 +/- 33 nM and a Vmax of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBankTM accession number Z69637) and Archaeoglobus fulgidus (GenBankTM accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.     

Lack of TEL/AML1 fusion in pediatric AML: further evidence for lineage specificity of TEL/AML1

The percutaneous cardiopulmonary support system (PCPS) was used in a 64-year-old woman with cardiogenic shock due to sustained ventricular fibrillation (Vf) caused by severe aortic stenosis and regurgitation. The Vf attack was resistant to cardioversion and adrenaline for lack of left ventricular support by PCPS. She was transported to the operation theater with PCPS in situ and emergency aortic valve replacement was performed. Although preoperative cardiac resuscitation time was long (35 minutes), she was discharged from the hospital on foot without any neurological complications on 84th postoperative day. Because PCPS does not decrease left ventricular systolic stress in poorly contracting dilated heart, early surgical treatment is needed in patients with severely damaged heart.     

Protein turnover in the hypertrophying kidney

To establish whether altered proteolysis contributes to the increase in protein content in hypertrophying kidneys, we studied protein turnover in proximal renal tubules isolated from rats with three forms of renal hypertrophy, diabetes mellitus (DM), ammonium chloride-induced acidosis and compensatory renal growth (CRG). We found that in DM and in chronic acidosis the normal balance in protein turnover is altered due to attenuated proteolysis and accelerated protein synthesis. Together this favors an increase in kidney protein content. In contrast, in CRG, the increase in protein content is entirely due to increased protein synthesis. Thus, the changes in protein turnover leading to the net gain in kidney protein content in renal hypertrophy depends on the cause of hypertrophy.     

Thioether adducts of a new imine reactive intermediate of the pneumotoxin 3-methylindole

Cytochrome P450 enzymes can potentially oxygenate 3-methylindole to form 2,3-epoxy-3-methylindoline which could rearrange to the stable metabolite 3-methyloxindole or open to form 3-hydroxy-3-methylindolenine, a putative electrophilic imine. The purpose of the current work was to determine if the imine was formed, and to characterize it via its adducts with thiol nucleophiles. Thiols were added to incubations of goat lung microsomes with 3-methylindole and deuterated analogues of 3-methylindole to trap the imine intermediate as its thioether conjugates. The N-acetylcysteine conjugate of 3-hydroxy-3-methylindolenine was detectable by LC/MS, but a molecular ion was not observed because the adduct rapidly dehydrated to form the 2-substituted indole. However, the imine was S-alkylated, and the intermediate carbinol was intramolecularly trapped using thioglycolic acid as a trapping agent that induced cyclocondensation to a lactone. The retention of one atom of deuterium from [2-2H]-3-methylindole and three from 3-[2H3-methyl]indole substantiated the mechanism in which the lactone adduct was produced by sulfur addition to either 3-hydroxy-3-methylindolenine or the epoxide. Tandem mass spectrometry of the lactone adduct produced a daughter ion spectrum consistent with this adduct. These studies demonstrated the existence of a new reactive intermediate of 3-methylindole, 3-hydroxy-3-methylindolenine, which may play a role in the pneumotoxicity of this chemical.     

Renal changes induced by nitric oxide and prostaglandin synthesis reduction: effects of trandolapril and verapamil

The benefits of the simultaneous administration of low doses of a calcium antagonist and a converting enzyme inhibitor in the treatment of hypertension and renal vasoconstriction are well established. The objective of this study was to evaluate whether the administration of low doses of a calcium antagonist and a converting-enzyme inhibitor have beneficial effects in treating the renal alterations induced by the acute administration of a cyclooxygenase inhibitor when nitric oxide synthesis is reduced. These effects were examined in anesthetized dogs before and during an acute sodium load. It was found that the intrarenal infusion of meclofenamate (5 microg x kg[-1] x min[-1]), simultaneously with a low dose of NG-nitro-L-arginine methyl ester (1 microg x kg[-1] x min[-1]), produced a 40% decrease of renal blood flow and glomerular filtration rate and a reduction in the renal excretory response to the sodium load. In a second group of dogs, intrarenal verapamil (0.5 microg x kg[-1] x min[-1]) was effective in blocking the effects of nitric oxide and prostaglandin synthesis inhibition on sodium excretion and glomerular filtration rate but did not modify the effects on renal blood flow. An intrarenal infusion of trandolapril (0.3 microg x kg[-1] x min[-1]) was effective in a third group of dogs in reducing the renal hemodynamic effects but not in preventing the antinatriuretic effect observed in the first group. Finally, in a fourth group, the simultaneous administration of verapamil and trandolapril was effective in treating all the renal changes induced by the cyclooxygenase inhibitor when nitric oxide synthesis was reduced. These results suggest that the combination of low doses of trandolapril and verapamil has additive effects in treating the renal vasoconstriction and antinatriuresis induced by the acute administration of a cyclooxygenase inhibitor, when nitric oxide synthesis is reduced.     

Cancer genetics, cytogenetics--defining the enemy within

BACKGROUND/AIMS: International comparisons of clinical practice may help in assessing the magnitude and possible causes of variation in cross national healthcare utilisation. With this aim, the indications for cataract surgery in the United States, Denmark, the province of Manitoba (Canada), and the city of Barcelona (Spain) were compared. METHODS: In a prospective multicentre study, patients scheduled for first eye cataract surgery and aged 50 years or older were enrolled consecutively. From the United States 766 patients were enrolled; from Denmark 291; from Manitoba 152; and from Barcelona 200. Indication for surgery was measured as preoperative visual status of patients enlisted for cataract surgery. Main variables were preoperative visual acuity in operative eye, the VF-14 score (an index of functional impairment in patients with cataract) and ocular comorbidity. RESULTS: Mean visual acuity were 0.23 (USA), 0.17 (Denmark), 0.15 (Manitoba), and 0.07 (Barcelona) (p < 0.001). When restricting the sample to eyes with normal retina and macula, no significant difference between United States and Denmark was observed (p > 0.05). Mean VF-14 scores were 76 (USA), 76 (Denmark), 71 (Manitoba), and 64 (Barcelona) (p < 0.001). CONCLUSION: Similar indications for cataract surgery were found in the United States and Denmark. Significantly more restricted indications were observed in Manitoba and Barcelona. Possible explanations for the results are discussed, including differences in sociodemographic characteristics, access to care, surgeons' willingness to operate, and patient demand.     

Preparation and in vitro characterization of gentamycin-impregnated biodegradable beads suitable for treatment of osteomyelitis

A new method for preparing poly(L-lactide) (PLA) biodegradable beads impregnated with an ionic aminoglycoside, gentamycin, is described. The process employs hydrophobic ion pairing to solubilize gentamycin in a solvent compatible with PLA, followed by precipitation with a compressed antisolvent (supercritical carbon dioxide). The resulting precipitate is a homogeneous dispersion of the ion-paired drug in PLA microspheres. The microspheres are approximately 1 microm in diameter and can be compressed into beads (3-6 mm in diameter) strung on surgical sutures for implantation. The bead strings exhibit no significant change in release kinetics upon sterilization with a hydrogen peroxide plasma (Ster-Rad). The kinetics of gentamycin release from the PLA beads are consistent with a matrix-controlled diffusion mechanism. While nonbiodegradable poly(methyl methacrylate) (PMMA) beads initially release gentamycin in a similar manner, the drug release from PMMA ceases after 8 or 9 weeks, while the PLA beads continue to release drug for over 4 months. Moreover, only 10% of the gentamycin is released from the PMMA beads, while PLA beads release more than 60% of their load, if serum is present in the release medium. The PLA system displays improved release kinetics relative to PMMA, is biodegradable, is unaltered by gas sterilization, can be used for a range of antibiotics, and can be manipulated without disintegration. These are all desirable properties for an implantable drug delivery system for the prevention or treatment of osteomyelitis.     

Autonomous epicardial and endocardial boundary detection in echocardiographic short-axis images

An autonomous endocardial and epicardial boundary detection (ABD) method is reported. One hundred ten cycles from 55 clinical studies were selected retrospectively. Image sequences were digitized at 512 x 480 pixel resolution. The point-by-point boundary positions of the ABD and the areas enclosed were compared with positions and enclosed areas drawn by three independent observers. Correlation coefficients for epicardial end-diastolic (ED) and end-systolic (ES) areas, endocardial ED and ES areas, muscle area, and fractional area change were 0.970, 0.976, 0.951, 0.985, 0.887, and 0.878, respectively. Bland-Altman analysis showed negligible biases with standard deviations comparable to those of the observers. The mean difference between the ABD border and the consensus observer border positions in 64 directions falls within the mean range of interobserver border positions, suggesting that shape is also well defined by the ABD.     

Changes in fetal plasma corticotropin-releasing hormone during androstenedione-induced labor in the rhesus monkey: lack of an effect on the fetal hypothalamo-pituitary-adrenal axis

Androstenedione infusion to pregnant monkeys leads to premature labor and live delivery. Androstenedione-induced labor also increased placental CRH messenger RNA and peptide to concentrations observed at term in pregnant monkeys. Placental CRH may modulate fetal pituitary-adrenal function during pregnancy in primates. This study tested the hypothesis that androstenedione-induced premature delivery in pregnant monkeys results from androstenedione-induced increases in placental CRH, which stimulate premature activation of the fetal pituitary-adrenal axis. The hypothesis was tested by comparing fetal umbilical vein (FUV) plasma CRH, ACTH, dehydroepiandrosterone sulfate, and cortisol concentrations at cesarean section in fetuses from mothers undergoing spontaneous, term labor (group I), with those in fetuses from mothers undergoing androstenedione-induced, premature labor (group II) and with those from mothers not in labor (group III). In addition, gestation-related changes in maternal plasma CRH concentrations were investigated, and CRH immunoactivity was characterized by Sephadex G50 chromatography in pooled maternal plasma extracts. FUV CRH concentrations were similarly elevated in group I and group II fetuses, compared with group III fetuses. Despite similar FUV blood gases in all fetuses, FUV ACTH and dehydroepiandrosterone sulfate concentrations were higher in group I fetuses than in group II or group III fetuses. The majority of CRH immunoactivity coeluted with synthetic human CRH. Maternal plasma CRH concentrations showed a modest increase with gestation in the rhesus monkey. These data: 1) demonstrate that androstenedione treatment of pregnant monkeys at 0.8 of gestation elevates fetal plasma CRH to similar concentrations measured at term; 2) do not support the hypothesis that androstenedione-induced delivery in the monkey results from premature activation of the fetal pituitary-adrenal axis by placental CRH; but 3) do support a role for activation of the fetal hypothalamo-pituitary-adrenal axis in association with spontaneous term labor in the monkey; and 4) demonstrate important interprimate species differences in maternal CRH physiology.