Lipid peroxidation-dependent chemiluminescence from the cyclization of alkylperoxyl radicals to dioxetane radical intermediates

This work reveals a novel mechanism for triplet carbonyl formation (and hence chemiluminescence) during lipid peroxidation, whose chemiluminescence has been attributed to both triplet carbonyls and singlet oxygen. As a model for polyunsaturated fatty acid hydroperoxides, we have synthesized 3-hydroperoxy-2,3-dimethyl-1-butene by photooxygenation of tetramethylethylene. One-electron oxidation of this hydroperoxide with heme proteins and peroxynitrite to the corresponding alkylperoxyl radical results in chemiluminescence, both direct and 9,10-dibromoanthracene-2-sulfonate-sensitized, the latter attributed to the formation of triplet acetone. It is postulated that triplet acetone results from the cyclization of the alkylperoxyl radical to a dioxetane radical intermediate followed by its thermolysis. This is supported by EPR spin-trapping experiments in which discrimination between carbon-centered radicals derived from the alkyloxyl and alkylperoxyl radicals is achieved through the use of one-electron oxidants and reductants, e.g., FeII- and TiIII.     

Electrophysiologic changes in preterm neonates: auditory brain stem response and distortion product otoacoustic emissions

The purposes of this investigation were to determine 1) if auditory peripheral maturity is present in the newborn; 2) if not, at what age maturational changes occur in the peripheral auditory system from preterm to full-term; and 3) how results of tests used to identify auditory dysfunction in neonates, such as distortion product otoacoustic emissions (DPEs) and auditory brain stem responses (ABRs), change during this period. Longitudinal DPE amplitude and ABR wave I latency measurements were obtained from a single ear of 18 preterm neonates. The DPEs were evoked at f2s of 2, 3, 4, and 5 kHz. The longitudinal data revealed that in general, DPE amplitude increased and ABR wave I latency decreased as a function of postconceptional age. These findings suggest that 1) the peripheral auditory system has not reached maturity in the preterm neonate; 2) maturational changes continue from preterm to full-term; and 3) these changes are reflected in ABR and DPE measurements.     

Arterial remodeling after balloon angioplasty or stenting in an atherosclerotic experimental model

The actions of the endogenous ORL1-receptor ligand nociceptin on the membrane properties and synaptic currents in rat periaqueductal gray (PAG) neurons were examined by the use of whole-cell patch-clamp recording in brain slices. Nociceptin produced an outward current in all neurons tested, with an EC50 of 39 +/- 7 nM. The outward current was unaffected by naloxone. Outward currents reversed polarity at -110 +/- 3 mV in 2.5 mM extracellular potassium, and the reversal potential increased when the extracellular potassium concentration was raised (slope = 66.3 mV/log[K+]o mM). Thus, the nociceptin-induced outward current was attributable to an increased K+ conductance. Nociceptin inhibited evoked fast GABAergic (IP-SCs) and glutamatergic (EPSCs) postsynaptic currents and increased paired-pulse facilitation in a subpopulation of PAG neurons. Nociceptin inhibited evoked IPSCs and EPSCs in approximately 50% of neurons throughout the PAG, except in the ventrolateral PAG, where nociceptin inhibited evoked IPSCs in most neurons. Nociceptin decreased the frequency of spontaneous miniature postsynaptic currents (mIPSCs and mEPSCs) in a subpopulation of PAG neurons but had no effect on their amplitude distributions. Thus, nociceptin had a presynaptic inhibitory effect on transmitter release. These findings suggest that nociceptin, via its pre- and postsynaptic actions, has the potential to modulate the analgesic, behavioral, and autonomic functions of the PAG.     

Bacteria in the prostate tissue of men with idiopathic prostatic inflammation

PURPOSE: Although antibiotics represent the first line of treatment for prostatitis syndromes, physicians can document infection in remarkably few cases. We examined the relationship of genitourinary infection to inflammatory prostatitis in 85 subjects without bacteriuria. MATERIALS AND METHODS: Evaluation consisted of cultures of urethra, urine and transperineal prostate biopsies, specifically for commensal and fastidious organisms, and leukocyte counts of expressed prostatic secretions. RESULTS: Men with inflamed expressed prostatic secretions (25) were more likely to have any bacterial isolation (p = 0.01), positive cultures for anaerobic bacteria (p = 0.03), higher total bacterial counts (p = 0.02) and more bacteria, species isolated (p = 0.02) in prostate biopsy cultures than men without expressed prostatic secretion inflammation (60). CONCLUSIONS: Bacterial colonization/invasion of the prostate may be associated with inflammatory prostatitis in some cases.     

Subcellular localization of myosin-V in the B16 melanoma cells, a wild-type cell line for the dilute gene

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the alpha-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.     

Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.     

Combined analysis of randomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis

BACKGROUND & AIMS: Long-term ursodeoxycholic acid (UDCA) therapy slows the progression of primary biliary cirrhosis. This study examined the effect of UDCA therapy on survival free of liver transplantation in a large group of patients. METHODS: Data from three clinical trials were combined in which patients with primary biliary cirrhosis were randomly assigned to receive UDCA (n = 273) or placebo (n = 275). After 2 years, patients from French and Canadian studies received UDCA for up to 2 years. Patients from the American study remained on their assigned treatment for up to 4 years. RESULTS: Survival free of liver transplantation was significantly improved in the patients treated with UDCA compared with the patients originally assigned to placebo (P < 0.001; relative risk, 1.9; 95% confidence interval, 1.3-2.8). Subgroup analyses showed that survival free of liver transplantation was significantly improved in medium- and high-risk groups (serum bilirubin level, 1.4 to 3.5 or > 3.5 mg/dL; P < 0.0001 and P < 0.03, respectively) and histological stage IV subgroup (P < 0.01). CONCLUSIONS: Long-term UDCA therapy improves survival free of liver transplantation in patients with moderate or severe disease. An effect in patients with mild disease is probably not found because they do not progress to end-stage disease in 4 years.     

What is core? Guidelines for the core curriculum in paediatrics

This multicentre, randomised, double-blind, double-dummy, parallel group study was investigated in order to compare on 3 days the efficacy and the safety of the 16 mg once a day (od) ondansetron suppository (suppository group) with the recommended ondansetron treatment, i.e. 8 mg intravenous (i.v.) ondansetron on day 1 followed by 8 mg tablet (p.o.) twice daily (i.v. + p.o. group) on days 2 and 3 in patients receiving cisplatin (> or = 50 mg/m2) containing chemotherapy. In the 420 patients included in the intent-to-treat population, 209 received the 16 mg suppository and 211 the i.v. + p.o. treatment. The number of emetic episodes and the nausea score were recorded each day. Concerning the primary criterion, both treatments provided good anti-emetic control with 87% of all patients having a complete or major response (0-2 emetic episodes) on day 1 in the suppository group and 92% in the i.v. + p.o. group (P = 0.058). The 90% confidence interval for the difference between the two treatments for complete or major control was included in the interval (-15%, 15%) and showed that the treatment groups could be regarded as equivalent. Small differences in favour of the i.v. + p.o. group were observed concerning the secondary parameters. Both treatments were well tolerated. The results of this study show that both treatments are equivalent in the prevention of cisplatin-containing chemotherapy induced emesis for the primary efficacy criteria and that the ondansetron suppository is efficient and well tolerated and is a suitable alternative to the anti-emetic treatment combining the intravenous and oral routes.     

Helix bending as a factor in protein/DNA recognition

Normal vectors perpendicular to individual base pairs are a powerful tool for studying the bending behavior of B-DNA, both in the form of normal vector plots and in matrices that list angles between vectors for all possible base pair combinations. A new analysis program, FREEHELIX, has been written for this purpose, and applied to 86 examples of sequence-specific protein/DNA complexes whose coordinates are on deposit in the Nucleic Acid Data Base. Bends in this sample of 86 structures almost invariably follow from roll angles between adjacent base pairs; tilt makes no net contribution. Roll in a direction compressing the broad major groove is much more common than that which compresses the minor groove. Three distinct types of B-DNA bending are observed, each with a different molecular origin: (1) Localized kinking is produced by large roll at single steps or at two steps separated by one turn of helix. (2) Smooth, planar curvature is produced by positive and negative roll angles spaced a half-turn apart, with random side-to-side zigzag roll at intermediate points, rather than a tilt contribution that might have been expected theoretically. (3) Three-dimensional writhe results from significant roll angles at a continuous series of steps. Writhe need not change the overall direction of helix axis, if it is continued indefinitely or for an integral number of helical turns. A-DNA itself can be formally considered as possessing uniform, continuous writhe that yields no net helix bending. Smooth curvature is the most intricate deformation of the three, and is least common. Writhe is the simplest deformation and is most common; indeed, a low level of continuous writhe is the normal condition of an otherwise unbent B-DNA helix of general sequence. With one exception, every example of major kinking in this sample of 86 structures involves a pyrimidine-purine step: C-A/T-G, T-A, or C-G. Purine-purine steps, especially A-A, show the least tendency toward roll deformations.