8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo

BACKGROUND: Myocardial reperfusion is believed to be associated with free radical injury. However, indexes of oxidative stress in vivo have been limited by their poor specificity and sensitivity. Isoprostanes are stable products of arachidonic acid formed in a nonenzymatic, free radical-catalyzed manner. We have developed a sensitive and specific assay for one of these compounds, 8-epi prostaglandin (PG) F2 alpha. METHODS AND RESULTS: To address its utility as an index of oxidative stress during coronary reperfusion, we measured urinary levels by gas chromatography/mass spectrometry in a canine model of coronary thrombolysis, in patients with acute myocardial infarction treated with thrombolytic therapy, and in patients after elective coronary artery bypass surgery. Urinary 8-epi PGF2 alpha was unchanged after circumflex artery occlusion in a canine model of coronary thrombolysis (n = 13; 437.2 +/- 56.4 versus 432.7 +/- 55.2 pmol/mmol creatinine) but increased significantly (P < .05) immediately after reperfusion (553.8 +/- 64.7 pmol/mmol). Urinary levels were increased (P < .001) in patients (n = 12) with acute myocardial infarction given lytic therapy (265.8 +/- 40.8 pmol/mmol) compared with age-matched control subjects (n = 20; 91.5 +/- 11.8 pmol/mmol) and patients with stable coronary disease (n = 20; 95.7 +/- 6.3 pmol/mmol). Preoperative levels rose from 113.2 +/- 11.8 to 248.2 +/- 86.3 pmol/mmol at 30 minutes into revascularization to 332.2 +/- 82.6 pmol/mmol by 15 minutes after global myocardial reperfusion (P < .05) and dropped to 181.2 +/- 50.4 pmol/mmol at 30 minutes and 120.2 +/- 9.9 pmol/mmol at 24 hours after bypass surgery (n = 5). Corresponding changes in spin adduct formation, found with electron paramagnetic resonance, were noted in 2 patients. CONCLUSIONS: These data support the hypothesis that free radical generation occurs during myocardial reperfusion. Measurement of isoprostane production may serve as a noninvasive index of oxidative stress.     

Metastatic renal cell carcinoma presenting as an aural polyp

Renal cell carcinoma may metastasize to the head and neck region at different stages of its evolution. We present a case of an undiagnosed renal cell carcinoma presenting as an ear polyp, and discuss the difficulties of the diagnosis and the management of these tumours.     

Retained fetal lung liquid in congenital lobar emphysema: a possible predictor of polyalveolar lobe

The purpose of this study was to determine whether retention of fetal lung liquid is more prevalent in polyalveolar congenital lobar emphysema than in conventional congenital lobar emphysema. Two patients with congenital lobar emphysema were prospectively identified in a 3-year period. Twenty-five such patients were identified in a retrospective study covering 39 years. Medical records were available for 22 patients who had 23 emphysematous lobes. Both babies from the prospective study and six subjects from the retrospective group had respiratory symptoms and underwent chest X-ray in the first day of life. Six of the eight babies with respiratory symptoms and chest imaging in the first day of life had retention of fetal lung liquid in an emphysematous lobe. All six of these lobes were polyalveolar. The lobe in one child was a polyalveolar lobe but without retained fetal lung liquid, and one child exhibited conventional lobar emphysema also without retained fetal lung liquid. One polyalveolar lobe caused no neonatal symptoms and was not imaged until the child was 3 months old. No baby with conventional lobar emphysema was shown to have retained fetal lung liquid. There seems to be a correlation between polyalveolar lobe and onset of respiratory symptoms in the first day of life. Retention of fetal lung liquid within the affected lobe was documented only in cases of polyalveolar lobe.     

Effect of constant administration of a gonadotropin-releasing hormone agonist on reproductive activity in mares: induction of ovulation during seasonal anestrus

The potential of a gonadotropin-releasing hormone (GnRH) agonist (goserelin acetate), delivered constantly for 28 days via a subcutaneous depot, to induce ovulation in seasonally anestrous mares, was investigated. Two experiments were conducted, in which a range of doses (30 to 240 micrograms/mare/d) was examined. Mares were selected on the basis of lack of substantial follicular development (follicle diameter < 20 mm determined ultrasonically) and low serum concentrations of luteinizing hormone (LH) and progesterone. Constant administration of the GnRH agonist-induced ovulation in anestrous mares, but a dose-response relation was not observed. Furthermore, with identical doses tested in consecutive or alternate years, considerable variation was observed in the ovulatory response. In general, ovulation in all treated mares was accompanied by increased circulating concentrations of LH and a decrease in follicle-stimulating hormone values. Ovulation was preceded by an increase in estradiol and LH concentrations. In mares in which ovulation did not occur, concentration of LH increased during agonist treatment, whereas that of follicle-stimulating hormone either increased or did not change. It was concluded that constant administration of GnRH agonists may induce ovulation in mares during seasonal anestrus; however, percentage of mares ovulating and the lack of reproducibility of effect indicate that this approach is inappropriate for use as a reliable method to manipulate breeding activity in commercial broodmares.     

Non-functional variants of yeast mitochondrial ATP synthase subunit 8 that assemble into the complex

Myocardial and pulmonary beta-adrenoceptors can be imaged with 2-(S)-(-)-(9H-carbazol-4-yl-oxy)-3-[1-(fluoromethyl)ethyl]amino-2- propanol (S-1'-[18F]fluorocarazolol, I). Quantification of unmodified fluorocarazolol in plasma is necessary for analysis of PET images in terms of receptor densities. We have determined I and its radioactive metabolites in rat, sheep and human plasma, using (1) solid-phase extraction (C18) followed by reversed-phase HPLC and (2) direct injection of untreated plasma samples on an internal-surface reversed-phase (ISRP) column. The two methods were in good agreement. Unmodified I decreased from over 99% initially to less than 5%, 5-10% and 20% at 60 min post-injection in rats, sheep and human volunteers, respectively. Protein binding in sheep and human plasma was determined by ultrafiltration. The fraction of total plasma radioactivity bound to protein and the fraction representing unmodified radioligand were linearly correlated, suggesting that fluorocarazolol was more than 70% protein-bound, whereas its metabolites showed negligible protein binding. Direct injection of plasma on an ISRP column seems a convenient method for quantification of lipophilic radioligands such as fluorocarazolol.     

Value of transcranial Doppler indices in predicting raised ICP in infantile hydrocephalus. A study with review of the literature

Cerebral hemodynamic changes in infants with progressive hydrocephalus have been studied with the transcranial Doppler (TCD) technique. Several authors have referred to the correlation between the hemodynamic changes and increased intracranial pressure (ICP). Despite conflicting conclusions on the value of pulsatility index (PI) and resistance index (RI) measurements for monitoring infantile hydrocephalus, these pulsatility indices are the most commonly used for this purpose. Although clinical signs of raised ICP are highly variable and unreliable in infants, assumptions have been made in most of the studies about the presence of elevated ICP on the basis of the patient's clinical state. Few studies have reported on actual ICP values, however, and a direct relationship between ICP and TCD changes has never been adequately demonstrated. In the present study, this relationship was investigated in long-term simultaneous TCD/ICP measurements, in an attempt to develop a noninvasive method of monitoring the effect of ICP on intracranial hemodynamics. Two groups of data sets were established. Group I consisted of pre- and postoperative (shunt implantation) TCD/ICP measurements. Group II were long-term simultaneous TCD/ICP recordings showing significant ICP variations. In most of the postoperative measurements there was a decrease in the average PI and RI values. The correlation between PI or RI and ICP in the long-term simultaneous recordings, however, was generally poor. The risk of obtaining false positive or false negative PI or RI values in short-term measurements was also demonstrated. It can be concluded from our results, besides the wide range of reference values for the Doppler indices and extracranial influences upon them, that the present Doppler indices are inadequate for monitoring the complex intracranial dynamic responses in patients with raised ICP.     

Diazepam alters caffeine-induced effects on beta-endorphin levels in specific rat brain regions

The present study was conducted to evaluate the effects of caffeine and the benzodiazepine agonist diazepam, and a combination of both on beta-endorphin (beta-EN) levels in specific rat brain regions. Male Sprague-Dawley rats (150-200 g) adapted to a 12-hour light: 12-hour dark illumination cycle were used in this study. Caffeine (10 mg/kg), diazepam (2 mg/kg) or a combination of caffeine (10 mg/kg) and diazepam (2 mg/kg) were administered intraperitoneally to rats at 11:00 hr. Control animals were injected with saline. Animals were sacrificed by decapitation 1 h after injection, the brains were immediately removed; the cortex, hippocampus, hypothalamus and midbrain were dissected and their B-EN levels measured by radioimmunoassay. Caffeine administration significantly increased B-EN levels in the cortex. Similarly, administration of diazepam alone resulted in a significant increase of B-EN levels in cortex. However, concurrent administration of diazepam and caffeine resulted in higher increase of B-EN levels in cortex. No significant changes in B-EN levels were detected in hippocampus and midbrain after administration of either caffeine or diazepam alone. On the other hand, when diazepam and caffeine were concurrently administered a significant increase of B-EN levels were observed in the midbrain. Moreover, administration of diazepam alone resulted in a significant increase of B-EN levels in hypothalamus. This increase was still observed following concurrent administration of diazepam and caffeine. These results clearly indicate that diazepam alters caffeine-induced effects on B-EN in specific rat brain regions.     

Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.