A high concentration of melatonin inhibits in vitro LDL peroxidation but not oxidized LDL toxicity toward cultured endothelial cells

The pineal hormone, melatonin, was recently found to be a potent free scavenger for hydroxyl and peroxyl radicals. Melatonin also inhibits neuronal and thymocyte damage due to oxidative stress. Atherosclerosis development is mediated by low-density lipoprotein (LDL) oxidation and the endocytosis of oxidized LDL by resident macrophages in the subendothelial vascular wall. Furthermore, the cytotoxic effect of oxidized LDL increases atherogenicity. The goal of this study was to compare the antioxidant activities of melatonin and vitamin E against in vitro LDL oxidation and their cytoprotective actions against oxidized LDL-induced endothelial cell toxicity. An attempt at loading LDL with melatonin by incubating human plasma with an ethanolic melatonin solution gave only low protection against Cu2+-induced LDL oxidation in comparison with vitamin E and gave no detectable incorporation of melatonin into LDL, measured by high-performance liquid chromatography (HPLC) coupled to UV detection. High concentrations of melatonin (10-100 microM) added to the oxidative medium induced a clear inhibition of Cu2+-induced LDL oxidation, characterized as an increase in the lag-phase duration of conjugated diene formation and decreases in the maximal rate of the propagation phase and in the maximal amount of conjugated diene formation. Determination of the median efficacious dose (ED50) of melatonin and vitamin E by their ability to increase lag-phase duration showed that melatonin was less active than vitamin E (ED50, 79 vs. 10 microM, respectively). Melatonin was also less active than vitamin E in limiting the formation of thiobarbituric acid-reactive substances (TBARS) and LDL fluorescence intensity increase in the medium during Cu2+-induced LDL oxidation. Cu2+-induced LDL oxidation in the presence of 100 microM melatonin produced oxidized LDLs that were less recognizable for the scavenger receptors of J774 macrophages than were untreated LDLs. Vitamin E, 10 microM, was more active than 100 microM melatonin in inhibiting LDL oxidation and the resulting lipoprotein alterations leading to binding internalization and degradation by the J774 macrophages. Vitamin E, 100 microM, inhibited the pursuit of the oxidation of oxidized LDL mediated by bovine aortic endothelial cells (BAECs) in a culture medium containing Cu2+, whereas 100 microM melatonin had no antioxidant effect. Melatonin, 100 microM, as well as 100 microM vitamin E inhibited intracellular TBARS formation during the incubation of BAECs with highly oxidized LDL but had no influence on the increase in glutathione (GSH) concentration during this lengthy exposure (16 h) of BAECs to highly oxidized LDL. During this period, the same dose of vitamin E but not of melatonin tended to limit the decrease in adenosine triphosphate (ATP) concentration. Vitamin E, 100 microM, did not significantly reduce cellular lactate dehydrogenase (LDH) release in the culture medium during the incubation of oxidized LDL with BAECs, whereas 100 microM melatonin dramatically increased this release. These data show that melatonin is less active than vitamin E in inhibiting in vitro LDL oxidation and does not inhibit the cytotoxicity of oxidized LDL toward cultured endothelial cells. The concentrations necessary to inhibit LDL oxidation are far beyond those found in human plasma (100 microM vs. 100 pM). Therefore our results indicate that the pineal hormone melatonin per se appears to have little antiatherogenic property in the in vitro oxidation of LDL and the cytoprotective action against the toxicity of oxidized LDL. Nevertheless, in vivo LDL oxidation takes place in the subendothelium of the artery wall, and nothing is known about the concentration of melatonin or its catabolites in this environment.     

Incidence, timing, and causes of cerebral ischemia during carotid endarterectomy with regional anesthesia

PURPOSE: Controversy exists regarding the best technique to identify cerebral ischemia during carotid endarterectomy (CEA). Regional anesthesia allows continuous evaluation of neurologic function and therefore can help determine the incidence, timing, and causes of cerebral ischemia. METHODS: The timing and clinical manifestations of any neurologic event during CEA and as long as 30 days afterward was determined by review of operative reports, hospital charts, and outpatient records of consecutive patients who underwent CEA under regional anesthesia over a 68-month period. RESULTS: Two hundred patients underwent CEA; indications were asymptomatic stenosis > 60% in 25%, transient ischemic attack with stenosis > 50% in 52%, and prior stroke with stenosis > 50% in 23%. Eight patients (4%) were converted to general anesthesia for non-ischemic reasons. Of the remaining 192 patients, 183 (95.5%) underwent the procedure with regional anesthesia and no shunt, 2% had cerebral ischemia and underwent shunt placement, and 2.5% had cerebral ischemia, were converted to general anesthesia, and underwent shunt placement. Cerebral ischemia developed in nine patients after carotid cross-clamping, manifested by loss of consciousness in four, confusion in two, dysarthria and confusion in one, and decreased contralateral motor strength in two. Immediate cerebral ischemia developed in four of the nine patients within 1 minute of cross-damping; all four underwent shunt placement. In five of the nine patients, cerebral ischemia occurred between 20 and 30 minutes after cross-clamping; all occurred during relative intraoperative hypotension (average reduction of 35 mm Hg in the systolic pressure). All awake patients in whom ischemic symptoms developed immediately regained and maintained normal neurologic function with shunt placement. Five of 26 patients (19%) with contralateral occlusion required a shunt; none had postoperative ischemia. The mean carotid cross-clamp time was 27 minutes. Postoperative (30 day) complications included a 0.5% stroke rate, a 0.5% rate of postoperative transient ischemic attack, a 0.5% rate of worsening of preexisting acute stroke, and a 0.5% rate of myocardial infarction (no deaths). Of the nine patients who had intraoperative ischemic changes, none had a postoperative neurologic deficit; the three patients who had postoperative neurologic changes had no intraoperative ischemic symptoms. CONCLUSIONS: CEA with regional anesthesia allows continuous neurologic monitoring and can be performed safely even when contralateral occlusion coexists; intraoperative shunting for ischemia is necessary in 4.5% of all cases and in 19% of patients with contralateral occlusion. Intraoperative ischemia was flow-related in our patients; it occurred early from ipsilateral carotid clamping and late from reduced collateral flow as a result of hypotension. Monitoring should be continued throughout cross-clamping to identify late cerebral ischemia. Postoperative cerebral ischemia is not associated with intraoperative ischemia, if corrected.     

Symptoms of depression among community-dwelling elderly African-American and white older adults

BACKGROUND: Few studies have explored the variance in individual symptoms by race in older adults. METHODS: Data were analysed from the Duke site of the Established Populations for Epidemiologic Studies of the Elderly (EPESE), a community sample of persons 65 years-of-age and older, 54% of whom were African-Americans. Of the 3401 subjects with adequate data on depressive symptomatology, confirmatory factor analysis and LISREL were first used to confirm the presence of the factor structure previously reported for the CES-D. Next, bivariate analysis was performed to determine the prevalence of individual symptoms by race. Finally, LISREL analysis was performed to control for potential confounding variables. RESULTS: When bivariate comparisons of specific symptoms by race were explored, African-Americans were more likely to report less hope about the future, poor appetite, difficulty concentrating, requiring more effort for usual activities, less talking, feeling people were unfriendly, feeling disliked by others and being more 'bothered' than usual. When LISREL analyses were applied to these data (controlling for education, income, cognitive impairment, chronic health problems and disability and other factors) racial differences in somatic complaints and life satisfaction disappeared, yet differences in interpersonal relations persisted. CONCLUSIONS: This study confirms earlier findings of minimal overall differences in symptom frequency between African-American and non-African-American community-dwelling older adults in controlled studies.     

Purification and properties of a novel chloroplast stromal peptidase. Processing of polyphenol oxidase and other imported precursors

Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the DeltapH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.     

Use of somatostatin analogue scintigraphy in the localization of recurrent medullary thyroid carcinoma

Detection of recurrence of medullary thyroid carcinoma (MTC) remains a diagnostic problem. Increased serum tumour marker levels frequently indicate recurrence while conventional imaging techniques (CIT) are non-diagnostic. In this study, we performed indium-111 octreotide scintigraphy and CIT in a series of 20 patients with MTC presenting with elevated serum tumour markers after surgery. 111In-octreotide whole-body studies detected 15 pathological uptake foci in 11 of the 20 patients studied and CIT detected 17 lesions in 11 of the 20 patients. Ten patients underwent reoperation, five of them with positive 111In-octreotide scintigraphy and CIT and two with positive isotopic exploration and negative CIT. Surgical findings demonstrated that the results of isotopic study and CIT had been false-positive for MTC in one case (sarcoidosis). The six patients with true-positive 111In-octreotide studies had significantly higher basal calcitonin (CT) and carcinoembryonic antigen (CEA) levels than the patients with negative isotopic studies. The expression of somatostatin receptor (SSTR) subtypes by PC-PCR could be investigated in four cases with a positive isotopic study. Among the three cases with a true-positive study, SSTR2, the SSTR subtype that preferentially binds to the somatostatin analogue octreotide, was detected in two, SSTR5 was demonstrated in the three, and SSTR3 was detected in one. No subtype of SSTR was detected in the case with a final diagnosis of sarcoidosis. We conclude that 111In-octreotide has limited sensitivity in detecting recurrence in patients with MTC, although its sensitivity may improve with high serum CT levels. This radionuclide imaging technique should be employed when conventional imaging techniques are negative or inconclusive or when the presence of somatostatin receptors may provide the basis for treatment with somatostatin analogues.     

Sensitivity and reproducibility of RT-PCR to detect Borna disease virus (BDV) RNA in blood: implications for BDV epidemiology

Borna disease virus (BDV) infection of domestic animals and humans appears to have a worldwide distribution. There is evidence suggesting an association of BDV with certain psychiatric disorders. However, more comprehensive epidemiological studies are required to establish rigorously a link between BDV and human mental disorders, and to evaluate the role of carrier animals as potential source of BDV for human infection. The use of RT-PCR to detect BDV RNA in peripheral blood mononuclear cells (PBMCs) of infected individuals is a powerful tool to address these questions. The comparison of discrepant results reported by different investigators using this approach is hampered by the lack of controls to assess the sensitivity and reproducibility of the assays. Procedures are now described that allow the establishment of standardized controls to evaluate the performance of the RT-PCR assays. This RT-PCR assay detected reproducibly 100 copies of BDV p40 RNA in 5 microg of RNA. The data illustrate that the number of PBMCs used for RNA preparation, rather than the amount of RNA, has a critical influence on the outcome of the RT-PCR assay. Evidence is provided that levels of BDV in blood do not necessarily reflect viral load in brain.     

Human immunodeficiency virus type 1 protease inhibitors: evaluation of resistance engendered by amino acid substitutions in the enzyme's substrate binding site

The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication. A number of specific, active-site inhibitors for this enzyme have been described. Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes. An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity. The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82. Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions. The substitution at position 32 resulted in a significant adverse effect on inhibitor potency. However, this substitution also mediated a noted increase in the Km of the substrate. Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility. Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.     

Effects of gallstone solvents on commonly used catheters

PURPOSE: To determine what interaction and effect different cholesterol gallstone solvents have on catheters used for gallstone chemolysis. MATERIALS AND METHODS: Five types of catheters used for biliary procedures were chosen: polyethylene, Percuflex, silicon, Silitek, and polyurethane. The solvents chosen were methyl tert-butyl ether, ethyl propionate, isopropyl acetate, and N-propyl acetate. After incubation of the catheters in the solvents for 72 hours, they were air dried. Weight and area changes were determined for each catheter. Additionally, carbon-13 nuclear magnetic resonance (NMR) spectroscopy was performed for analysis of composition changes. RESULTS: Three catheters--silicone, Silitek, and polyurethane--showed changes in their physical characteristics that would make them less desirable for stone chemolysis. The silicone catheter showed changes in elastic texture as well as marked weight reduction. The Silitek and polyurethane catheters had similar, but less dramatic changes. C-13 NMR analysis of collected solvents showed that commonly used plasticizers were leached out of some catheters. CONCLUSION: These results suggest that all catheters are not suitable for use with all solvents. The choice of catheter should be made based on the solvent in use. The polyethylene catheter performed best under the conditions and endpoints used in this study.     

Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.