Superoxide as an intracellular radical sink

BACKGROUND: A major complication of unvalved drainage tube implants is hypotony. Occluding stents to internally block the tube, but which lie exposed in the inferior fornix, have been described. The authors report modifications of this technique. METHODS: A nonexposed 3-0 Supramid suture is used to internally block the tube, with the end placed subconjunctivally. Small venting slits are made in the extrascleral portion of the tube to provide early control of intraocular pressure. Molteno implants were placed in 32 eyes of 29 patients using these modifications. RESULTS: Mean preoperative intraocular pressure was 30.3 mmHg on an average of 2.6 medications. The stent was pulled without difficulty in the office treatment room a mean of 26 days postoperatively. Intraocular pressure from postoperative day 2 until the day of stent removal ranged from 2 to 26 mmHg, with 72% of eyes having intraocular pressure between 5 and 19 mmHg. There were 3 (9.4%) minor complications in the 32 eyes, with 1 case of early anterior chamber shallowing and 2 cases of moderate choroidal detachments, which settled spontaneously. With an average follow-up of 10.2 months, mean intraocular pressure at the final visit was 13.4 mmHg on an average of 1 medication. CONCLUSION: The "occluding stent" described here can be a helpful adjunct to unvalved drainage tube surgery in preventing early postoperative hypotony.     

Electron-beam melting with an intermediate container as an efficient way for the improvement of the mechanical properties of molybdenum

The ways of improving the mechanical properties of molybdenum at low temperatures are studied. Positive effects of electron-beam melting with an intermediate container (EBMIC) on the structure of molybdenum ingots, in particular, on refining from impurities and microstructure refinement are shown. The effect of EBMIC on the mechanical properties of molybdenum in the ductile-brittle transition temperature range is studied.     

Potential application of p53 as an intermediate biomarker in Barrett's esophagus

Diagnosis of the neoplastic progression in Barrett's esophagus using the histologic classification of dysplasia is frequently difficult. The tumor suppressor protein p53, when mutated, confers a promoter effect on cell growth. The purpose of this study was to evaluate the applicability of p53 as an intermediate biomarker of malignancy in Barrett's esophagus. Archival analysis of 100 biopsy specimens of Barrett's esophagus and 10 esophageal adenocarcinomas were compared with 35 chronic esophagitis biopsy specimens. Immunocytochemistry using an anti-p53 monoclonal antibody was performed and elevated immunoreactivity quantitated microscopically. Data were analyzed using a logistic regression model. Significant p53 immunoreactivity occurred as follows: chronic esophagitis (0%), Barrett's esophagus without dysplasia (10%), with low-grade dysplasia (60%), with high-grade dysplasia (100%), and adenocarcinoma (70%). All cases of Barrett's esophagus were significantly immunoreactive when compared with the chronic esophagitis cases (p = 0.001). There was an increase in p53 immunoreactivity as the histologic classification progressed toward adenocarcinoma (p = 0.001). Progression to high-grade dysplasia may be predicted based on p53 immunoreactivity. These findings suggest a role for p53 as an intermediate biomarker in Barrett's esophagus.     

ADP-ribosylation as an intermediate step in inactivation of rifampin by a mycobacterial gene

Mycobacterium smegmatis DSM43756 inactivates rifampin, and the inactivated antibiotic product recovered from culture medium was ribosylated on the 23-OH group. To study this process, the gene responsible for the inactivation was expressed at high levels by the lac promoter in Escherichia coli conferring resistance to >500 microg of antibiotic per ml. Cell homogenates generated a novel derivative designated RIP-TAs; in this study, we determined that RIP-TAs is 23-(O-ADP-ribosyl)rifampin. Our results indicated that RIP-TAs is an intermediate in the pathway leading to ribosylated rifampin and that the previously characterized gene encodes a mono(ADP-ribosyl)transferase which, however, shows no sequence similarity to other enzymes of this class.     

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.     

Superoxide anion reacts with enzyme intermediate in the bacterial luciferase reaction competitive with intramolecular electron transfer

The aims of this study were to identify the sexual problems of elderly people institutionalised in 2 nursing-homes and to find out how much the caregivers employed at these homes know about the sexuality-related problems affecting the elderly people they care for. Two questionnaires were issued: one to the residents of the home, the other to the caregivers. Results showed that the caregivers had very little awareness of sexuality in elderly people and of the particular problems and discomfort of those living in institutions. Discomfort was mainly due to loneliness, difficulty in communicating with caregivers about sentimental and sexual needs, finding new partners and lack of communication between the elderly person and his relatives regarding sexuality-related problems.     

Superoxide generation by neutrophils as defence mechanisms in elderly

We investigated superoxide generation by polymorphonuclear leukocytes (PMNs) stimulated with various species of bacteria in order to clarify the species-specific immunocompromised status in the elderly. We studied 66 elderly patients (ranged 66 to 89 year-old; mean 77-year-old) and 26 young healthy volunteers (ranged 25 to 3-year-old; mean 31-year-old) as a control group. The elderly patients were divided into two groups, i.e., the malignancy group and the benign group. Peripheral blood was prepared from each host and diluted. The activity of superoxide generation by PMNs was determined by means of chemiluminescence method. PMNs were stimulated with the standard stimulants; i.e., phorbol myristate acetate (PMA) and zymosan (ZY), and four bacterial species; i.e., Escherichia coli, Psudomona aeruginosa, Staphylococcus aureus and Candida albicans. The activity of superoxide generation by PMNs in the malignancy group was significantly lower than that in the control group (p < 0.05). Furthermore, the activity of superoxide generation by PMNs in the malignancy group and the benign group stimulated with C. albicans were significantly lower than those in the control group (p < 0.05). These results indicate that the elderly are in an immunocompromised status especially against fungus such as C. albicans.     

Environmental noise as a cause of sleep disruption in an intermediate respiratory care unit

Our laboratory previously reported continuously monitored peak sound levels in several areas at Rhode Island Hospital. The number of sound peaks greater than 80 A-weighted decibels (dBA) was found to be high in the intensive and intermediate respiratory care unit (IRCU) areas, even at night. Environmental noise of this magnitude is potentially sleep-disruptive. Therefore, we hypothesized that nocturnal peak sound levels of > or = 80 dBA would be associated with an increase in EEG arousals from sleep in patients in the IRCU. Six patients underwent sleep monitoring while environmental peak sound levels were continuously recorded. Each 8-hour period (2200 to 0600 hours) was broken down into 30-minute segments. If there were 10 minutes or more of wakefulness in a segment, that segment was dropped from further analysis. Of the remaining 61 segments, there was a very strong correlation (r = 0.57, p = 0.0001) between the number of sound peaks of > or = 80 dBA and arousals from sleep. These 61 periods were then classified as quiet, moderately loud, and very loud based on the number of sound peaks (< or = 5, 6-15, and > 15, respectively). Analysis of variance revealed a significant difference between the number of arousals (p = 0.001) in quiet periods and that in very loud periods. We conclude that environmental noise may be an important cause of sleep disruption in the IRCU.     

Hemichorea-hemiballism: an explanation for MR signal changes

Studies were performed to determine if the nonsteroidal anti-inflammatory drug ibuprofen alters bone and mineral metabolism in female rats. In experiment 1, four groups of growing rats underwent either sham operation or ovariectomy (OVX). One week later, controlled-release pellets with ibuprofen or placebo were implanted subcutaneously at the back of the neck. Following 3 weeks of treatment, rats were sacrificed and blood and bone samples were removed for serum assays and histomorphometric analysis. Body growth rate and the static cortical bone measurements made at the tibial diaphysis did not change in response to OVX. OVX, however, did increase radial bone growth, lowered serum 17beta-estradiol, reduced uterine weight, and decreased the cancellous bone area of the tibial metaphysis in the rats. Ibuprofen did not alter serum 17beta-estradiol or uterine weight but reduced radial bone growth as well as cancellous bone area of the tibial metaphysis in both sham-operated and OVX animals. In experiments 2 and 3, we tested the influence of ibuprofen on the effects of the tissue-selective estrogen agonist tamoxifen and of exogenous 17beta-estradiol in the OVX rat. Ibuprofen completely blocked the effects of tamoxifen and partially blocked the effects of 17beta-estradiol to prevent cancellous osteopenia. In contrast, ibuprofen did not influence the effects of tamoxifen and 17beta-estradiol to reduce radial bone growth. Besides the skeletal effects, ibuprofen suppressed estrogen-induced uterine growth. Our data suggest that ibuprofen blocks selective estrogen receptor-mediated activities in the rat.     

Green fluorescent protein as a signal for protein-protein interactions

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.     

A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.     

A function for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in fibroblasts during mitogenesis: requirement for insulin- and lysophosphatidic acid-mediated signal transduction

We have previously shown that phosphatidylinositol 3-kinase alpha (PI 3-Kalpha) (p85alpha-p110alpha) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185-9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kbeta (p85alpha-p110beta) during mitogenesis. By using antibodies specific to p110beta we showed that PI 3-Kbeta is expressed in NIH 3T3 cells. PI 3-Kbeta and PI 3-Kalpha have common features: PI 3-Kbeta is tightly associated with a protein serine kinase that phosphorylates p85alpha, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kbeta was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kalpha was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110beta into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kbeta plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kalpha and PI 3-Kbeta, suggesting that Ras uses other effectors for DNA synthesis.     

Kinetic evidence for an on-pathway intermediate in the folding of cytochrome c

An early folding event of cytochrome c populates a helix-containing intermediate (INC) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U <-->INC <--> N, or whether the I must first move back through the U and then fold to the N through some alternative path (INC <--> U <--> N). By means of a kinetic test, it is shown here that the cytochrome c I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I <--> U <--> N option.     

Evidence for an obligatory intermediate in the folding of interleukin-1 beta

We investigated the susceptibility of three clinically isolated strains of Cryptococcus neoformans with different virulences to reactive nitrogen and oxygen intermediates (RNI and ROI, respectively), representing two important mediators of macrophage microbicidal activity. All mice infected with the highly virulent strain of C. neoformans, YC-11, died within 3 to 6 weeks because of rapid multiplication of the organism in the lungs and dissemination to the brain. In contrast, a weakly virulent strain, YC-13, was almost completely eradicated from the lungs and did not disseminate to the brain, leading to survival of all infected animals during the period of observation (15 weeks). The virulence of the third strain, YC-5, was intermediate between the other two strains. To examine the susceptibility of C. neoformans to the fungicidal effect of nitric oxide (NO) and superoxide anions (O2-), the organisms were exposed to these oxidants, which were chemically generated in a cell-free system. Interestingly, the number of live YC-13 yeast cells was markedly reduced after exposure to NO and O2-. In contrast, YC-11 was almost completely resistant to the killing effect of these oxidants. YC-5 showed an intermediate susceptibility. Our results demonstrate that the resistance of C. neoformans to the fungicidal effects of RNI and ROI is related to virulence, and suggest that the resistance to nitrogen- and oxygen-derived oxidants may be one of the factors to determine the outcome of infection with C. neoformans.