Membrane protein alterations in rodent erythrocytes and synaptosomes due to aging and hyperoxia.

We have applied the technique of electron paramagnetic resonance (EPR) protein-specific spin labeling to the study of membrane protein alterations occurring during age and exposure to isobaric hyperoxia. Cortical synaptosomes and erythrocyte membranes (ghosts) were isolated from young rodents (Fisher 344 rats or mongolian gerbils, 3-4 months of age) and aged rodents (age 22-27 months for rats, greater than 15 months for gerbils). Membrane proteins were spin labeled with the thiol-specific spin label MAL-6 (2,2,6,6,-tetramehtyl-4-maleimido-piperdin-1-oxyl). The relevant EPR spectral parameter of MAL-6 labeled membranes, the W/S ratio, decreased significantly with age of animal in both synaptosomes and ghosts (P < 0.001). As a paradigm for accelerated oxidative stress, young and aged gerbils were exposed to an atmosphere of 90-100% O2 for 0-48 h. In both young and aged gerbils, the W/S ratio decreased significantly with hyperoxic stress (P < 0.003). The W/S ratio of synaptosomes isolated from aged gerbils decreased continually from 0-48 h hyperoxia, whereas the W/S ratio of synaptosomes from young animals demonstrated a pronounced rebound effect from 24-48 h. The results are discussed with reference to membrane protein oxidation in aging.

Effects of mitochondrial respiratory stimulation on membrane lipids and proteins: an electron paramagnetic resonance investigation.

Previous studies have implicated mitochondria-derived reactive oxygen species (ROS) in both the aging process and age-related diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease etc. The current study, utilizing electron paramagnetic resonance (EPR) spectrometry, was designed to determine if mitochondrial respiratory stimulation, under state 4 conditions, caused extensive oxidative modifications to membrane cytoskeletal proteins and lipids in the brain. A mixed population of cortical synaptosomes and mitochondria, prepared by centrifugation techniques using rat brain cortex from adult (4-6 months) female Brown Norway rat brains, were labeled with the lipid-specific spin probe, 5-nitroxyl stearate (5-NS). Stimulation of the mitochondrial electron transport chain was accomplished using 20 mM succinate at 25 degrees C for 3 h. Mitochondrially derived free radicals, when reacted with the paramagnetic center of the spin probe, result in a loss of paramagnetism resulting in loss of intensity. A significant lowering (23%, P<0.0001) in the signal amplitude (B0) of 5-NS, indicative of generation of oxyradicals, was found. The order parameter, an inverse EPR-measure of membrane fluidity of the 5-NS spin labeled mitochondrial and synaptosomal membranes, also decreased following mitochondrial respiratory stimulation (P<0.005). Changes in the physical state of cytoskeletal and transmembrane proteins due to succinate oxidation were measured using MAL-6 (2,2,6, 6,-tetramethyl-4 maleimidopiperdin-1-oxyl), a thiol-specific nitroxide spin label. The ratio of the amplitudes of the weakly to strongly immobilized spin label reaction sites (W/S ratio) in the low-field region of the spectrum was used to determine any alteration in protein conformation. Previous studies in our laboratory have established that increased protein oxidation is associated with a decreased W/S ratio. In the current study, our results indicated significant lowering of the W/S ratio in cortex (30%, P<0.0001) upon stimulation of the mitochondria with 20 mM succinate. Thus, we conclude that respiratory stimulation of mitochondria, due to a hypermetabolic stress with succinate, caused significant oxidative modifications of cortical membrane lipids and proteins.

Detection of HIV-1 nucleic acid and HIV-1 antibodies in needles and syringes used for non-intravenous injection.

BACKGROUND: HIV antibodies and HIV DNA have been detected in needles and syringes that have been used for intravenous injections in HIV-infected persons. During intravenous injection, blood is typically aspirated into the lumen of the syringe. During intramuscular or subcutaneous injection, however, blood is not usually introduced into the syringe. OBJECTIVES: To investigate the presence of HIV antibodies, HIV proviral DNA, HIV RNA, and human DNA in needles and syringes that had been used for intramuscular or subcutaneous injection in persons known to have HIV infection. METHODS: Discarded disposable needles and syringes used by health-care personnel for medically indicated intramuscular or subcutaneous injections of HIV-infected patients were collected. Residual material was extracted from the syringes. The extracts were analyzed by enzyme immunoassay for the presence of HIV antibodies. PCR was conducted to detect HIV and human DNA, as well as HIV RNA. RESULTS: HIV antibodies were detected in 16 (6.2%) out of 260 syringes. Human DNA or HIV-specific DNA were not detected. A second set of 80 syringes was collected to examine the presence of HIV RNA. HIV RNA was detected in three (3.8%) out of 80 syringes. CONCLUSION: This analysis demonstrates that the risk of transmitting HIV from syringes that have been used for intramuscular or subcutaneous injection may be low, but is not zero.

Cutaneous tissue repair following CO2 laser irradiation.

We studied the mechanism of repair following exposure of normal skin to the CO2 laser in a focused mode. Exposed areas were biopsied at 0, 24, 48 h; 1, 2 weeks; 1, 2 1/2 months (pulse width varying from 0.1 to 1.0 s) after irradiation. The initial pattern was a V-shaped zone of cauterized collagen with a central crevice, the depth of which correlated with the total energy applied. The epidermal changes consisted of transepidermal cauterization and basal vacuolar changes lateral to the site of impact. Over a period of 1 week, the wound crevice decreased in depth and width and the central margins of the zone of cauterized collagen approximated. The cauterized collagen was extruded and was noted in the epidermal crust; minimal granulation tissue was present. Biopsies at later time periods showed formation of granulation tissue and retention of small amounts of necrotic collagen; the process of collagen extrusion was largely prevented by suturing. These observations show that dermal contraction and necrotic collagen extrusion are important components of initial tissue repair following limited dermal destruction produced by CO2 irradiation.

Oxidative alterations in Alzheimer's disease.

There is increasing evidence that free radical damage to brain lipids, carbohydrates, proteins, and DNA is involved in neuron death in neurodegenerative disorders. The largest number of studies have been performed in Alzheimer's disease (AD) where there is considerable support for the oxidative stress hypothesis in the pathogenesis of neuron degeneration. In autopsied brain there is an increase in lipid peroxidation, a decline in polyunsaturated fatty acids (PUFA) and an increase in 4-hydroxynonenal (HNE), a neurotoxic aldehyde product of PUFA oxidation. Increased protein oxidation and a marked decline in oxidative-sensitive enzymes, glutamine synthetase and creatinine kinase, are found in the brain in AD. Increased DNA oxidation, especially 8-hydroxy-2'-deoxyguanosine (8-OHdG) is present in the brain in AD. Immunohistochemical studies show the presence of oxidative stress products in neurofibrillary tangles and senile plaques in AD. Markers of lipid peroxidation (HNE, isoprostanes) and DNA (8-OHdG) are increased in CSF in AD. In addition, inflammatory response markers (the complement cascade, cytokines, acute phase reactants and proteases) are present in the brain in AD. These findings, coupled with epidemiologic studies showing that anti-inflammatory agents slow the progression or delay the onset of AD, suggest that inflammation plays a role in AD. Overall these studies indicate that oxidative stress and the inflammatory cascade, working in concert, are important in the pathogenetic cascade of neurodegeneration in AD, suggesting that therapeutic efforts aimed at both of these mechanisms may be beneficial.

Modulation of ornithine decarboxylase mRNA following transient ischemia in the gerbil brain.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme that catalyzes the synthesis of polyamines from ornithine and is thought to be involved in the cellular response to growth, differentiation, and stress. Previous studies have demonstrated that transient cerebral ischemia results in an increase in ODC activity and polyamine synthesis. We have used the Mongolian gerbil as a model system to test the hypothesis that the cellular response to ischemia induces a distinct pattern of ODC gene expression. Our results indicate that transient ischemia, induced by bilateral carotid occlusion, elevates ODC mRNA within 1-4 h after reperfusion, which correlates with increased ODC activity and polyamine synthesis. Increased ODC mRNA can be detected in the forebrain, striatum, hippocampus, and midbrain but not the cerebellum, which is not subject to ischemic injury. In contrast, c-fos mRNA increased by 15 min after reperfusion and actin mRNA did not demonstrate alterations in level after ischemia. Pentobarbital prevented the increase in ODC mRNA, whereas the glutamate antagonist MK-801 had no effect on the elevation of ODC gene expression after ischemia. We conclude that the ischemia-induced increase in ODC enzyme activity may be attributed in part to transcriptional activation of the ODC gene.

Basic FGF, NGF, and IGFs protect hippocampal and cortical neurons against iron-induced degeneration.

Iron is believed to contribute to the process of cell damage and death resulting from ischemic and traumatic insults by catalyzing the oxidation of protein and lipids. Exposure of cultured rat hippocampal neurons to iron (FeSO4) caused a dose-dependent reduction in neuronal survival, which was potentiated by ascorbate. Damage to neurons was associated with a significant level of oxygen radical in the culture medium. The iron chelator desferal prevented both the neuronal degeneration caused by FeSO4 and the production of oxygen radical, demonstrating that ionic iron was responsible for the cell damage. Iron neurotoxicity was associated with an elevation of [Ca2+]i and was attenuated by NMDA receptor antagonists. Since recent findings demonstrated neuroprotective effects of growth factors in cell culture and in vivo models of ischemia, we examined the effects of growth factors on iron-induced damage. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and insulin-like growth factors (IGF-I and IGF-II) each protected neurons against iron-induced damage. Both rat hippocampal and human cortical neurons were protected by these growth factors. Taken together, the data suggest that the neuroprotective effects of growth factors against excitotoxic/ischemic insults may result, in part, from a prevention or attenuation of oxidative damage.

Age effect on brain pH during ischemia/reperfusion and pH influence on peroxidation.

Older gerbils are more sensitive to ischemia/reperfusion injury (IRI) than younger ones. Utilizing 31P-NMR to monitor in vivo pH and high energy phosphates of brain cortex undergoing an IRI showed that cortical intracellular pH decreased to 6.35 after ischemia and then increased with reperfusion, but older gerbils required significantly more time to recover than younger animals. Brain high energy phosphates dropped during ischemia but rebounded within 20 min reperfusion in younger gerbil brain but remained significantly lower in older gerbil brain for 50 min. These data suggest that IRI-induced brain acidification may enhance oxidative damage. In an in vitro system it was shown in both young and old brain homogenate that peroxidation rate increased when the pH of the incubation medium was decreased from 7.4 to 6.4. This was true in the presence or absence of an ADP/Fe/Ascorbate system in both young and old brain homogenate. Enhancement of peroxidation rate by ADP/Fe/Ascorbate addition was much more pronounced at pH 7.4 (30- to 40-fold increase) as compared to pH 6.4 (7.8- to 9.5-fold increase). This data can be interpreted to indicate that the lower pH makes endogenous Fe more available to catalyze oxidative damage. The fact that brain pH and high energy phosphates remain lower in older gerbil brains during IRI suggests that brain mitochondria from older animals are less capable of responding to a large oxidative stress brought on by an IRI.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain creatine kinase with aging in F-344 rats: analysis by saturation transfer magnetic resonance spectroscopy.

We measured in vivo forward flux of the creatine kinase reaction in rat forebrain in young (Y: 6 month, n = 13), mid-aged (M: 12 month, n = 7) and aged (O: 27 month, n = 10) animals using 31P magnetic resonance saturation transfer. Forward flux was reduced in the aged rats (Y: 0.42 +/- 0.08; M: 0.41 +/- 0.10; O: 0.31 +/- 0.03 s(-1) +/- SD; p = 0.008 O vs. Y). In vitro studies in a subset of the same rats showed a parallel decline in CK activity (Y: 2.16 +/- 0.40; M: 2.17 +/- 0.25; O: 1.56 +/- 0.06 IU +/- S.D.; p = 0.002 O vs. Y). The in vivo spectroscopic and in vitro biochemical measures were significantly correlated. Reduced creatine kinase activity could account for the observed decreased forward flux in aging brain. Intracellular pH, phosphocreatine/inorganic phosphate ratio, and phospocreatine/gamma-adenosine triphosphate ratio did not differ between groups. Forward flux may represent a better measure of brain energy function than relative phosphocreatine or adenosine triphosphate levels observable in vivo.

Aging and caloric restriction affect mitochondrial respiration and lipid membrane status: an electron paramagnetic resonance investigation.

Previous studies have indicated that reactive oxygen species (ROS) are likely involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). ROS, generated by succinate-stimulated mitochondria, have been reported to be spin trapped and detected by electron paramagnetic resonance (EPR). Our aim in the current study was to study the impact of aging on the effect of increased metabolic stimuli on mitochondrial respiration in terms of oxy-radical generation and possible lipid peroxidative changes in brain neocortical membranes. A mixed population of brain synaptosomes and mitochondria from brown norway male rats of differing ages being fed either ad lib (AL) or a caloric-restricted diet (DR) was prepared and labeled with 5-nitroxyl stearate (5-NS), a membrane lipid-specific spin label. The changes in anisotropic motion of the intercalated 5-NS spin probe also allows one to evaluate the status of the membrane fluidity in the lipid microenvironment via the order parameter. Upon succinate stimulation of mitochondria, the ROS generated resulted in a decrease in the EPR signal amplitude of the 5-NS reporter molecule indicative of the flux of oxy-radicals produced and possible peroxidation-induced changes in the synaptosomal lipid membrane. The line width remained constant, indicating that the overall intensity was reduced. The results showed a significant overall age effect in the ability to generate oxygen-derived radicals following metabolic stimulation (p < .0001). Stimulation of state 4 mitochondrial respiration with 20 mM succinate resulted in greater oxy-radical production in 25-month-old animals as compared to younger animals, suggesting increased mitochondrial leakage with age. Free radical stress induced by metabolic stimulation also causes a concomitant increase in membrane fluidity (p < .0001). There was also a significant age effect (p < .0007) on the order parameter of the mixed population of membranes. Although caloric restriction attenuated the membrane rigidization caused by aging, it was found to play a role in limiting the oxy-radical production following metabolic stimulation of mitochondria. The overall effect of age on membrane spin-label intensities EPR signal upon succinate stimulation suggests that progressive mitochondrial dysfunction may be a key factor in the aging process and in development of age-associated diseases.

Protein oxidation and enzyme activity decline in old brown Norway rats are reduced by dietary restriction.

The effect of aging and diet restriction (DR) on the activity of creatine kinase (CK), glutamine synthetase (GS) and protein carbonyl formation in the cerebellum, hippocampus and cortex of male and female brown Norway (BN) rats has been investigated. It was demonstrated that CK activity in three different regions of the rat brain declines with age by 30%. Age-related decrease of GS activity was only 10-13% and did not reach statistical significance. Consistent with previously published studies, age-related increase of protein carbonyl content in each brain area studied has been observed. Preventive effects of a caloric restricted diet on the age-associated protein oxidation and changes of the activity of CK and GS in the brain was observed for both aging male and female BN rats. DR delayed the accumulation of protein carbonyls. Age-related changes of CK activity in rat brain were abrogated by DR. The activity of GS in the brain of old rats subjected to the caloric restricted diet was higher than that in the brain of young animals fed ad libitum. The results are consistent with the notion that DR may relieve age-associated level of oxidative stress and lessen protein damage.

Effects of selected analogs of adenosine on schedule-controlled behavior in rats.

Acute administration of N6(L-phenylisopropyl)adenosine (L-PIA; 0.01-0.178 mg/kg, s.c.), N6-cyclohexyladenosine (CHA; 0.01-0.178 mg/kg, s.c.), 2-chloroadenosine (2-CA; 0.32-0.56 mg/kg, s.c.), N6-cycloheptyladenosine (CHPA; 0.032-1.0 mg/kg, s.c.), N6-phenyladenosine (PA; 0.1-1.0 mg/kg, s.c.), N6(D-phenylisopropyl)adenosine (D-PIA; 0.32-1.0 mg/kg, s.c.) and N6-benzyladenosine (BA; 1.0-17.8 mg/kg, s.c.) produced dose-related decreases in responding under a fixed-ratio (FR) schedule of food reinforcement. Dose-effect curves were determined by administering cumulative doses (s.c.) during periods that preceded the sequential components of the schedule. Neither 2-chloroadenine arabinoside (2-CAB) nor 2',5'-dideoxyadenosine (2',5'-DDA) altered fixed-ratio responding at the doses studied (0.1-3.2 and 1.0 and 3.2, respectively). Caffeine (0.32 and 3.2 mg/kg) antagonized the behavioral effects of L-PIA in a dose-related, surmountable manner. Daily administration of L-PIA (0.1 mg/kg) resulted in the development of tolerance to the effects of L-PIA decreasing rate and cross-tolerance to other analogs of adenosine (CHA, BA, D-PIA). No cross-tolerance was produced to caffeine, levorphanol or chlordiazepoxide. The activity and order of potency of the analogs of adenosine tested were consistent with the effects of agonists at A1 adenosine receptors. Furthermore, the results demonstrate that the behavioral effects of N6- and 2-chlorine-substituted analogs of adenosine probably act through similar CNS mechanism(s).

Selective effects of behaviorally active doses of methamphetamine on mRNA expression in the gerbil brain.

Administration of methamphetamine results in neuronal damage that may be mediated through the production of oxygen-free radicals and modulations in levels of calcium and glutamate in the brain. These changes have been associated with alterations in gene expression, which may play a role in cell damage. To assess the differences in gene expression related to treatment with methamphetamine, levels of mRNA were evaluated for the proto-oncogene c-fos, heat-shock protein (HSP 70) and actin. It was found that c-fos mRNA expression increased in a dose-dependent manner after administration of methamphetamine. Whereas, levels of HSP mRNA dropped at small doses of methamphetamine and increased dramatically at large doses. In addition, both c-fos and HSP mRNA showed increased levels throughout the brain. Actin mRNA expression was unaffected by any dose of the drug. At the doses that altered gene expression, methamphetamine produced dose-related behavioral changes. Spontaneous locomotor activity was increased at 1.0 mg/kg of methamphetamine, while, larger doses did not alter activity. The data demonstrated that effects of methamphetamine on gene expression occurred within the behaviorally-active dose range and might correlate with the degree of neuronal damage. Selective modulation of gene expression may have a role in determining the acute quantitative and qualitative effects of methamphetamine and the long-term changes that occur after administration of methamphetamine.

Phencyclidine-induced desensitization of striatal dopamine release.

Increased release of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) from slices of striatum of DBA/2J mouse in response to administration of phencyclidine (PCP) in vitro has been observed to be transient, despite continued exposure to PCP. To determine whether this transient response was a result of depletion of releasable pools, toxicity or an adaptive response (desensitization), the recovery of the response to PCP was evaluated. Slices in the control condition were exposed to PCP (300 microM) during test-exposure only. Slices in the PCP pre-exposure condition, were exposed first to PCP (30 microM; pre-exposure) and subsequently to PCP (300 microM; test-exposure). During a washout period (0, 30, 60 or 120 min) between exposures to PCP, the slices were superfused in the absence of PCP. Pre-exposure to PCP diminished the subsequent response to test-exposure to PCP (49 and 37% of control for DA and DOPAC, respectively) after 0 min washout. Overflow of DA evoked by PCP returned towards control values but remained decreased (66% of control) after up to 120 min washout. However, overflow of DOPAC did not return to control values after 60 min washout. Thus, the diminished dopaminergic response, resulting from continued exposure to PCP was not due to depletion of the releasable pool or cytotoxicity but rather to a dynamic adaptive response of the dopaminergic neuron to PCP.

Prevention of ischemia/reperfusion-induced alterations in synaptosomal membrane-associated proteins and lipids by N-tert-butyl-alpha-phenylnitrone and difluoromethylornithine.

Previous studies in our laboratory demonstrated the alteration in the physical state of synaptosomal membrane lipids and proteins in ischemia/reperfusion injury using selective spin labels and electron paramagnetic resonance spectroscopy [Hall et al. (1995) Neuroscience 61, 84-89]. Since many investigations have provided evidence for free radical generation during ischemia/reperfusion injury, we investigated whether a free radical scavenger would prevent the membrane damage, in gerbils. Further, experiments to determine if a secondary effect of polyamine generation at 14 h reperfusion could be blocked by this free radical scavenger or by an inhibitor of ornithine decarboxylase were also carried out. The alterations in synaptosomal membrane integrity observed during ischemia/reperfusion injury were selectively neutralized by treatment with the free radical spin trap N-tert-butyl-alpha-phenylnitrone or an inhibitor of ornithine decarboxylase, difluoromethylornithine. Administration of N-tert-butyl-alpha-phenylnitrone prior to ischemia totally abrogated both lipid and protein alterations observed at 1 and 14 h reperfusion. Pretreatment with difluoromethylornithine neutralized only the 14 h change in lipid label motion. Treatment with N-tert-butyl-alpha-phenylnitrone at 6 h post ischemia showed only a slight attenuation of the 14 h lipid effect and no change in the protein effect. Difluoromethylornithine treatment at 6 h post ischemia negated the 14 h ischemia/reperfusion injury-induced lipid effect and had no effect on the protein change. These data support previous suggestions that free radicals and polyamines play a critical role in neuronal damage and cell loss following ischemia/reperfusion injury and that the polyamine effect is dependent upon free radical generation during ischemia/reperfusion injury.

Effect of 2-cyclohexene-1-one-induced glutathione diminution on ischemia/reperfusion-induced alterations in the physical state of brain synaptosomal membrane proteins and lipids.

Glutathione is able to protect membrane proteins from oxidative stress. In ischemia/reperfusion injury, free radicals cause synaptosomal membrane protein and lipid oxidation that is prevented by the free radical scavenger N-tert-butyl-alpha-phenylnitrone (Hall N. C. et al. (1995) Neuroscience 64, 81-89; 69, 591-600). We wondered if diminution of glutathione would lead to further membrane alterations. Accordingly, the effects of glutathione depletion, by intraperitoneal administration of 2-cyclohexene-1-one, on the physical state of cortical synaptosomal membrane proteins and lipids, with and without global ischemia/reperfusion, were studied in vivo and in vitro in adult and aged gerbils utilizing electron paramagnetic resonance spectrometry. 2-Cyclohexene-1-one (100 mg/kg, i.p.) was administered 30 min prior to 10-min ischemia followed by 1 or 14 h reperfusion. This glutathione reduction agent was also administered to gerbils under the same temporal schedule in the absence of ischemia and compared to untreated controls. Synaptosomal membranes were labeled with a protein-specific spin label, 2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl, or a lipid-specific spin probe, 5-doxylstearic acid. There were no significant changes in the physical state of the lipid portion of synaptosomal membranes when comparing ischemia reperfusion and 2-cyclohexene-1-one-treated ischemia reperfusion in either the adult or aged gerbils. However, glutathione depletion without ischemia/reperfusion caused significant changes in the physical state of the protein portion of cortical synaptosomal membranes in both the adult and aged models. Glutathione depletion, without ischemia/reperfusion, in the adult model showed a maximum change at 3 h that returned to control values by 14 h. In contrast, the aged model showed significant changes at 1 h reperfusion, which did not return to control values by 14 h reperfusion. Glutathione depletion combined with ischemia/reperfusion caused initial protein change in both adult and aged models at 1 h reperfusion, which did not return toward control values by 14 h reperfusion. The results of this study suggest that glutathione depletion increases the severity of membrane protein damage associated with ischemia/reperfusion injury.

Ischemia/reperfusion-induced changes in membrane proteins and lipids of gerbil cortical synaptosomes.

The effects of transient bilateral carotid occlusion on the physical state of synaptosomal membrane proteins and lipids were studied in adult and aged gerbils employing electron paramagnetic resonance. Transient ischemia was produced in adult and aged gerbils by bilateral occlusion of the common carotid arteries with reperfusion times ranging from 0 to 24 h. Synaptosomes of the cerebral cortices were isolated and labeled with a protein-specific spin probe (2,2,6,6-tetramethyl-4-maleimido-piperidine-1-oxyl) and a lipid-specific spin probe (5-doxylstearic acid). Changes in the physical state of the protein peaked at 60 min reperfusion for both adult and aged gerbil models, with a more intense change in aged, but did not return to control values by 24 h. A biphasic change occurred with the lipid-specific label in both the aged and adult models. The onset of the first phase of change occurred at an earlier time (30 min reperfusion) for aged gerbil tissue than for adult tissue (between 3 and 6 h reperfusion), while the second phase of change occurred at 12 h reperfusion for both adult and aged. These results are consistent with the hypothesis that protein oxidation and lipid peroxidation are direct results of free radicals produced during the reperfusion following ischemia and that protein oxidation may be intensified by peroxidation of the surrounding lipids. Phospholipase A2 activation is implicated to cause changes in membrane phospholipid organization as seen in these studies.

Candida esophagitis and laryngitis in chronic mucocutaneous candidiasis.

Five children (aged 11 to 19 years) with lifelong chronic mucocutaneous candidiasis had 12 episodes of esophageal and/or laryngeal candidiasis documented by endoscopy. Symptoms included hoarseness (8/12), dysphagia (6/12), and hemoptysis (1/12). There was poor correlation between oral lesions and esophageal or laryngeal involvement. On fiberoptic endoscopy, the esophagus was involved alone in four episodes (33%), the larynx in two episodes (17%), and both structures in six episodes (50%). In six of eight instances, the esophagram was nondiagnostic or markedly underestimated the extent of inflammation. Intravenous amphotericin B or miconazole resulted in the resolution of these infections for variable periods of time. Repeat endoscopy was used to follow the course of the disease. Aerosolized amphotericin B was effective on one occasion in clearing candidal lesions of the larynx and one small area of the left mainstem bronchus. Oral topical therapy was not beneficial. Since the signs and symptoms of laryngitis or esophagitis are often minimal or absent and complications, including strictures, may arise from chronic inflammation, periodic endoscopy and systemic therapy may be necessary.

Effects of caffeine, theophylline and theobromine on scheduled controlled responding in rats.

1 Rats were trained to respond under a variable interval 30 s (VI 30) schedule of food reinforcement. Caffeine (0.32-32 mg/kg), theophylline (1.0-56 mg/kg) and theobromine (10-320 mg/kg) in general produced dose-related decreases in operant responding. At relatively low doses, caffeine (1.0 mg/kg) and theophylline (3.2 mg/kg) produced slight but nonsignificant increases in VI 30 responding. 3 The rank order of potency for producing decreases in responding was caffeine greater than theophylline greater than theobromine. 4 Daily caffeine injections (32 mg/kg, i.p.) resulted in the development of caffeine tolerance. This tolerance was characterized by a 6 fold shift to the right in the caffeine dose-effect curve. Saline substitution for the 32.0 mg/kg caffeine maintenance dose resulted in a substantial decrease in responding.

Stereospecific dextrorphan tolerance in rats.

1 Levorphanol was 20 times more potent than dextrorphan in decreasing food-reinforced fixed ratio 15 responding in male Sprague Dawley rats. 2 Chronic dextrorphan (100 mg/kg, i.p.; every 8 h) resulted in the development of dextrorphan tolerance. The dextrorphan dose-effect curve was shifted to the right three fold. 3 In contrast to dextrorphan, nontolerance developed to the effects of levorphanol. 4 These data support the hypothesis that (+)-isomers of opioids produce pharmacologically distinct CNS effects.