Neuronal nitric oxide synthase: expression in rat parietal cells.

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.

Telepathology between Richmond and Beckley Veterans Affairs Hospitals: report on the first 1000 cases.

OBJECTIVE: A telepathology connection between Richmond VAMC and Beckley VAMC using dynamic robotic telepathology to provide pathology services remotely was established. MATERIALS AND METHODS: This study reports a 14-month experience using telepathology to diagnose surgical specimens obtained from patients at the Beckley VA Medical Center and viewed in Richmond 250 miles away. Over 14 months, 2325 slides representing 1000 cases were viewed. RESULTS: Discrepancies were observed in 20 of 2325 slides, or 0.86% of the total. None of the patients, where a discrepancy was found, were adversely affected by the preliminary report given. CONCLUSIONS: This study demonstrates that telepathology is a reliable and cost-effective alternative to on-site pathology services and reviews advantages and disadvantages of the system.

Corneal ulcer and adverse reaction rates in premarket contact lens studies.

We analyzed clinical data on 22,739 contact lens wearers who were studied and whose lenses were approved under 48 manufacturer-sponsored studies for the Food and Drug Administration between 1980 and 1988. The incidence of corneal ulcers was low in the cosmetic (nontherapeutic) daily-wear soft and rigid gas-permeable lens wearers (1/1,923 and 1/1,471 patient-years, respectively). Corneal ulcers and severe adverse reactions occurred two to four times more frequently in extended-wear cosmetic soft and rigid gas-permeable lens wearers than in cosmetic daily-wear lens wearers. Aphakic extended-wear soft lens users were nine times more likely to develop a corneal ulcer when compared to the soft daily-wear cosmetic group. Corneal abrasions and keratitis accounted for 81 of 159 severe adverse reactions, whereas corneal ulcers accounted for 28 of 159 adverse reactions. The data indicate that overnight extended wear of contact lenses is associated with a greater risk of serious, sight-threatening complications than daily wear.

The FDA role in contact lens development and safety.

The Food and Drug Administration (FDA) exercises a multifaceted role in fulfilling its mission of enforcing the Federal Food, Drug and Cosmetic Act (Act), functioning not only as industry regulator and consumer protector, but also as scientific advisor and consumer educator regarding medical devices, drugs, foods, cosmetics, and veterinary medicine. Medical devices are regulated within the Center for Devices and Radiological Health. Contact lenses are regulated under the authority of the medical device amendments. The Center is responsible for promulgating regulations, publishing guidelines, and developing written guidance in enforcing the Act, and also for guiding manufacturers of medical devices in safe and effective product development. Other components deal with the compliance of manufacturers with the marketing of medical devices within the meaning of the Act, and through labeling requirements of the Act and consumer education and informational activities. As for contact lenses, the process of updating product development regulations and guidelines is an ongoing activity. The most recent version of the Contact Lens Guideline Document, issued in April 1988, contains two major revisions involving preclinical and clinical testing. The first redefines plastics into one materials category, thus reducing testing requirements with respect to animal toxicology studies and other preclinical areas. The second revision restricts clinical testing requirements to allow confirmatory trials in applications for new daily wear lenses. The intention was to maintain the ability of studies to detect major material or design flaws in lenses, thus boosting confidence in their performance while eliminating unnecessary trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Attenuation of decompressive hypoperfusion and cerebral edema by superoxide dismutase.

This study tested the hypothesis that ischemia-reperfusion injury initiated by the superoxide anion radical is a major component of postdecompression hypoperfusion and cerebral edema, and could be attenuated by superoxide dismutase (SOD). A supratentorial extradural balloon was placed in 20 fasting, lightly anesthetized, mechanically ventilated dogs and inflated in 0.5-ml increments (0.07 ml/sec) at 15-minute intervals. The end-point of balloon expansion was the onset of an isoelectric electroencephalogram, near-arrest of hemispheric cerebral blood flow (CBF) (measured by H2 clearance), and the appearance of a suprainfratentorial intracranial pressure gradient, which was held for 15 minutes. The in vivo development of brain edema was detected by measuring brain elastic response (BER) extradurally, and was correlated with postmortem measurement of brain water content (gravimetry); blood-brain barrier integrity was tested by Evans blue dye given after the insult. After decompression, the dogs were randomly assigned to one of four treatment groups: Group I received hyperventilation (PaCO2 28 +/- 1 mm Hg, mean +/- standard deviation); Group II received furosemide (2.4 mg/kg) and pentobarbital (10 mg/kg) every 8 hours; Group III received 20% mannitol in a 1.4-gm/kg bolus plus furosemide, 0.5 mg/kg; and Group IV received SOD, 15,000 U/kg every 15 minutes for 3 hours. At 4 hours of decompression Group IV had significantly greater recovery in local CBF and BER than Groups I, II, and III (p less than 0.05). The 24-hour survival rate was 20% for Group I, 60% for Group II, 80% for Group III, and 100% for Group IV. The survival rate appeared to correlate with a variable degree of postmortem intraparenchymal hemorrhages, blood-brain barrier disruption, and moderate to severe brain edema for Groups I, II, and III. In contrast, Group IV had the least brain edema (p less than 0.05) and Evans blue dye extravasation (p less than 0.05) and the fewest intraparenchymal hemorrhages. These data support the hypothesis that, under the experimental conditions described here, the superoxide anion plays a major role in the pathophysiology of postdecompression ischemic edema.

Anterior horn changes of motor neuron disease associated with demyelinating radiculopathy.

Morphologic study of the spinal cord of a patient with generalized motor deficits revealed changes in the anterior horns characterized by the selective loss of large motor neurons, gliosis and the abnormal accumulation of 10 nm filaments which appeared as argyrophilic spheroids in the perikarya and axons of motor neurons. The ventral roots were predominantly affected and showed a variable loss of axons. The remaining axons displayed prominent onion-bulb formations, frequent axonal sprouting and occasionally evidence of active demyelination. The coexistence of a demyelinating motor radiculopathy and anterior horn changes simulating those of amyotrophic lateral sclerosis (ALS) may contribute to our understanding of the unresolved question of whether the neuronal perikaryon or its axon is the primary target in the pathogenesis of ALS. These observations also indicate that a rigid separation of pathogenetic mechanisms into neuronopathy, axonopathy and myelinopathy may not be always possible.

Rapid in vivo quantification and comparison of hydroperoxides and oxidized collagen in aging mice, rabbits and man.

A new method is described for the measurement of hydroperoxides and oxidized collagen in epidermal skin of mice, rabbits and man using reflective, near-infrared spectroscopy. Slight decreases in levels of hydroperoxides and oxidized collagen occur during the non-senescent phase of life. On the other hand, dramatic increases are experienced during the senescent phase. These increases are smallest in man, who has the most efficient defence system against peroxides and other active oxygens. Preliminary clinical work suggests that significantly reduced levels of hydroperoxides and oxidized collagen can be obtained with antioxidant diet-supplementation in middle-aged mice (approximately 90%, approximately 34%) and man (approximately 19%), respectively.

A new method that investigates superoxide versus respiration in vitro using bioluminescence and Sepharose-bound adenosine derivatives.

In vitro reactions between superoxide and phosphate derivatives of adenosine yielded quantitative amounts of stable ozonide-products. ADP-ozonide was formed in an optimized in vitro synthesis from ADP + O-(2) and affected by inhibitors and uncouplers in a similar manner to in vivo, oxidative-phosphorylation results. ADP-ozonide was further reacted with phosphoric acid to form ATP. Superoxide and ADP-ozonide may be important carrier-intermediates between respiration's electron-transport chain and nodule ATP formation in vivo.

A new method which investigates lysosomal sensitivity to superoxide with endocytotic, site-specific, chemiluminescent probes.

The end result of cellular autophagocytosis and lysosomal digestion is incompletely understood in regard to the roles of the superoxide anion radical (O2-.). Cultured glial cells sequestered endocytotically two probes that were site-specific to the lysosome vacuome and sensitive to free-radical activities. The Sepharose-4B-polyisoluminol probe emitted chemiluminescent light in proportion to externally injected O2(-).. Bioluminescence measurements of beta-glucuronidase activity in intact glial cell lysosomes was achieved with the second site-specific, enzyme-specific Sepharose-4B-(dodecanate)5'-luciferinylglucuronate probe. Considerable activity was found in the lysosomal vacuome. In conclusion, we suggest that the use of site-specific, chemiluminescent probes can be of importance in the study of free radicals.

Application of chemiluminescent probes in investigating lysosomal sensitivity to superoxide versus suspected radical scavengers.

The role of the superoxide anion radical (O2(-).) relative to catalytic/inhibitory substances in lysosomes is poorly understood. Cultured glial cells sequestered endocytotically two probes that were site-specific to the lysosome vacuome and sensitive to radical activities. The Sepharose-4B-isoluminol probe emitted chemiluminescent light in proportion to externally injected O2(-). and was inhibited or catalysed by various radical scavengers, transition metals and other substances which may affect lysosomal metabolism and lipofuscin formation. We conclude that lysosomal radical activities may be inhibited by butylated hydroxytoluene, hydrocortisone, ACF, RNA, alpha-tocopherol, and, in special circumstances, with fully metabolized iron. Choline and oxidized copper and iron cations in overload concentrations in incubated freshly in lysosomes may catalyse radical activities, and they may be important factors in lipofuscin formation and its role in aging.

The prolongation of life: a comparison of antioxidants and geroprotectors versus superoxide in human mitochondria.

Metabolically-active human mitochondria were impregnated with a site-specific, chemiluminescent probe and different mixtures of known or suspected antioxidants and/or geroprotectors. Superoxide flux from the endomitochondrial respiratory chain and from exomitochondrial sources were measured in regard to the degree of protection from different geroprotective mixtures. In human mitochondria, mixtures of choline, RNA, ACF 223, BHT, copper, alpha-tocopherol, ascorbic acid and mercaptans showed some indication of superoxide scavenger effect, especially when compared to hydroxy-group substances such as mannitol, glucose and ethanol. Both in rat and human mitochondria, strong synergetic effects were evidenced by formulations incorporating alpha-tocopherol, BHT, ACF 223 and mercaptoamino acids.