Nitric oxide synthase activity is inducible in rat,but not rabbit alveolar macrophages,with a concomitant reduction in arginase activity

Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 × 10⁶ cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of ³H-citrulline (NO synthase activity) and ³H-ornithine (arginase activity) were determined.During incubation of rat alveolar macrophages with ³H-arginine clear amounts of ³H-citrulline and ³H-ornithine (3.8 and 4.6% of the added ³H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra-/intracellular of 17 and 70 for ³H-citrulline and ³H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of ³H-citrulline was increased about 30-fold and this was accompanied by a reduction in ³H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 mol/1). Inhibition of NO synthase by N^G-monomethyl-l,-arginine (l-NMMA, 100 mol/1) in LPS treated alveolar macrophages reduced the formation ³H-citrulline by more than 90% and restored the ³H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of ³H-citrulline and ³H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive. During incubation of rabbit alveolar macrophages a marked formation of ³H-ornithine (about 5.3% of the added ³H-arginine), but no significant formation of ³H-citrulline could be detected. Pretreatment with LPS tended to enhance the formation of ³H-ornithine (by 50%) without effects on ³H-citrulline. Rabbit-interferon and/or tumor necrosis factor- present together with LPS during the culture period did not result in a significant ³H-citrulline formation. Under all conditions tested, culture media of rabbit alveolar macrophages did not contain significant amounts of nitrite (less than 0.5 nmol) whereas in culture media of untreated rat alveolar macrophages 22 nmol nitrite (per 18 h) were detected, and LPS induced a 3-fold nitrite accumulation, an effect prevented by dexamethasone.In conclusion, in rabbit alveolar macrophages NO synthase activity was not detectable and could also not be induced by LPS and different cytokines, whereas in rat alveolar macrophages NO synthase was readily inducible. Alveolar macrophages of both species showed marked arginase activity. After induction of marked NO synthase activity, ornithine formation was largely reduced possibly by concomitant inhibition of arginase and/or withdrawn of arginine from arginase.     

Ophthalmic findings in GAPO syndrome

BACKGROUND: The main manifestations of GAPO syndrome are growth retardation (G), alopecia (A), pseudoanodontia (P), and optic atrophy (O). CASES: This syndrome has been described in 21 patients from 16 different families. Four cases are from Turkey and have been presented by Sayli and Gül. The purpose of our study is to document the cases from Turkey and discuss the ophthalmological and neuro-ophthalmolgical findings of these and other reported GAPO cases. OBSERVATIONS: All patients in the literature and our 4 cases have severe growth retardation with delayed bone age in infancy, characteristic facial appearance (high and bossed forehead, midface hypoplasia), alopecia or severe hypotrichosis, and pseudoanodontia. Optic atrophy was present in 1 of our cases and in 5 previous cases. Glaucoma was present in 5 cases, including 2 of ours. Buphthalmia and keratopathy secondary to glaucoma were also observed. White eyelashes, seen only in our cases, may be a sign of "early senility." CONCLUSIONS: Optic atrophy is not a constant finding in GAPO syndrome. Glaucoma may accompany the ocular findings. This syndrome has been attributed to either ectodermal dysplasia or the accumulation of extracellular connective tissue matrix, due to an enzyme deficiency involved in its metabolism. Current studies show that an elastin defect and secondary changes in collagen may be important in the pathogenesis of the disease.     

The effects of acetylcholine and propolis extract on corneal epithelial wound healing in rats.

PURPOSE: To investigate the effects of topical acetylcholine and topical administration of propolis, a natural beehive product, on corneal epithelial wound healing. METHODS: The whole corneal epithelium was debrided in 42 eyes of 21 rats by mechanical scraping with a dulled scalpel blade. Animals were divided into three groups. Group 1 received topical 1% water extract of propolis (WEP), group 2 received topical acetylcholine (ACh), and group 3 (control group) received topical phosphate-buffered saline, 6 times a day for 3 days, starting immediately after debridement. The area of the corneal epithelial defect was stained with fluorescein, photographed, and then measured every 12 h. The mean epithelial defect area and the mean percentage of epithelial defect remaining at each follow-up were compared between the groups. RESULTS: The mean epithelial defect area and the mean percentage of epithelial defect remaining at each time were significantly smaller (p < 0.001, p < 0.05, respectively) in the ACh and propolis groups as compared with control groups. There was no statistically significant difference between the propolis or ACh groups at any time (p > 0.05). At 72 h, the mean percentage of defect remaining was 2.58% in the ACh group, 1.3% in the propolis-treated group, and 8.68% in the control group. CONCLUSIONS: This study demonstrated that ACh and propolis facilitated corneal epithelial wound healing of rats. Although the mechanisms of the effect of propolis on wound healing and its clinical use still remain to be determined, ACh may have a place in the treatment of corneal epithelial injuries.     

Dose-dependent alteration of rat cardiac sodium current by isoproterenol: Results from direct measurements on multicellular preparations

Conflicting results have been reported in literature about the influence of-adrenergic stimulation on the fast cardiac sodium current (I _Na ⁺). To elucidate these mechanisms in multicellular preparations we used the loose-patch-clamp technique to evaluate the effect of the-adrenergic agonist isoproterenol 1–1000 nmol/l. Isoproterenol enhancedI _Na+ at all membrane potentials by elevation of the maximal availableI _Na ⁺. Only at the high concentration of 1 mol/l wasI _Na ⁺ slightly depressed after depolarizing conditioning clamps. The most marked increase of the maximal availableI _Na ⁺ was 30 ± 9 % after application of 100 nmol/l isoproterenol. To learn about the mechanisms in view of sodium channel modulation we combined isoproterenol with the sodium channel blocker lidocaine (47 mol/l). Under these circumstances the effects of both drugs were completely independent. This investigation shows clearly that low concentrations of isoproterenol increaseI _Na+ in multicellular preparations by a gating-independent mechanism.     

Central Nervous System Effects of Ginkgo Biloba, a Plant Extract

Extracts of Ginkgo biloba (EGb) are among the most prescribed drugs in France and Germany. EGb is claimed to be effective in peripheral arterial disorders and in "cerebral insufficiency." The mechanism of action is not yet well understood. Three of the ingredients of the extract have been isolated and found to be pharmacologically active, but which one alone or in combination is responsible for clinical effects is unknown. The recommended daily dose (3 x 40 mg extract) is based more on empirical data than on clinical dose-findings studies. However, despite these, according to double-blind, placebo-controlled clinical trials, EGb has therapeutic effects, at least, on the diagnostic entity of "cerebral insufficiency," which is used in Europe as synonymous with early dementia. To determine whether EGb has significant pharmacological effects on the human brain, a pharmacodynamic study was conducted using the Quantitative Pharmacoelectroencephalogram (QPEEG(R)) method. It was established that the pharmacological effects (based on a predetermined 7.5--13.0-Hz alpha frequency band in a computer-analyzed electroencephalogram = CEEG(R)) of EGb on the central nervous system (CNS) are significantly different than placebo, and the high and low doses could be discriminated from each other. The 120-mg, but particularly the 240-mg, single doses showed the most consistent CNS effects with an earlier onset (1 h) and longer duration (7 h). Furthermore, it was established that the electrophysiological effects of EGb in CNS are similar to those of well-known cognitive activators such as "nootropics" as well as tacrine, the only marketed "antidementia" drug currently available in the United States.     

Application of the Electrotopological State Index to QSAR Analysis of Flavone Derivatives as HIV-1 Integrase Inhibitors

Purpose. A QSAR study based on electrotopological state (E-state) indices was conducted for a series of flavone HIV-1 integrase inhibitors to guide drug design. Methods. E-state indices formulated to encode electronic and topological information for each skeletal atom in a molecule (Kier and Hall Pharm. Res. 7:801–807 (1990)) were calculated using the Molconn-X program, and partial least squares (PLS) multivariate regression was used to derive QSAR models. Results. Predictive models with correlation coefficients (r²) of 0.98 (3 PLS components) and 0.99 (5 PLS components) and corresponding cross-validated correlation coefficients (c.v. r²) of 0.51 and 0.73, were obtained for inhibition of cleavage and integration, respectively, with one molecule omitted from the analysis. Conclusions. E-state indices at C6, C3, C5, C5, and O4 were found to be more important for prediction of activity than those for any of the other 12 flavone skeletal atoms that are common to the molecules in the data set.     

Significance of wet autoclave pretreatment in immunohistochemistry

Until recently the only way to rescue masked epitopes in routinely processed surgical pathological material was enzymatic digestion. The use of heat for antigen retrieval, first by microwave irradiation, represents an important breakthrough in immunohistochemistry. With the acceptance of microwave oven pretreatment, various modified techniques and alternative heating methods have also been proposed. Wet autoclave pretreatment for tissue proteolysis is a highly reliable alternative to the microwave antigen retrieval technique. It provides uniform heating of the slides, hence an even enhancement of staining intensity in a variety of formalin-sensitive antigens, and it also offers consistent interlaboratory results. The method has been introduced in routine diagnostic immunohistochemistry for the detection of estrogen-and progesterone receptors, L26-, Ki-67- and bcl-2 antigens and variable types of cytokeratins (1/5/10/11, 8, 13, 19). Experimentally, wet autoclaving can be used very successfully for the immunophenotyping of p53 and mdm2 expression, for the detection of adhesion molecules (CD44, integrins) and some anti-inflammatory molecules (annexins), among others. It has produced a substantial improvement in the visualisation of silver-stained nucleolar organizer regionsassociated proteins (AgNORs) in routine paraffin sections and along with modified silver staining and standardized AgNOR parameters assessed by image analysis. Wet autoclaving-based AgNOR staining has been proposed by a European multicentric study group as the standardized method for AgNOR analysis in archival material.     

Proliferative (mib1, mdm2) versus anti-proliferative (p53) markers in head and neck cancer. An Immunohistochemical study

Formalin fixed and paraffin embedded samples from 36 squamous cell carcinomas of the larynx and the oral cavity (pT₂N₀M₀, R₀) surrounded by non-tumorous mucosa were studied immunohistochemically using a panel of four different anti-p53 antibodies (CM1, PAbl801, D07, PAb240), a monoclonal anti-mdm2 antibody and MIB1, following wet autoclave antigen retrieval. P53 immunoreactivity was detected in 11/14 laryngeal and in 9/22 oral carcinomas. All p53 positive oral, and all but one laryngeal tumors revealed mdm2 positivity as well, whereas in p53 negative tumors 4/12 and 1/3 mdm2 immunopositive cases were demonstrated, respectively. MIB1 labeling indices of the tumors ranged between 18% – 64% in p53 positive cases, and 10% – 53% in p53 negative ones. The difference was not statistically significant. Close spatial coexpression of p53, mdm2 and MIB1 immunoreactivity was observed at the invasive front of the carcinomas and in the basal and suprabasal layers of the non-tumorous epithelium in all p53 positive cases. However, the MIB1 expression was similarly increased at the invasive margins in carcinomas lacking immunohistochemically detectable p53 alterations. Our results strongly suggest that p53 overexpression does not necessarily correspond to increased rate of proliferation, but rather to mdm2 overexpression and is largely dependent on the anatomical site in case of small and localized squamous cell carcinomas of the head and neck region.     

Assessment of left ventricular function: comparison between radionuclide angiography and semiquantitative two-dimensional echocardiographic analysis

Measurement of global left ventricular function is important in the follow-up of cardiac patients and is a good prognostic indicator in acute cardiac situations. We compared quantitative measurements of global left ventricular function made with radionuclide angiography (RNA) and contrast cardiac ventriculography (CVG) to visual semiquantitative estimates from two-dimensional echocardiographic images (2D-echo). Three hundred and thirty-nine consecutive patients who underwent RNA were assessed with 2D-echo within 3 months. In addition, 92 of these patients also underwent CVG (correlation of ejection fraction between CVG and RNA:r=0.82;P<0.0001). The RNA mean ejection fractions in the four 2D-echo groups (0=normal, 1=slightly, 2=moderate, or 3=severe reduced left ventricular function) differed markedly (P<0.0001); however, there was overlapping among the groups (2D-echo score/RNA ejection fraction: 0=57.3%±12.8%; 1=46.0%±12.9%; 2=29.6%±12.2%; and 3=24.6%±11.5%) and the difference between 2D-echo scores 2 and 3 was not significant. 2D-echo showed a good concordance in RNA classes (0=505; 1=35%–49%; 2=21%–34%; and 3=520% ejection fraction) 0 (133/166; 80%) and 3 (18/30; 60%) but low concordance in classes 1 (27/82; 33%) and 2 (21/61; 34%). For accurate assessment of global left ventricular ejection fraction, visual semiquantitative judgement of a 2D echocardiographic image is limited in comparison to CVG or RNA, especially in patients with a slight or moderate reduction in left ventricular ejection fraction.