Aminotriazole effects on lung and heart H2O2 detoxifying enzymes and TBA-RS at two pO2

In order to clarify the physiological role in vivo of H2O2-detoxifying enzymes at low and high levels of O2 tension we studied catalase (CAT), glutathione peroxidases (GP), and in vivo peroxidation (TBA-RS) in the lung and heart of Rana perezi frogs chronically treated with hyperoxia, aminotriazole (AT) -a CAT inhibitor-, or both. Hyperoxia did not change CAT, GP or TBA-RS. Aminotriazole caused an almost complete depletion of CAT, a 30% decrease of GP and a 132% (lung) to 200% (heart) increase of TBA-RS. Changes similar to these were found in the group treated with AT in hyperoxia. No mortality or changes in total or organ weight occurred in the experimental groups. Main conclusions are: (1) The maximal hyperoxia tolerance showed by frogs among vertebrates does not need antioxidant enzyme induction from lung or heart and is probably related to the presence of high constitutive levels of GP in relation to metabolic rate. (2) Even in normoxia the tissues present significant amounts of H2O2, and CAT is needed to avoid oxidative damage. GP does not compensate its absence. The implications of these results in relation to oxygen toxicity in man is discussed.     

Prospective study of antigenemia,plasma viremia and lymphocytic viremia in HIV-infected hemophiliacs

A total of 186 blood samples from 24 HIV-1 seropositive hemophiliac patients, monitored every four months for 29 months, were investigated for the presence of viral antigen in plasma. In addition, peripheral blood mononuclear cells (PBMC) were cultured for HIV-1, using normal PBMC as a target for replication. Antigenemia was detected in 51 % of the patients and from PBMC in 87.5 % of the patients. The incidence of HIV isolation in asymptomatic patients (42.8 %) was similar to that found in symptomatic patients (51.4 %). Patients with opportunistic infections had a higher incidence of lymphocytic viremia (p<0.05). Plasma viremia was closely associated (p<0.05) with low CD4+ counts and infection progression. The persistence of antigenemia was also a marker of a poor clinical course. In treated patients, plasma viremia was the marker that better correlated with the clinical course, and it did not appear during the first nine months of therapy. Zidovudine doses of >500 mg/day significantly lowered the appearance of antigenemia and lymphocytic viremia (p<0.05).     

A rapid and sensitive method for measuring monooxygenase activities in hepatocytes cultured in 96-well plates

Summary Measurement of biotransformation activities in cells is of great importance for drug metabolism and toxicologic studies. It is currently done by measuring the enzymatic activities in partially purified microsomes. In the present work we report on a rapid, easy, sensitive, and reproducible fluorimetric assay for quantifying cytochrome P450-dependent monooxygenase activities (P450IA1, P450IIB1) in hepatocytes cultured in 96-well plates. The procedure involves the direct determination of enzymatic activities in intact hepatocytes while avoiding cell homogenization, thereby permitting use of a the reduced number of cells and allowing cultured cells to be used in later experiments. Substrates (7-ethoxyresorufin, 7-pentoxyresorufin) are added to culture medium and metabolized by hepatocytes. After enzymatic deconjugation, the fluorescent resorufin present in culture medium is quantified by means of a microplate fluorimetric reader. Major advantages of this technique, as compared to other available methods, are: a) no cell disruption is required; b) activity can be measured with a very small number of cells; c) rapid processing time; and d) possibility of performing repeated assays with the same cell monolayer.     

Autoregulation of beat frequency in respiratory ciliated cells. Demonstration by viscous loading

As opposed to the virtually constant load exerted by water on the cilia of ciliated protozoa, the hydrodynamic load on respiratory cilia can undergo broad variations because of changes in the rheologic properties of mucus. When water-rowing ciliated cells are exposed to increased viscosity (1 to 50 cP), their beat frequency decreases exponentially. According to Newton's fluid dynamic theory, this outcome is expected for an engine that generates constant force. However, the findings reported here indicate that when mucus-propelling respiratory ciliated cells are exposed to high viscous loads, ranging from 12 to 150 cP, the frequency of ciliary beat decreases only slightly, whereas the beat amplitude remains virtually constant. These observations suggest that ciliated cells of the respiratory tract have a functional reserve that allows them to autoregulate their mechanical output in response to the changes in viscosity to which they are normally exposed in the airway.     

Stimulation of phagocytic function in mouse macrophages by neurotensin and neuromedin N

The neuropeptides neurotensin and neuromedin N (from 10⁻¹² M to 10⁻⁹ M) have been showed in this study to stimulate significantly in vitro several steps of the phagocytic process: adherence to substrate, chemotaxis, ingestion of inert particles (latex beads) and production of superoxide anion measured by nitroblue tetrazolium reduction in resting peritoneal macrophages from BALB/c mice. A dose-response relationship was observed, with a maximal stimulation of the phagocytic process at 10⁻¹¹ M. The two neuropeptides induced no change of intracellular cyclic AMP in murine macrophages. Moreover, adherence and chemotaxis decreased significantly in the presence of EGTA (1 mM), a chelator of extracellular Ca²⁺, or ryanodine (0.5 mM), a blocker of a Ca²⁺-gated channel from the endoplasmic reticulum, in both controls and samples with the addition of neurotensin or neuromedin N. These results suggest that there is no relation between the cAMP messenger system and the phagocytic process stimulation in murine peritoneal macrophages by neurotensin or neuromedin N. In addition, the results observed with EGTA and ryanodine could indicate that these two neuropeptides produce their effects through an increase of intracellular Ca²⁺ concentration.