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.     

Intralbugineous testicular prosthesis a new technique summary of 30 implants

A study of 16 patients who underwent intralbugineous testicular implants during the practice of orchiectomy is presented. In 14 cases of prostatic carcinoma, after bilateral subcapsular orchiectomy intralbugineous prostheses were implanted and in 2 other cases of testicular torsion unilateral prosthesis was implanted. With this new, easily executed technique the size, mobility and testicular sensibility are maintained.     

Significance of the diagnosis and treatment of arterial injuries of the wrist

Authors report on arterial injuries in a 2-year material of hand injured. The combination of injuries observed and the methods of treatment are described. Attention is called to the fact, that because of anatomical characteristics of these regions, the diagnosis of arterial injuries is not always easy. Four cases of their own are briefly described in whom the arterial injury could be stated only at the development of complications (pulsating haematoma, false aneurysm, later bleeding). The importance of the primary careful treatment and the significance of the examination of the circulation (Allen's test, Doppler) are stressed.     

Conventional viral cultures and shell vial assay for diagnosis of apparently culture-negativeCoxiella burnetii endocarditis

A patient with culture-negative endocarditis was diagnosed with Q fever endocarditis based on the results of serological tests and positive leukocyte cultures obtained using conventional viral cultures and the shell vial technique. This case report suggests that isolation ofCoxiella burnetii from blood may allow better diagnostic and therapeutical evaluation of patients with Q fever endocarditis. The use of both conventional and shell vial viral cultures is recommended for the isolation ofCoxiella burnetii from the blood of patients with apparently culture-negative endocarditis.     

In vivo evaluation of EPO-secreting cells immobilized in different alginate-PLL microcapsules.

Alginates are the most employed biomaterials for cell encapsulation due to their abundance, easy gelling properties and apparent biocompatibility. However, as natural polymers different impurities including endotoxins, proteins and polyphenols can be found in their composition. Several purification protocols as well as different batteries of assays to prove the biocompatibility of the alginates in vitro have been recently developed. However, little is known about how the use of alginates with different purity grade may affect the host immune response after their implantation in vivo. The present paper investigates the long-term functionality and biocompatibility of murine erythropoietin (EPO) secreting C2C12 cells entrapped in microcapsules elaborated with alginates with different properties (purity, composition and viscosity). Results showed that independently of the alginate type employed, the animals presented elevated hematocrit levels until day 130, remaining at values between 70-87%. However, histological analysis of the explanted devices showed higher overgrowth around non-biomedical grade alginate microcapsules which could be directly related with higher impurity content of this type of alginate. Although EPO delivery may be limited by the formation of a fibrotic layer around non-biomedical grade alginate microcapsules, the high EPO secretion of the encapsulated cells together with the pharmacodynamic behaviour and the angiogenic and immune-modulatory properties of EPO result in no direct correlation between the biocompatibility of the alginate and the therapeutic response obtained.