A methylcellulose microculture assay for the in vitro assessment of drug toxicity on granulocyte/macrophage progenitors (CFU-GM)

In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a reduction of the amount of drug needed for testing, which is crucial for screening new molecules, when many different toxicological tests have to be carried out. The microassay is therefore a useful and reproducible tool for screening compounds (chemicals, drugs and xenobiotics) for potential haematotoxicity directly on human myeloid progenitors, and could contribute significantly to reducing the use of animals in toxicity testing.     

Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS-PAGE.     

Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.     

Nylon versus polydioxanone in the correction of rectus diastasis

Nylon and polydioxanone are two sutures commonly used to correct rectus diastasis. Polydioxanone, as an absorbable suture, has the advantage of not being palpable in thin patients. Because several forces act against the plication, an absorbable suture would not be efficient in these cases. In this study, two groups of 10 patients each were studied. These patients underwent abdominoplasty and correction of rectus diastasis. In the control group, 2-0 nylon was used to plicate the anterior aponeurosis and 0-polydioxanone was used in the experimental group. The tension of the abdominal wall was measured with a dynamometer in both groups. The width of rectus diastasis was measured 3 cm above and 2 cm below the umbilicus, using a computed tomography (CT) scan before the operation and 3 weeks and 6 months after surgery. The width of rectus diastasis was measured intraoperatively at the same levels. The data were analyzed by Student's t test.Both groups had similar abdominal wall tension on both levels. The diastasis recti was completely corrected at both levels, as confirmed by the 3-week postoperative CT scan and the 6-month CT scan. At the superior level, the width of the rectus diastasis on the preoperative CT scan (2.6 +/- 0.7 cm) was similar to the values obtained intraoperatively (2.7 +/- 0.6 cm), showing no significant statistical difference. At the inferior level, the largest difference between the preoperative CT scan and the intraoperative finding was 0.3 cm.In conclusion, the correction of rectus diastasis with 2-0 nylon and 0-polydioxanone was achieved and maintained after 6 months. CT scans are an accurate method for studying rectus diastasis and other muscles of the abdominal wall.     

Factors affecting the uptake of DMSO by the eggs and embryos of medaka,Oryzias latipes

High performance liquid chromatography (HPLC) was used to assess the uptake dynamics of the cryoprotectant DMSO by intact unfertilized eggs (stage 0), 8-cell (stage 5) and eyed embryos (stage 30) of medaka, Oryzias latipes, the relation of the internal concentration (Cin) of DMSO with fertilization and survival rates, and the effects of several factors on these processes. The factors examined were: cryoprotectant concentration (0.6, 1.2, 1.9 and 2.5 M), impregnation time (1, 3, 5, 10, 15 and 20 min), temperature (0, 5 and 20 degrees C), hydrostatic pressure (0 and 50 atm), and the osmotic conditions of the materials (normal or partially dehydrated). Cryoprotectant permeation, estimated from the initial rates of DMSO uptake, was higher in embryos than in eggs and increased with embryonic development; however, the DMSO Cin in eyed embryos reached a plateau at 1-5 min and could not be increased by prolonging impregnation. The highest fertilization and survival rates for any given DMSO Cin were obtained with high concentrations and short times of impregnation rather than low concentrations and long impregnation times. Application of hydrostatic pressure (50 atm) and exposure for 3 min to a 1 M trehalose solution prior to impregnation induced a substantial increase in the DMSO Cin of 8-cell embryos in comparison to untreated controls with no significant effect on survival. Hydrostatic pressure also promoted DMSO uptake in unfertilized eggs, but with rapid loss of viability, and was ineffective in eyed embryos. The uptake of DMSO and its toxicity to 8-cell embryos were directly proportional to the temperature of impregnation. The results of this study reveal important interactions between cryoprotectant concentration, impregnation time and the developmental stage (or type) of the materials and provide evidence that hydrostatic pressure, temperature of impregnation and the osmotic conditions of the materials can be manipulated to increase the uptake of cryoprotectant by fish eggs and embryos.     

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

In vitro tests for haematotoxicity: prediction of drug-induced myelosuppression by the CFU-GM assay

In a prevalidation study, a standard operating procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggest promising applications for developing and validating other in vitro methods for use in haematotoxicology.     

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.     

Temperature and meal size effects on the postprandial metabolism and energetics in a boid snake

We investigated the combined effect of meal size and temperature on the aerobic metabolism and energetics of digestion in Boa constrictor amarali. Oxygen uptake rates (Vd2;o2) and the duration of the digestion were determined in snakes fed with meals equaling to 5%, 10%, 20%, and 40% of the snake's body mass at 25 degrees and 30 degrees C. The maximum Vd2;o2 values attained during digestion were greater at 30 degrees C than at 25 degrees C. Both maximal Vd2;o2 values and the duration of the specific dynamic action (SDA) were attained sooner at 30 degrees C than at 25 degrees C. Therefore, the temperature effect on digestion in Boa is characterized by the shortening of the SDA duration at the expense of increased Vd2;o2. Energy allocated to SDA was not affected by meal size but was greater at 25 degrees C compared to 30 degrees C. This indicates that a postprandial thermophilic response can be advantageous not only by decreasing the duration of digestion but also by improving digestive efficiency. Maximal Vd2;o2 and SDA duration increased with meal size at both temperatures.     

Morphological and biochemical modifications induced by a static magnetic field on Fusarium culmorum

The effects of the exposure to a static magnetic field (sMF) of 0.3 +/- 0.03 T on the Fusarium culmorum were investigated in vitro. sMF inhibition of mycelia growth was accompanied by morphological and biochemical changes. Fungal conidia germination and cell viability were also reduced. We provide evidence of the influence of sMF on Ca(2+)-dependent signal transduction pathways involved in conidia germination. Perturbation of these pathways by adding different compounds (i.e. CaCl(2), phorbol 12-myristate 13-acetate, neomycin, EGTA, LiCl) to the medium, suggested that exposed conidia are unable to mobilise calcium from intracellular stores and that the hindered mechanism may be IP(3)-dependent.