The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.

Leukocyte adhesion - a fundamental process in leukocyte physiology

Leukocyte adhesion is of pivotal functional importance. The adhesion involves several different adhesion molecules, the most important of which are the leukocyte ß2-integrins (CD11/CD18), the intercellular adhesion molecules, and the selectins. We and others have extensively studied the specificity and binding sites in the integrins and the intercellular adhesion molecules for their receptors and ligands. The integrins have to become activated to exert their functions but the possible mechanisms of activation remain poorly understood. Importantly, a few novel intercellular adhesion molecules have been recently described, which seem to function only in specific tissues. Furthermore, it is becoming increasingly apparent that changes in integrins and intercellular adhesion molecules are associated with a number of acute and chronic diseases.

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

Effect of hyperbaric oxygenation on the regeneration of the liver after partial hepatectomy in rats

The aim of the present study was to assess the influence of hyperbaric oxygenation (HBO) on rat liver regeneration before and after partial hepatectomy. Rats were sacrificed 54 h after 15% hepatectomy, liver and body weights were measured, and serum alanine transaminase (ALT) and aspartate transaminase (AST) activity and albumin levels were determined. The lipid peroxide level, as indicated by malondialdehyde production in the remnant liver was measured, and liver sections were analyzed by light microscopy. Five groups of 10 rats in each group were studied. The preHBO and pre-hyperbaric pressure (preHB) groups were treated before partial hepatectomy with 100% O2 and 21% O2, respectively, at 202,650 pascals, daily for 3 days (45 min/day). The control group was not treated before partial hepatectomy and recovered under normal ambient conditions after the procedure. Groups postHBO and postHB were treated after partial hepatectomy with HBO and HB, respectively, three times (45 min/day). The preHBO group presented a significant increase in the initiation of the regeneration process of the liver 54 h postoperatively. The liver/body weight ratio was 0.0618 ± 0.0084 in the preHBO compared to 0.0517 ± 0016 g/g in the control animals (P = 0.016). In addition, the preHBO group showed significant better liver function (evaluated by the lowest serum ALT and AST activities, P = 0.002 and P = 0.008, respectively) and showed a significant decrease in serum albumin levels compared to control (P < 0.001). Liver lipid peroxide concentration was lowest in the preHBO group (P < 0.001 vs control and postHBO group) and light microscopy revealed that the composition of liver lobules in the preHBO group was the closest to normal histological features. These results suggest that HBO pretreatment was beneficial for rat liver regeneration after partial hepatectomy.

Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Agmatine has neuroprotective effects on retinal ganglion cells (RGCs) as well as cortical and spinal neurons. It protects RGCs from oxidative stress even when it is not present at the time of injury. As agmatine has high affinity for various cellular receptors, we assessed protective mechanisms of agmatine using transformed RGCs (RGC-5 cell line). Differentiated RGC-5 cells were pretreated with 100 μM agmatine and consecutively exposed to 1.0 mM hydrogen peroxide (H2O2). Cell viability was determined by measuring lactate dehydrogenase (LDH), and the effects of selective alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM) and N-methyl-D-aspartic acid (NMDA) receptor agonist NMDA (0-100 µM) were evaluated. Agmatine’s protective effect was compared to a selective NMDA receptor antagonist MK-801. After a 16-h exposure to H2O2, the LDH assay showed cell loss greater than 50%, which was reduced to about 30% when agmatine was pretreated before injury. Yohimbine almost completely inhibited agmatine’s protective effect, but NMDA did not. In addition, MK-801 (0-100 µM) did not significantly attenuate the H2O2-induced cytotoxicity. Our results suggest that neuroprotective effects of agmatine on RGCs under oxidative stress may be mainly attributed to the alpha 2-adrenergic receptor signaling pathway.

Protection against neurotoxicity by an autophagic mechanism

The objective of the present study was to investigate the effects of 3-n-butylphthalide (NBP) on a 1-methyl-4-phenylpyridinium (MPP+)-induced cellular model of Parkinson’s disease (PD) and to illustrate the potential mechanism of autophagy in this process. For this purpose, rat PC12 pheochromocytoma cells were treated with MPP+ (1 mM) for 24 h following pretreatment with NBP (0.1 mM). Cell metabolic viability was determined by the MTT assay and cell ultrastructure was examined by transmission electron microscopy. The intracellular distribution and expression of α-synuclein and microtubule-associated protein light chain 3 (LC3) were detected by immunocytochemistry and Western blotting. Our results demonstrated that: 1) NBP prevented MPP+-induced cytotoxicity in PC12 cells by promoting metabolic viability. 2) NBP induced the accumulation of autophagosomes in MPP+-treated PC12 cells. 3) Further study of the molecular mechanism demonstrated that NBP enhanced the colocalization of α-synuclein and LC3 and up-regulated the protein level of LC3-II. These results demonstrate that NBP protects PC12 cells against MPP+-induced neurotoxicity by activating autophagy-mediated α-synuclein degradation, implying that it may be a potential effective therapeutic agent for the treatment of PD.

Propofol pretreatment attenuates lipopolysaccharide-induced acute lung injury in rats by activating the phosphoinositide-3-kinase/Akt pathway

The aim of this study was to investigate the effect of propofol pretreatment on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the role of the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway in this procedure. Survival was determined 48 h after LPS injection. At 1 h after LPS challenge, the lung wet- to dry-weight ratio was examined, and concentrations of protein, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) were determined using the bicinchoninic acid method or ELISA. Lung injury was assayed via lung histological examination. PI3K and p-Akt expression levels in the lung tissue were determined by Western blotting. Propofol pretreatment prolonged survival, decreased the concentrations of protein, TNF-α, and IL-6 in BALF, attenuated ALI, and increased PI3K and p-Akt expression in the lung tissue of LPS-challenged rats, whereas treatment with wortmannin, a PI3K/Akt pathway specific inhibitor, blunted this effect. Our study indicates that propofol pretreatment attenuated LPS-induced ALI, partly by activation of the PI3K/Akt pathway.

Prevention of etomidate-induced myoclonus during anesthetic induction by pretreatment with dexmedetomidine

Myoclonus induced by etomidate during induction of general anesthesia is undesirable. This study evaluated the effect of dexmedetomidine (DEX) pretreatment on the incidence and severity of etomidate-induced myoclonus. Ninety patients undergoing elective surgical procedures were randomly allocated to three groups (n=30 each) for intravenous administration of 10 mL isotonic saline (group I), 0.5 µg/kg DEX in 10 mL isotonic saline (group II), or 1.0 µg/kg DEX in 10 mL isotonic saline (group III) over 10 min. All groups subsequently received 0.3 mg/kg etomidate by intravenous push injection. The incidence and severity of myoclonus were recorded for 1 min after etomidate administration and the incidence of cardiovascular adverse events that occurred between the administration of the DEX infusion and 1 min after tracheal intubation was recorded. The incidence of myoclonus was significantly reduced in groups II and III (30.0 and 36.7%), compared with group I (63.3%). The incidence of severe sinus bradycardia was significantly increased in group III compared with group I (P<0.05), but there was no significant difference in heart rate in groups I and II. There were no significant differences in the incidence of low blood pressure among the 3 groups. Pretreatment with 0.5 and 1.0 µg/kg DEX significantly reduced the incidence of etomidate-induced myoclonus during anesthetic induction; however, 0.5 µg/kg DEX is recommended because it had fewer side effects.

A morphometric study of the lumbar spinous process in the Chinese population

Our goal was to analyze the anatomical parameters of the lumbar spine spinous process for an interspinous stabilization device designed for the Chinese population and to offer an anatomical basis for its clinical application. The posterior lumbar spines (T12-S1) of 52 adult cadavers were used for measuring the following: distance between two adjacent spinous processes (DB), distance across two adjacent spinous processes (DA), thickness of the central spinous processes (TC), thickness of the superior margin of the spinous processes (TS), thickness of the inferior margin of the spinous processes (TI), and height of the spinous processes (H). Variance and correlation analyses were conducted for these data, and the data met the normal distribution and homogeneity of variance. DB decreased gradually from L1-2 to L5-S1. DA increased from T12-L1 to L2-3 and then decreased from L2-3 to L4-5. The largest H in males was noted at L3 (25.45±5.96 mm), whereas for females the largest H was noted at L4 (18.71±4.50 mm). Usually, TS of the adjacent spinous process was lower than TI. Based on the anatomical parameters of the lumbar spinous processes obtained in this study, an “H”-shaped coronal plane (posterior view) was proposed as an interspinous stabilization device for the Chinese population. This study reports morphometric data of the lumbar spinous processes in the Chinese population, which provides an anatomical basis for future clinical applications.

SEPARATION OF SATURED AND UNSATURATED FATTY ACIDS FROM PALM FATTY ACIDS DISTILLATES IN CONTINUOUS MULTISTAGE COUNTERCURRENT COLUMNS WITH SUPERCRITICAL CARBON DIOXIDE AS SOLVENT: A PROCESS DESIGN METHODOLOGY

In this work the separation of multicomponent mixtures in counter-current columns with supercritical carbon dioxide has been investigated using a process design methodology. First the separation task must be defined, then phase equilibria experiments are carried out, and the data obtained are correlated with thermodynamic models or empirical functions. Mutual solubilities, Ki-values, and separation factors aij are determined. Based on this data possible operating conditions for further extraction experiments can be determined. Separation analysis using graphical methods are performed to optimize the process parameters. Hydrodynamic experiments are carried out to determine the flow capacity diagram. Extraction experiments in laboratory scale are planned and carried out in order to determine HETP values, to validate the simulation results, and to provide new materials for additional phase equilibria experiments, needed to determine the dependence of separation factors on concetration. Numerical simulation of the separation process and auxiliary systems is carried out to optimize the number of stages, solvent-to-feed ratio, product purity, yield, and energy consumption. Scale-up and cost analysis close the process design. The separation of palmitic acid and (oleic+linoleic) acids from PFAD-Palm Fatty Acids Distillates was used as a case study.

Influence of carbon source and the fermentation process on levan production by Zymomonas mobilis analyzed by the surface response method

The aim of this study is to assess sugar cane juice and sucrose as substrates, the batch and fed batch processes and their interaction in the levan production using a complete factorial design. Zymomonas mobilis was cultivated in different sugar cane juice and sucrose concentrations in two fermentation processes at 25 °C for 20 h. A complete factorial design (2³) was used to analyze the effects of the type and concentration of the substrate, as well as the batch and fed batch processes. A complete second factorial design (2²) was used to observe the importance of sugar cane juice. The results indicated that the batch process improved the levan production reaching 40.14 g/L. The addition of sugar cane juice was not statistically significant for levan formation, however sugar cane juice stimulated biomass, sorbitol and ethanol production. The best medium for levan production was 150 g/L sucrose in batch.

Modifications in the chemical compounds and sensorial attributes of Engraulis anchoita fillet during marinating process

Marinated fish are fish products preserved by the combined action of salt and organic acids. The objective of this work was to analyze the variations in the chemical compounds of anchovy fillets that give them sensorial characteristics during the marinating process of Engraulis anchoita. The protein content decreased slightly and the TVB-N level decreased significantly in both the brining and marinating stages. In the marinating stage an increase in the total free aminoacids was observed. The NBV level in the brining and marinating solutions increased during these stages due to the solubilization of the non-protein nitrogenous compounds and the degradation of some protein compounds.The decrease of the contents of protein and TVB-N, and the increase of the acidity and the free aminoacids content during the marinating process give the marinated fillets the characteristic texture and aroma.

The effect of irradiation and thermal process on beef heme iron concentration and color properties

The aim of this study was to evaluate the influence of irradiation and thermal process on the heme iron (heme-Fe) concentration and color properties of Brazilian cattle beef. Beef samples (patties and steaks) were irradiated at 0-10 kGy and cooked in a combination oven at 250 ºC for 9 minutes with 70% humidity. Total iron and heme iron (heme-Fe) concentrations were determined. The data were compared by multiple comparisons and fixed- effects ANOVA. Irradiation at doses higher than 5 kGy significantly altered the heme-Fe concentration. However, the sample preparation conditions interfered more in the heme-Fe content than did the irradiation. Depending on the animal species, meat heme iron levels between 35 and 52% of the total iron are used for dietetic calculations. In this study the percentage of heme-iron was, on average, 70% of the total iron showing that humidity is an important factor for its preservation. The samples were analyzed instrumentally for CIE L*, a*, and b* values.

Drying of sicilian lemon residue: influence of process variables on the evaluation of the dietary fiber produced

In this study, the effect of the process variables of the air-drying of Sicilian lemon residues on some technological properties of the fibers produced was studied. The determination and modeling of desorption isotherms were used to establish the equilibrium moisture content at 60, 75, and 90 °C using the static method with 8 saturated salt solutions. The best fit was obtained with BET and GAB models. The drying process was conducted in a vertical tray dryer and delineated according to a central composite experimental design (2²) using the following as factors: air velocity (0.5, 0.75 and 1 m/s) and temperature (60, 75, and 90 °C), and it presented a good fit to the exponential model (R² > 99.9%). The experimental design responses evaluated were the technological properties of the fibers: water-holding, oil-holding, and swelling capacity. Since these properties were present in high levels, the lemon residues could be used to increase content of fibers in foods resulting in the addition of nutritional benefits for the consumers.

Study of clarification process of sugar cane juice for consumption

Sugar cane juice or garapa darkens quickly after extraction due to the oxidation of some of its constituents harming its commercialization thus requiring rapid consumption. The objective of this study was to develop a mild process for sugar cane clarification, obtaining a cloudy, greenish-yellow beverage. The following parameters were combined to aiming at this objective: heat treatment at 65 ºC/50 minutes; pH change (to 7.0, 7.5, and 8.0); addition of flocculant (0, 30, and 60 ppm Aluminum polychloride or APC - "Panclar P-1010"), and clarifier aid (0, 2, or 4 ppm of positively charged polyelectrolyte - "Magnafloc LT-27"). The decantation time was 45 minutes and the supernatant liquid was removed with a vacuum pump. The treatments were defined using the Response Surface Methodology and were submitted to physicochemical analysis for turbidity (%), total polysaccharide content (µg.mL-1), dextran content (µg.mL-1), and sensory analysis (acceptance test) for the attributes of color, appearance, and turbidity. It was concluded that the addition of 60 ppm APC, pH 8, and 0 ppm polyelectrolyte represented the best treatment to obtain a low polysaccharide content, 90% turbidity, and high scores for color, appearance, and turbidity. The beverage was sensorially well accepted by consumers.