Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 µM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

Changes in types of muscle fibers induced by transcutaneous electrical stimulation of the diaphragm of rats

The objective of the present study was to assess the effect of transcutaneous electrical diaphragmatic stimulation (TEDS) on different types of diaphragm muscle fibers. Male Wistar rats (8-12 weeks old) were divided into 2 experimental groups (N = 8 in each group): 1) control, 2) animals submitted to TEDS [frequency = 50 Hz; T ON/T OFF (contraction/relaxation time) = 2/2 s; pulse duration = 0.4 ms, intensity = 5 mA with a 1 mA increase every 3 min for 20 min] for 7 days. After completing this treatment period, the I, IIA, IIB, and IID diaphragm muscle fibers were identified using the mATPase technique. Statistical analysis consisted of the normality, homoscedasticity and t-tests (P < 0.05). There was a 19.6% (P < 0.05) reduction in the number of type I fibers and a 49.7% increase (P < 0.05) in type IID fibers in the TEDS group compared with the control group. An important result of the present study was that electrical stimulation with surface electrodes was efficient in altering the distribution of fibers in diaphragm muscle. This therapeutic resource could be used in the treatment of respiratory muscle alterations.

Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Quality of minimally processed guava with different types of cut, sanification and packing

The purpose of this project was to evaluate the sanitization effect on the quality of minimally processed guava. Initially, research was carried out with consumers in a supermarket to verify preferences of packaging for guava. Following this, the guava cv. Paluma underwent two sanitization sequences using dehydrated sodium dichloroisocyanurate compound, in 50 ppm concentration, sanitization prior to (S1) and after (S2) being cut; removal of excess water; conditioning in PET packaging and PSPVC and storage at 3 ºC ± 1 ºC. Physicochemical analysis - [pH, total soluble solids (SST), total labeled acidity (ATT), ascorbic acid (AA), total sugars (AT) and reducers (AR)], textural sensorial and microbiological analyses were used to monitor the quality of the products. The consumers preferred the guava cut in halves with pulp and packed in PET, although this packaging promoted condensation of water vapor on the inner surface of the lid, compromising the appearance of the product. The two sanitization sequences and the two kinds of packaging did not significantly affect the pH, SST, ATT, SST/ATT, texture and AA values. The AT and AR tenors increased significantly in the MP guavas stored in the PSPVC package. Both sanitizations were efficient in the bacterial control of the indicators of the hygienicsanitary conditions, although the S1 sanitization proved to be more efficient in the control of autochthonous aerobic microbiota (aerobic mesophylic microorganisms). It can be concluded that guava cv. Paluma packed in PSPVC can be conserved for 6 days when stored at 3 ºC.

Effect of different sweetener blends and fat types on ice cream properties

The development of processed foods requires the understanding of the phenomena that dictate the ingredient interactions normally used in food formulations, as well as the effects of the numerous operations involved in the processing of the final product. In ice creams, sugars are responsible for taste, but they also affect the freezing behavior and viscosity of processed mixes. Components such as fats influence mechanical properties, melting resistance, and palatability of final products. The objective was to study the technological effects of different sugars and fats on the structure of ice cream formulations. Fructose syrup was used as a substitute for glucose syrup in blends with sucrose, and palm fat was employed as an alternative to hydrogenated vegetable fat. The analysis of variance showed significant differences in chemical compositions. Hygroscopicity of fructose syrup increased the solids content in the formulations. Melting rate and overrun were higher in products added with this sugar. Palm fat caused changes in melting ranges of formulations, and higher melting rate was observed in the combination of palm fat and fructose syrup.

Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions

Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM). The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC), the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

Oxidative stability of cereal bars made with fruit peels and baru nuts packaged in different types of packaging

Food industries have been concerned about managing the waste generated by their production processes in order to minimize environmental impacts and also about the development of formulations with different and innovative ingredients such as fruits from the Brazilian savanna. Seeking to meet the expectations of consumers who desire healthy and practical products, this study aimed to evaluate the oxidative stability and the variations in chemical composition and antioxidant potential of cereal bars made with fruit peels and baru nuts packaged in different types of packaging. The bars formulated were packed in four different types of packaging: laminated without vacuum (LWV), transparent without vacuum (TWV), transparent under vacuum (TV), and laminated under vacuum (LV); they were subsequently analyzed for proximate composition, fatty acid profiles, antioxidant activity, and oxidative capacity. The results showed that the cereal bars made with fruit peel and baru are sources of protein, dietary fiber, and fat, especially unsaturated fatty acids such as oleic and linoleic acids. The cereal bars exhibited oxidative stability up to 120 days of storage, and the type of packaging was not significant for the variables evaluated; therefore, they can be stored in low cost packaging such as transparent packaging without vacuum for a period of 120 days.

Effect of preparation practices and the cowpea cultivar Vigna unguiculata L.Walp on the quality and content of myo-inositol phosphate in akara (fried bean paste)

Akara is one of Brazil's national treasures prepared from cowpea (Vigna unguiculata L.Walp), grated onions and salt and deep-fried in crude palm oil. The results of this study on akara preparation methods showed that, in general, cowpeas were soaked for up 3 hours at room temperature, and the seed coats were then removed. The akara makers preferred the olho de pombo cultivar, because of its cream hue, or the macassar cultivar because it produces a crispier paste. The seeds purchased from street markets had lower ranges of InsP6, InsP5, and InsP4 (1.03-7.62 ∝mol.g- 1; 0.14-1.31 ∝mol.g- 1; and 0.0-0.10 ∝mol.g- 1, respectively) than both the paste and akara (6.72-19.24 ∝mol.g- 1; 1.29-4.57 ∝mol.g- 1; 0.0-0.76 ∝mol.g- 1; 3.31-13.71 ∝mol.g- 1; 0.0-4.48 ∝mol.g- 1; and 0.0-1.32 ∝mol.g- 1). These results suggest that other beans or cowpea varieties have been used in the preparation of akara and that the phytate levels do not affect its nutritional quality.

Effect of an edible crosslinked coating and two types of packaging on antioxidant capacity of castilla blackberries

In order to increase the shelf life and maintain the quality and stability of the biological compounds with antioxidant activity present in Castilla blackberry fruits, a sodium alginate-based edible crosslinked coating was applied, and the fruits were packed in two different plastic containers and stored under refrigeration (3 ± 1 °C). Total antioxidant capacity and its relationship to physicochemical variables such as pH, Brix, and acidity were evaluated in six treatments: uncoated blackberry stored in a macroperforated container (T1) and thermosealed container (T2), without crosslinked coating in a macroperforated container (T3) and thermosealed container (T4), with crosslinked coating (calcium ions) packed in macroperforated container (T5) and thermosealed container (T6). The results indicated that factors such as gas permeability in the coatings, the packaging used, and physicochemical parameters significantly affected the fruit total antioxidant capacity, with the highest level in T1 (0.22 µgEAA/ml) at the end of the essay, which is related to the lowest levels of pH and direct exposure to air. On the other hand, the lowest value was obtained in T6 (0.16 µgEAA/ml) due to the crosslinked coating, packaging in the thermosealed container, and higher pH value. Variations in acidity, Brix, and pH indicate the presence of degenerative processes in the crosslinked coating treatments, which limited the physicochemical changes.