A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Cytotoxicity of Marchantia convoluta leaf extracts to human liver and lung cancer cells

The cytotoxicity of three extracts (petroleum ether, ethyl acetate and n-butanol) from a plant used in folk medicine, Marchantia convoluta, to human non-small cell lung carcinoma (H1299) and liver carcinoma (HepG2) cell lines was tested. After 72-h incubation of lung and liver cancer cell cultures with varying concentrations of extracts (15 to 200 µg/mL), cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and reported in terms of cell viability. The extracts that showed a significant cytotoxicity were subjected to gas chromatography-mass spectrometry analysis to identify the components. The ethyl acetate, but not the petroleum ether or n-butanol extract, had a significant cytotoxicity against lung and liver carcinoma cells with IC50 values of 100 and 30 µg/mL, respectively. A high concentration of ethyl acetate extract (100 µg/mL) rapidly reduced the number of H1299 cells. At lower concentrations of ethyl acetate extract (15, 30, and 40 µg/mL), the numbers of HepG2 cells started to decrease markedly. Gas chromatography-mass spectrometry analysis of the ethyl acetate extract revealed the presence of several compounds such as phytol (23.42%), 1,2,4-tripropylbenzene (13.09%), 9-cedranone (12.75%), ledene oxide (7.22%), caryophyllene (1.82%), and caryophyllene oxide (1.15%). HPLC analysis result showed that there were no flavonoids in ethyl acetate extract, but flavonoids are abundant in n-butanol extract. Further studies are needed regarding the identification, toxicity, and mechanism of action of active compounds.

A pyrazolyl-thiazole derivative causes antinociception in mice

The present study investigates the antinociceptive effect of the pyrazolyl-thiazole derivative 2-(5-trichloromethyl-5-hydroxy-3-phenyl-4,5-dihydro-1 H-pyrazol-1-yl)-4-(4-bromophenyl)-5-methylthiazole (B50) in mice. Male albino Swiss mice (30-40 g) were used in the acetic acid-induced abdominal writhes and tail-immersion tests. B50 caused dose-dependent antinociception (8, 23 and 80 µmol/kg, sc) in the acetic acid writhing assay (number of writhes: vehicle: 27.69 ± 6.15; B50 (8 µmol/kg): 16.92 ± 3.84; B50 (23 µmol/kg): 13.85 ± 3.84; B50 (80 µmol/kg): 9.54 ± 3.08; data are reported as means ± SEM for 9 animals per group). On the other hand, B50 did not cause antinociception in the tail immersion assay. Naloxone (2.75 µmol/kg, sc) prevented B50-induced antinociception (number of writhes: vehicle-saline: 31.11 ± 3.15; vehicle-naloxone: 27.41 ± 3.70; B50 (80 µmol/kg)-saline: 8.70 ± 3.33; B50 (80 µmol/kg)-naloxone: 31.84 ± 4.26; morphine-saline: 2.04 ± 3.52; morphine-naloxone: 21.11 ± 4.26; 8-9 animals per group). The removal of the methyl group of the thiazole ring of B50 or substitution of the bromo substituent with the methyl at position 4 of the phenyl group, which is attached to the thiazole ring of B50, resulted in loss of activity, suggesting that these substituents are important for antinociceptive activity. B50 had no effect on spontaneous locomotion or rotarod performance, indicating that the antinociceptive effect of B50 is not related to nonspecific motor effects. The antinociceptive profile of B50 seems to be closer to nonsteroidal anti-inflammatory drugs than to classic opioid agents, since it had no analgesic effect in a thermally motivated test.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Neuroprotective effect of ketamine/xylazine on two rat models of Parkinson's disease

There is a great concern in the literature for the development of neuroprotectant drugs to treat Parkinson's disease. Since anesthetic drugs have hyperpolarizing properties, they can possibly act as neuroprotectants. In the present study, we have investigated the neuroprotective effect of a mixture of ketamine (85 mg/kg) and xylazine (3 mg/kg) (K/X) on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA) rat models of Parkinson's disease. The bilateral infusion of MPTP (100 µg/side) or 6-OHDA (10 µg/side) into the substantia nigra pars compacta of adult male Wistar rats under thiopental anesthesia caused a modest (~67%) or severe (~91%) loss of tyrosine hydroxylase-immunostained cells, respectively. On the other hand, an apparent neuroprotective effect was observed when the rats were anesthetized with K/X, infused 5 min before surgery. This treatment caused loss of only 33% of the nigral tyrosine hydroxylase-immunostained cells due to the MPTP infusion and 51% due to the 6-OHDA infusion. This neuroprotective effect of K/X was also suggested by a less severe reduction of striatal dopamine levels in animals treated with these neurotoxins. In the working memory version of the Morris water maze task, both MPTP- and 6-OHDA-lesioned animals spent nearly 10 s longer to find the hidden platform in the groups where the neurotoxins were infused under thiopental anesthesia, compared to control animals. This amnestic effect was not observed in rats infused with the neurotoxins under K/X anesthesia. These results suggest that drugs with a pharmacological profile similar to that of K/X may be useful to delay the progression of Parkinson's disease.

Magnetic properties of defective graphene

In this thesis, the influence of the functionalization of graphene and graphite on their magnetic properties was investigated. The functionalization was performed by covalent attaching of a phenyl groups with three different radicals (4-bromoaniline, 4-chloroaniline and 4-nitroaniline). Magnetic properties were measured by SQUID magnetometer. Both pristine graphite and graphene showed strong diamagnetic behavior. For good quality graphite, diamagnetism was found to be temperature-dependent. All samples demonstrated noticeable paramagnetic contribution below 50 K. According to fitting experimental results with Brillouin function and Curie law, it was shown that paramagnetism is provided by small clusters of spins (superparamagnetic behavior). Moreover, the clusters size and spin concentrations were calculated. For the samples functionalized with nitroaniline the antiferromagnetic transition around 120 K was observed. To explain this behavior, a simple model was proposed. Additional analysis of the graphene quality, structure and composition of the samples was carried out by HRTEM, EDS mapping, Raman spectroscopy and X-ray diffraction techniques.

The inhibitory effect of photodynamic therapy and of an anti-VCAM-1 monoclonal antibody on the in vivo growth of C6 glioma xenografts

We investigated the effect of photodynamic therapy (PDT) and of an anti-vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibody on the in vivo growth of C6 glioma. Seven days after inoculation with C6 cells, adult male Wistar rats weighing 280-300 g with MRI-confirmed glioma were randomly assigned to 4 groups (N = 15 per group): PDT + VCAM-1 antibody group; PDT group; VCAM-1 antibody group; control group. Eight days after inoculation, hematoporphyrin monomethyl ether (HMME) was administered as a photosensitizer and PDT was performed at 630 nm (illumination intensity: 360 J/cm²) for 10 min. VCAM-1 antibody (50 µg/mL) was then administered (0.5 mL) through the tail vein every other day from day 8 to day 16. At day 21, 5 rats in each group were sacrificed and cancers were harvested for immunohistochemistry and Western blot assay for the detection of VCAM-1, and TUNEL assay was used to detect apoptosis. Survival and tumor volume were recorded in the remaining 10 rats in each group. In the PDT group, tumor growth was significantly suppressed (67.2%) and survival prolonged (89.3%), accompanied by an increase in apoptosis (369.5%), when compared to control. Furthermore, these changes were more pronounced in the PDT + VCAM-1 antibody group. After PDT, VCAM-1 expression was markedly increased (121.8%) and after VCAM-1 monoclonal antibody treatment, VCAM-1 expression was significantly reduced (58.2%). PDT in combination with VCAM-1 antibody can significantly inhibit the growth of C6 glioma and prolong survival. This approach may represent a promising strategy in the treatment of glioma.

Impairment of locomotor activity induced by the novelN-acylhydrazone derivatives LASSBio-785 and LASSBio-786 in mice

The N-acylhydrazone (NAH) analogues N-methyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-785) and N-benzyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-786) were prepared from 2-thienylidene 3,4-methylenedioxybenzoylhydrazine (LASSBio-294). The ability of LASSBio-785 and LASSBio-786 to decrease central nervous system activity was investigated in male Swiss mice. LASSBio-785 or LASSBio-786 (30 mg/kg, ip) reduced locomotor activity from 209 ± 26 (control) to 140 ± 18 (P < 0.05) or 146 ± 15 crossings/min (P < 0.05), respectively. LASSBio-785 (15 or 30 mg/kg, iv) also reduced locomotor activity from 200 ± 15 to 116 ± 29 (P < 0.05) or 60 ± 16 crossings/min (P < 0.01), respectively. Likewise, LASSBio-786 (15 or 30 mg/kg, iv) reduced locomotor activity from 200 ± 15 to 127 ± 10 (P < 0.01) or 96 ± 14 crossings/min (P < 0.01), respectively. Pretreatment with flumazenil (20 mg/kg,ip) prevented the locomotor impairment induced by NAH analogues (15 mg/kg, iv), providing evidence that the benzodiazepine (BDZ) receptor is involved. This finding was supported by the structural similarity of NAH analogues to midazolam. However, LASSBio-785 showed weak binding to the BDZ receptor. LASSBio-785 or LASSBio-786 (30 mg/kg,ip, n = 10) increased pentobarbital-induced sleeping time from 42 ± 5 (DMSO) to 66 ± 6 (P < 0.05) or 75 ± 4 min (P < 0.05), respectively. The dose required to achieve 50% hypnosis (HD50) following iv injection of LASSBio-785 or LASSBio-786 was 15.8 or 9.5 mg/kg, respectively. These data suggest that both NAH analogues might be useful for the development of new neuroactive drugs for the treatment of insomnia or for use in conjunction with general anesthesia.

An interleukin-33/ST2 signaling deficiency reduces overt pain-like behaviors in mice

Interleukin (IL)-33, the most recent member of the IL family of cytokines, signals through the ST2 receptor. IL-33/ST2 signaling mediates antigen challenge-induced mechanical hyperalgesia in the joints and cutaneous tissues of immunized mice. The present study asked whether IL-33/ST2 signaling is relevant to overt pain-like behaviors in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing responses in wild-type (WT) mice; this overt nociceptive behavior was reduced in ST2-deficient mice. In an antigen-challenge model, ST2-deficient immunized mice had reduced induced flinch and licking overt pain-like behaviors. In the formalin test, ST2-deficient mice also presented reduced flinch and licking responses, compared with WT mice. Naive WT and ST2-deficient mice presented similar responses in the rota-rod, hot plate, and electronic von Frey tests, indicating no impairment of motor function or alteration in basal nociceptive responses. The results demonstrate that IL-33/ST2 signaling is important in the development of overt pain-like behaviors.

Chemical characteristics of pequi fruits (Caryocar brasiliense Camb.) native of three municipalities in the State of Goiás - Brazil

In order to assure that the use of cerrado fruits occur in a sustainable way, studies to investigate their characteristics are extremely relevant. In this context, the present study aims to describe some chemical parameters of pequi fruits picked in three municipalities in southwestern Goiás State (Jataí, Rio Verde, and Serranópolis). In each city, two populations of pequi trees - pequizeiros, denominated areas, were selected. In each area, eight trees were selected for the fruit to be picked. The contents of phosphorus, potassium, calcium, magnesium, nitrogen, zinc, and ether extract were determined in the samples. The results demonstrate differences between the chemical characteristics studied for the fruits picked in different areas, which does not seem to vary in a significant way. Comparing the contents obtained in the present study with those required as human daily supply, further studies are recommended aiming at using the pequi fruit as a complementary alternative source of magnesium, manganese, and copper.

Changes in guava (Psidium guajava L. var. Paluma) nectar volatile compounds concentration due to thermal processing and storage

Guava nectars were formulated for approximately 10, 12, or 14 ºBrix, with 40% guava pulp. Sodium benzoate, 500 mg.kg-1 was used as preservative. The Brix value was adjusted with saturated sucrose syrup. The guava nectar was pasteurized (85 ºC/42 seconds) in tubular heat exchanger and then hot filled in 500 mL white glass bottles. The products were stored either at room temperature (25 ± 5 ºC) or refrigerated (5 ± 2 ºC) under fluorescent light exposure and analyzed on the day after processing (time zero) and also 40, 80, and 120 days of storage. Eight compounds were identified and quantified by Gas Chromatography (GC) -Mass Spectrometry (MS): hexanal, (E)-hex-2-enal, 1-hexenol, (Z)-hex-3-enol, (Z)-hex-3-enyl acetate, phenyl-3-propyl acetate, cinnamyl acetate, and acetic acid. There was no significant effect of thermal treatment on the volatile compound concentrations, except for a significant decrease (p = 0.0001) in hexanal and (Z)-hex-3-enyl acetate (p = 0.0029). As for the storage time, there was a much greater decrease in the esters contents, such as (Z)-hex-3-enyl and phenyl-3-propyl acetates. Cinnamyl acetate had the greatest decrease over storage time. Refrigeration was better than room temperature for guava nectar volatile compounds stability over storage time, mainly for esters compounds, which are important for the product aroma and flavor

Effect of genotype, finishing system, and sex on physiochemical characteristics of goat meat

Seventy-eight kids of both sexes and five genotypes were used: Alpine, ½ Boer + ½ Alpine (½ BA), ¾ Boer + ¼ Alpine, ½ Anglo-nubian + ½ Alpine and "tricross" (½ Anglo-nubian + ¼ Boer + ¼ Alpine) with initial average weight of 14.1 ± 2.5. The objective was to evaluate the effect of genotype, finishing system, and sex on the physiochemical characteristics of goat meat. Finishing systems were: ST1 - kid + dam in pasture and ST2 - weaned kid and feedlot. Kids in ST1 were kept in an area with Panicum maximum cv. Tanzania, and after grazing, water and mineral salt/mix were fed ad libitum to the animals. The animals in ST2 were confined in collective pens distributed according to genotypes and received diet with 16% CP and 73% TDN. The values of pH, a* (red content), Cooking Loss (CL), and Ether Extract (EE) percentage were influenced by genotype. Values for red content (a*) and L* (brightness), CL and percentages of moisture, protein, EE, and ash were influenced by the finishing system. Longissimus dorsi muscle from animals ½ BA exhibited better physiochemical characteristics. For greater tenderness and higher percentages of fat, consumers should choose female kid goat meat.

Use of colour parameters for roasted coffee assessment

Fast and non-destructive indicators were evaluated as tools to measure the technological quality of Arabica and Robusta coffee. Accordingly, considering the roasting intensity in highly valuable commercial samples, volume, mass, apparent density, moisture, total ash, ash insoluble in hydrochloric acid, and ether extract were characterized. The chromatic parameters L*, C*, Hº were measured using illuminants D65 and C. It was found that in roasted coffee beans, the parameters L*, C*, Hº, and coordinate b* had an antagonist interaction due to an increase in the roasting intensity, whereas after milling, only L* and Hº decreased progressively. Considering that the parameters L* and Hº followed similar patterns using both illuminants, D65 and C, it can be concluded that they are appropriate to evaluate coffee colour changes during roasting, enabling a relationship with coffee quality.

Phisicochemical, sensory, and microbiological evaluation and development of symbiotic fermented drink

The goal of this study was to develop a symbiotic lacteous drink, evaluate its physicochemical and sensory characteristics, and verify the viability of Lactobacillus acidophilus in the drink. The milk serum-based drink consisted of 50% milk serum containing 10% saccharose, 25% powdered milk, 15% yacon pulp, and cultures of Lactobacillus acidophilus-La 5E and Bifidobacterium bifidum BB12. It was stored for up to 21 days under refrigeration. The milk serum-based drink was analyzed for protein, ether extract, total dietary fiber, total frutans, carbohydrate content, color, pH, acidity, and contamination by coliforms, and Salmonella sp. Coliforms and Salmonella sp were not detected, and L. acidophilus and B. bifidum provided satisfactory probiotic counts up to 21 days of storage under refrigeration. Lactobacillus acidophilus resistance to gastric acids and bile salts was detected only up to seven days of storage when evaluated in vitro. Sensory analysis and purchase viability were evaluated by consumers at 0, 7, 14, and 21 days of storage. Based on the analytical results and consumer evaluation, the drink was acceptable up to seven days of storage.

Antioxidant activities of fractions from longan pericarps

The antioxidant activities of ethanolic crude extract (LPCE) and its four different solvent sub-fractions (namely, diethyl ether fraction (LPDF), ethyl acetate fraction (LPEF), n-butyl alcohol fraction (LPBF) and residue fraction (LPR)) from longan pericarps were investigated employing various systems including 2,2-diphenyl-1-picrylhydrazyl (DPPH)/ 2,2'-amino-di(2-ethyl-benzothiazoline sulphonic acid-6)ammonium salt (ABTS)/hydroxyl radical scavenging activity, total phenolic content and reducing power. Each extract showed concentration-dependent antioxidant activity. LPEF showed the highest scavenging activity against DPPH, ABTS and hydroxyl radicals with EC50 values of 0.506, 0.228 and 4.489 mg/mL, respectively. LPEF showed the highest reducing power with EC50 values of 0.253 mg/mL. The next was LPDF with EC50 values of 0.260 mg/mL. LPEF possessed the highest total phenolic content (230.816 mg/g, expressed as gallic acid equivalents), followed by LPDF, LPBF, LPCE and LPR. The results suggested that longan pericarp fractions possessed significant antioxidant activities and could be a promising source of natural antioxidant.