Differential biological activity of two extracts of ripe fruits of Carica candamarcensis Hook on human neutrophils / Actividad biologica diferencial de dos extractos de frutos maduros de Carica candamarcensis Hook en neutrófilos humanos

We evaluated the effect of the total macerate (TM) and seed oil (SO) of mature Carica candamarcensis fruits, on the release of Matrix metalloproteinase 9 (MMP9) and the phosphorylation of MAPK in neutrophils. The antioxidant capacity of these extracts was evaluated by ABTS assay. Neutrophils stimulated with different dilutions of TM or SO were analyzed for cytotoxicity, MMP9 release, and MAPK phosphorylation, using trypan blue exclusion assays, zymography, and immunoblotting, respectively. Both extracts show antioxidant activity, being higher in TM; none presented cytotoxic effect. The 5% and 2.5% dilutions of TM significantly reduced MMP9 release, and all decreased MAPK phosphorylation. SO significantly increased the release o f MMP9 and MAPK phosphorylation, the effect being greater when they were prestimulated with lipopolysaccharide.TM may have anti - inflammatory potential, while SO could have a priming effect that needs to be confirmed

Evaluamos el efecto del macerado total (MT) y aceite de semillas (AV) de frutos maduros de Carica candamarcensis , en la liberación de Matriz metaloproteinasa 9 (MMP9) y la fosfor ilación de MAPK en neutrófilos. La capacidad antioxidante de estos extractos se evaluó por ensayo ABTS. En neutrófilos estimulados con diferentes diluciones de MT o AV se analizó la citotoxicidad, liberación de MMP9 y fosforilación de MAPK, mediante ensayo s de exclusión con azul de tripano, zimografía e inmunotransferencia, respectivamente. Ambos extractos muestran actividad antioxidante, siendo mayor en MT; ninguno presentó efecto citotóxico. Las diluciones 5% y 2,5% de MT redujeron significativamente la l iberación de MMP9, y todas disminuyeron la fosforilación de MAPK. El AV incrementó significativamente la liberación de MMP9 y la fosforilación de MAPK, el efecto fue mayor cuando se preestimularon con lipopolisacárido. El MT puede tener potencial antiinfla matorio, mientras que el AV podría tener un efecto "priming" que necesita ser corroborado.

Cloning and prokaryotic expression analysis of squalene synthase CpSQS1 and CpSQS2 from Crataegus pinnatifida / 中国中药杂志

In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.

Purification and some properties of rose (Fructus cynosbati) hips invertase.

Invertase was purified from rose (Fructus cynosbati) hips by ammonium sulfate fractionation and hydroxyapatite column chromatography. The enzyme was obtained with a yield of 4.25% and about 10.48-fold purification and had a specific activity of 8.59 U/mg protein. The molecular mass of invertase was estimated to be 66.51 kDa by PAGE and 34 kDa by SDS-PAGE, indicating that the native enzyme was a homodimer. The enzyme was a glycoprotein and contained 5.86% carbohydrate. The Km for sucrose was 14.55 mM and the optimum pH and temperature of the enzyme were 4.5 and 40°C, respectively. Sucrose was the most preferred substrate of the enzyme. The enzyme also hydrolyzed D(+) raffinose, D(+) trehalose and inulin (activity 39.88, 8.12 and 4.94%, respectively of that of sucrose), while D(+) lactose, cellobiose and D(+) maltose showed no effect on the enzyme. The substrate specificity was consistent with that for a β-fructofuranoside, which is the most popular type in the higher plants. The enzyme was completely inhibited by HgCl2, MnCl2, MnSO4, FeCl3, Pb(NO3)2, ammonium heptamolybdate, iodoacetamide and pyridoxine hydrochloride. It was also inhibited by Ba(NO3)2 (86.32%), NH4Cl (84.91%), MgCl2 (74.45%), urea (71.63%), I2 (69.64%), LiCl (64.99%), BaCl2 (50.30%), Mg(NO3)2 (49.90%), CrCl3 (31.90%) and CuSO4 (21.45%) and but was activated by Tris (73.99%) and methionine (12.47%).

Behavior of Araujiain, a new cysteine phytoprotease, in organic media with low water content

In this paper we studied the effect of different organic solvents (1-octanol, trichloroethylene, ethanol, ethyl acetate, tetrahydrofuran, cyclohexane, propanone, acetonitrile, dichloromethane, chlorobenzene, N,N-dimethylformamide, acetophenone, diethyl ether, methanol, ethylene glycol and toluene) with low and constant water content on substrate preferences, thermostability and stability (caseinolytic activity retention after 4 h) of proteases of Araujia hortorum Fourn. (Asclepiadaceae). The stability of araujiain was high in N,N-dimethylformamide and ethanol at 40 ºC, but decreased at higher temperature. Araujiain substrates preferences in buffer Tris-HCl (pH 8), ethylene glycol and N,N-dimethylformamide exhibited different patterns, but the enzyme showed a high preference by glutamine derivative in all cases. According to FTIR spectroscopy studies, araujiain changed its secondary structure and as a consequence, it also changed its substrate preferences. This enzyme showed lower beta-helical character and greater beta-sheet folding in buffer than in organic media. A larger amount of antiparallel beta-sheet residues indicates the formation of tighter intermolecular hydrogen bonds and enzymatic aggregates. These facts could explain the higher esterolytic activities, the greater stability and good hydrolytic potential of araujiain in some organic media such as N,N-dimethylformamide.

Purificaçäo, caracterizaçäo parcial e expressäo da sacarose-fosfato sintase durante o amadurecimento da banana / Purification, partial caracterization and expression of sacarose-phosphate synthase during banana ripening

A enzima sacarose-fosfato sintase foi parcialmente purificada de bananas fisiologicamente imaturas (70 dias após antese), fisiologicamente maturas pré-climatéricas (110 dias após a antese) e climatéricas (130 dias após a antese). De acordo com os resultados apresentados a SPS de banana é uma enzima constituída de subunidade de 116kD, apresentando peso molecular nativo de 440 kD por filtraçäo em gel e bandas de 180, 240 e 686 kD por eletroforese em gel de poliacrilamida, nos três estágios estudados. Uma sequência parcial do gene da SPS foi amplificado através de PCR, clonado e seu sequenciamento indicou que a enzima de banana apresenta elevada homologia com as de outras fontes vegetais. A análise dos níveis de proteína e mRNA durante o desenvolvimento e amadurecimentro do fruto permitem correlacionar o aumento de atividade com o aumento na expressäo do gene da SPS. Näo foram observadas alteraçöes significativas no estado de ativaçäo, sugestivas de modificaçäo covalente como mecanismo de ativaçäo durante o amadurecimento

Aggregation of banana pyrophosphate fructose 6-phosphate 1-phosphotransferase by glycerol.

Addition of glycerol during purification of banana (Musaceae, Musa cavendishii) pyrophosphate fructose 6-phosphate 1-phosphotransferase [(PFP), EC 2.7.1.90] initiated molecular aggregation of the enzyme. The aggregation process was dependent on the glycerol concentration. The native enzyme (66 kDa molecular mass) showed enhanced activity at 3% (V/V) or less of glycerol concentration. Glycerol concentration between 4 and 5% (V/V) affected a gradual and sequential aggregation of native form of the enzyme. These aggregated forms had molecular masses of 135, 200 and 270 kDa. The 135 and 200 kDa forms were stable for about 72 hrs and prolonged storage over 2 weeks resulted in the formation of the 270 kDa form. Concentration over 5% could reduce the time required for aggregation. Fru2.6 bis P activated the enzyme over ten fold, but did not help in the aggregation process. Studies on the role of glycerol on PFP specific activity suggested a difference in the activation process compared to that by Fru2.6bis P. Replacement of Hepes buffer by Tris increased the Fru2.6 bis P requirement for maximum activation by around 10 fold. Removal of glycerol from the buffer media resulted in almost complete inactivation of the enzyme.

Quercetin glycosides in Psidium guajava L: leaves and determination of a spasmolytic principle

The traditional herbal remedy from Psidium guajava leaves had been medically proposed in mexico as effective treatment of acute diarrhea. A methanolic leaf extract was subjected to a bioassay-guided isolation of spasmolytic constituents. Six fractions were separated on a polyvinylpolypyrrolidine (PVPP) columm using a water methanol-gradient. The fraction containing flavonols inhibited peristalsis of guinea pig ileum in vitro. A trace of quercetin aglycone together with five glycosides was isolated from this active fraction and identified as quercentin 3-O-alpha-L-arabinoside (guajaravin); quercetin 3-O-ß-D-glucoside (isoquercetin); quercetin 3-O-ß-D-galactoside (hyperin); quercetin 3-O-ß-L-rhamnoside (quercitrin) and quercetin 3-O-gentobioside. Biological activity of each pure compound was studied in the same bioassay. Obtained results suggets that the spasmolytic activity of the Psidium guajava leaf remedy is mainly due to the aglycone quercetin, present in the leaf and in the extract mainly in the form of live flavonols, and whose effect is produced when these products are hydrolyzed by gastrointestinal fluid

Aislamiento y caracterización parcial de la enzima fenoloxidasa de manzana (Malus domestica, var. Anna) / Isolation and partial characterization of phenoloxidase from apple (Malus domestica, var. Anna)

El objetivo de este trabajo fue el aislamiento y la caracterización parcial de la enzima polifenoloxidasa de manzana (Malus domestica Var. Anna), cosechada en la región semidesértica de la Costa de Hermosillo, Sonora, México. Se estudió el efecto que tienen el pH, temperatura, especificidad hacia sustratos y separación bajo condiciones de cromatografía hidrofóbica. La enzima se aisló a partir de manzanas maduras tratadas con acetona fría. Del polvo residual obtenido se extrajo la enzima con regulador de fosfatos, y el extracto se utilizó para realizar caracterización, encontrándose que el pH y temperatura óptimos eran 5.36 y 35§C, respectivamente. La especificidad hacia sustratos mostró ser decreciente desde 4-metil catecol, ácido clorogénico, catecol y ácido cafeico hasta 3.4-dihidroxifenilalanina (DOPA). La enzima resultó ser más termoestable que la generalidad de las oxidasas en el intervalo de temperatura de 35§C a 60§C. El comportamiento del extracto a través de cromatografía hidrofóbica produjo un solo pico con actividad polifenolásica, lográndose una purificación de aproximadamente 300 veces. El contenido de compuestos con grupo fenólico fue de 1.16 g/100 g de fruta fresca. Las características polifenolásicas encontradas se asemejan a las de manzanas de regiones templadas, aunque éstas presentan una mayor termoestabilidad, lo que explica hasta cierto grado la gran influencia que la temperatura ejerce sobre el fenómeno del oscurecimiento enzimático en las condiciones tan cálidas...