Hops extract alleviates Aβ-injury to osteoblasts through antioxidant pathway / 药学实践杂志

Objective To explore the effects of Humulus lupulus L. extract (HLE) and its mechanism on improving bone formation of Aβ-injured osteoblasts. Methods Osteoblasts isolated from 24 h-old Wistar rats were injured by Aβ1-42 oligomer and intervened with HLE. The proliferation, differentiation and bone mineralization of osteoblasts were determined by MTT assay, alkaline phosphatase (ALP) activity assay and alizarin red staining, respectively. The apoptosis of osteoblasts was detected by flow cytometer. The expression levels of bone formation related proteins, and proteins of Nrf2 and FoxO1 pathways were measured by Western blotting analysis. The intranuclear expression of FoxO1 protein was detected by immunofluorescence. Results HLE significantly improved the cell proliferation, ALP activity and bone mineralization, and inhibited the apoptosis of Aβ-injured osteoblasts. HLE also significantly promoted the expressions of collagen type Ι (COL-I) and osteopontin (OPN) in Aβ-injured osteoblasts. HLE notably activated the Nrf2 and FoxO1 signaling pathways in Aβ-injured osteoblasts by promoting the expressions of related proteins and maintained bone metabolism through relieving oxidative stress. Conclusion This study confirms that HLE can alleviate Aβ-injury to osteoblasts, and preliminarily clarifies the mechanism being related to antioxidation, which provides a new reference for the mechanism research and drugs development for anti-osteoporosis.

Prokaryotic expression, protein purification and functional verification of human homotypic fusion and vacuole protein sorting complex subunit / 生物工程学报

Homotypic fusion and vacuole protein sorting(HOPS) is a protein complex consisting of VPS11, VPS16, VPS18, VPS33, VPS39, VPS41 and regulates membrane transport in vivo through membrane fusion mechanisms. The evidence suggests that HOPS complex as a fusion factor, facilitates autophagosome-lysosome fusion. To determine whether the HOPS complex directly interacts with the autophagic SNARE protein STX17 in vitro, the coding sequence of the six genes were amplified from the existing plasmids by PCR, and then ligated to the prokaryotic expression vector pGEX 4T-1-GST or pET-His-NusA. After identification through colony PCR and DNA sequencing, 6 recombinant plasmids were constructed and transferred into Escherichia coli BL21 (DE3). The recombinant proteins were purified by glutathione sepharose 4B and nickel column. We used the tobacco etch virus protease to cut off the GST-tag or His-NusA-tag, to obtain HA-VPS11 protein of about 105 kDa, Flag-VPS16 protein of about 97 kDa, HA-VPS18 protein of about 108 kDa, Flag-VPS33 protein of about 70 kDa, HA-VPS39 protein of about 97 kDa, and Flag-VPS41 protein of about 98 kDa. The function of the purified proteins was verified by in vitro glutathione S-transferases pull-down assay, confirming that autophagic SNARE protein STX17 interacted directly with HOPS components. Our findings provide experimental basis to further study the function and mechanism of HOPS complex in the process of autophagosome-lysosome fusion.

Efecto de antibióticos y un antimicrobiano sobre el control de bacterias acido lácticas en la fermentación alcohólica / Effect of antibiotics and an antimicrobial on the control of lactic acid bacteria in alcoholic fermentation

RESUMEN El propósito de esta investigación fue evaluar el efecto de antibióticos y un antimicrobiano para el control de bacterias ácido lácticas (BAL) en etapa fermentativa de tres ingenios (A, B y C) productores de alcohol en el Valle del Cauca (Colombia). Se establecieron dos ensayos de fermentación a escala de laboratorio por separado, tratados con cuatro antibióticos (8, 15 y 30 ppm), dos cocteles (4, 8 y 15 ppm) y un antimicrobiano (15 y 30 ppm); en el primero se cuantificó la producción de ácido láctico (AL), la población y viabilidad de la levadura, y en el segundo se determinó el crecimiento de BAL. En el primer ensayo, los tratamientos que controlaron los niveles de AL en los ingenios fueron virginiamicina (VIR) 15 ppm, maduramicina (MAD) 15 ppm, penicilina (PEN) 30 ppm y lúpulo (LUP) (30 ppm). Adicionalmente, los tratamientos PEN a 30 ppm, VIR, el coctel estreptomicina-penicilina-virginiamicina-monensina (EPVM) y LUP a 15 ppm no afectaron a la levadura en las condiciones evaluadas. En el ensayo dos, todos los tratamientos lograron controlar BAL, presentando un mayor control a las 24 horas pos-tratamiento en el ingenio A. En el ingenio B, MAD, monensina (MON), los cocteles estreptomicina-penicilina-virginiamicina (EPV) y EPVM, controlaron el crecimiento de BAL durante las primeras 6 horas, mientras que LUP controló la población de BAL a las 24 horas. En el ingenio C, LUP, MON y EPV lograron controlar las BAL en todas las concentraciones, principalmente a las 24 horas. Por tanto, se infiere que VIR en el ingenio A, EPVM en los ingenios B y C y el antimicrobiano LUP en los tres ingenios son eficientes en el control de BAL.

ABSTRACT The objective of this research was to evaluate the effect of antibiotics and an antimicrobial for the control of lactic acid bacteria (LAB) in the fermentative stage of three sugar mills producer (A, B and C), in "Valle del Cauca" (Colombia). Two fermentation assays on laboratory scale separately, treated with four antibiotics (8, 15 and 30 ppm), two cocktails (4, 8 and 15 ppm) and an antimicrobial (15 and 30 ppm) were established. In the first one, lactic acid (LA) production, population and yeast viability were quantified, and in the second the LAB growth was determined. In the first assay, the treatments that controlled LA levels in sugar mills were virginamicin (VIR) 15 ppm, maduramycin (MAD) 15 ppm, penicillin (PEN) 30 ppm and hop (HP) (30 ppm). Additionally, PEN treatments at 30 ppm, VIR, the streptomycin-penicillin-virginiamycin-monensin cocktail (SPVM) and HP at 15 ppm did not affect the yeast under the conditions evaluated. In the second assay, all treatments managed to control of LAB, with greater control at 24 hours in sugar mill A. In sugar mill B, MAD, monensin (MON), streptomycin-penicillin-virginiamycin (SPV) and SPVM cocktails controlled LAB growth during the first 6 hours, while HP controlled LAB population at 24 hours. In sugar mill C, HP, MON and EPV managed to control LAB in all concentrations mainly at 24 hours. Therefore, it is inferred that VIR in sugar mill A, SPVM in sugar mills B and C and HP antimicrobial in the three mills are efficient in LAB control.

Stability of Active Constituents of Hops (Humulus lupulus) Strobiles and their Ethanolic Extracts during Storage.

Aims: The purpose of this study was to evaluate the stability of three major active constituents (humulones, lupulones and xanthohumol) in dried hops (Humulus lupulus) strobiles (whole and ground) as well as their ethanolic extracts during storage. Methodology: A comparative study of humulones, lupulones and xanthohumol levels of H. lupulus strobiles during storage was carried out. Dried whole strobiles and cryogenically ground dried strobiles stored at -15ºC as well as ethanol extracts of the strobiles prepared using different ethanol concentrations (10%, 30%, 50%, 70%, and 95%) and stored at room temperature, were analyzed by HPLC to quantify each constituent. These hops samples were analyzed immediately after preparation, and then one year and two years later to determine the concentrations of the constituents. Results: HPLC analysis indicated that the amount of all three constituents in the ground strobiles and in the ethanol extracts decreased gradually during the storage period. The 10% and 30% ethanol extracts had very low amounts of constituents initially and were practically devoid of constituents at the end of two years. The 50% ethanol extract contained considerable amounts of humulones and xanthohumol, and low levels of lupulones initially, but lost substantial amounts over time. The 70% and 95% ethanol extracts showed higher levels of all three constituents, while the 95% H. lupulus ethanol extract contained the highest constituent levels throughout the experimental period. The ethanol content of the extract had a direct correlation to the constituent levels; the higher the ethanol level, the higher the initial and subsequent constituent levels. Conclusion: Both dried hops and ethanol extracts lose active components over storage time. When preparing extracts, at least 70% ethanol is necessary to extract the highest levels of three bioactive constituents and to retain them over a two-year period. Ethanol concentration is a critical factor to be considered in hops extraction process.

Vps1 in the late endosome-to-vacuole traffic.

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.