AT1 receptors activation enhances the expression of MMP-2, MMP-13 and VEGF but not MMP-9 in B16F10 melanoma cells

Melanoma is one of the most aggressive cancers of all solid tumors. The effect of angiotensin II on expression of three Matrix Metalloproteinases (MMPs) and Vascular Endothelial Growth Factor (VEGF) in B16F10 melanoma cells was evaluated. Also the blocking effect of losartan on angiotensin II induced effects was assessed. B16F10 murine melanoma cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 24 h prior to experiment the serum free medium was used. Angiotensin II (0 M, 10(-10) M, 10(-9) M or 10(-8) M) alone or in combination with Losartan (10(-6) M) in RPMI-1640 replaced the medium for experiments. After the incubation time (0, 1, 2, 6 and 12 h) cells were harvested using 0.05% (w/v) Trypsin and then recovered by centrifugation. The expression of MMP-2, MMP-13, MMP-9 and VEGF in B16F10 cell lysate was assessed by immunoblotting. Angiotensin II significantly enhanced the expression of MMP-2, MMP-13 and VEGF by concentrations as low as 0.1 nM. But angiotensin II could not stimulate any significant increase in MMP-9 expression by angiotensin II in B16F10 cells. Losartan abolished the enhancing effect of every concentration of angiotensin II on MMP-2, MMP-13 and VEGF expression completely and in all incubation times. As a result, angiotensin II through activation of AT1 receptors can stimulate the expression of MMP-2, MMP-13 and VEGF in B16F10 melanoma cells. This is an important conclusion because of the importance of these factors in melanoma invasiveness and the possible important role that angiotensin receptor blockers may play as cancer medications.     

Influence of E1-deleted recombinant adenoviruses on B7.1 and IL-2 expression in C1498 cells

BACKGROUND: Knowing that adenoviral vectors could initiate innate immunity, the ability of E1-deleted recombinant adenovirus (Ad-E1Delta) in induction of B7.1 and IL-2 molecules was studied. METHODS: The expression of green fluorescent protein in C1498 cells following transfection of these cells with adenovirus green fluorescent protein vector confirmed the ability of adenovirus vectors in infecting the cells and inducing the expression of the gene of interest. The expression of B7.1 molecule on the surface of the cells was assayed upon infection with Ad-E1Delta vector. Adenovirus-IL-2/B7.1 vector capable of inducing IL-2 and B7.1 expression in the cells was used as the positive control vector. RESULTS: According to the FACS results, about 4.17% of normal cells expressed B7.1 on their surface, while this level was increased in Ad-E1Delta transduced cells up to 14.43%. These results demonstrate that Ad-E1Delta vector considerably (about 3 folds) increases the expression of B7.1 on the cells. No detectable IL-2 was secreted into the medium of non-transduced and Ad-E1Delta transduced cells. CONCLUSION: Data indicate that the infection of C1498 cells with recombinant adenoviruses stimulates expression of B7.1 on the cell surface rather than secretion of IL-2 into the medium.     

Physicochemical,Nutritional and In Vitro Antidiabetic Characterisation of Blue Whiting (Micromesistius poutassou) Protein Hydrolysates

Protein hydrolysates from low-value underutilised fish species are potential sources of high-quality dietary protein and health enhancing peptides. Six blue whiting soluble protein hydrolysates (BW-SPH-A_F), generated at industrial scale using different hydrolysis conditions, were assessed in terms of their protein equivalent content, amino acid profile and score and physicochemical properties in addition to their ability to inhibit dipeptidyl peptidase IV (DPP-IV) and stimulate the secretion of insulin from BRIN-BD11 cells. Furthermore, the effect of simulated gastrointestinal digestion (SGID) on the stability of the BW-SPHs and their associated in vitro antidiabetic activity was investigated. The BW-SPHs contained between 70–74% (w/w) protein and all essential and non-essential amino acids. All BW-SPHs mediated DPP-IV inhibitory (IC₅₀: 2.12–2.90 mg protein/mL) and insulin secretory activity (2.5 mg/mL; 4.7 to 6.4-fold increase compared to the basal control (5.6 mM glucose alone)). All BW-SPHs were further hydrolysed during SGID. While the in vitro DPP-IV inhibitory and insulin secretory activity mediated by some BW-SPHs was reduced following SGID, the activity remained high. In general, the insulin secretory activity of the BW-SPHs were 4.5–5.4-fold higher than the basal control following SGID. The BW-SPHs generated herein provide potential for anti-diabetic related functional ingredients, whilst also enhancing environmental and commercial sustainability.     

不同土壤N_2O排放的研究

以森林和蔬菜土壤作对照,采用实验室培养研究了高产、中产和低产等三种茶园土壤N2O的排放水平,试验设不加氮(对照)与加氮[200mg/kg,(NH4)2SO4]二处理,在25℃恒温培养0、1、3、7和14d时分别取样检测N2O释放量。另外,选择两种茶园土壤研究了土壤含水量与加氮对N2O排放影响的交互作用。结果表明,对于不加氮的对照土壤组,高产茶园具有较高的N2O排放量,14d内平均日排放量高达11.26mg/(kg·d)(以纯氮计),显著高于其它四种土壤;但对于加氮处理组,以菜园土壤的N2O排放水平最高,极显著高于茶园和林地土壤;所有五种土壤加氮后,N2O排放量均有明显提高。茶园土壤N2O排放水平随着土壤含水量的提高而增加,并与施氮存在显著的正交互作用,当土壤含水量较高时施氮具有刺激N2O排放的作用。文章根据土壤培养期间NH4+和NO3-含量的变化就N2O形成机理进行了讨论。     

Augmenting Anti-Cancer Natural Products with a Small Molecule Adjuvant

Aquatic microbes produce diverse secondary metabolites with interesting biological activities. Cytotoxic metabolites have the potential to become lead compounds or drugs for cancer treatment. Many cytotoxic compounds, however, show undesirable toxicity at higher concentrations. Such undesirable activity may be reduced or eliminated by using lower doses of the cytotoxic compound in combination with another compound that modulates its activity. Here, we have examined the cytotoxicity of four microbial metabolites [ethyl N-(2-phenethyl) carbamate (NP-1), Euglenophycin, Anabaenopeptin, and Glycolipid 652] using three in vitro cell lines [human breast cancer cells (MCF-7), mouse neuroblastoma cells (N2a), and rat pituitary epithelial cells (GH4C1)]. The compounds showed variable cytotoxicity, with Euglenophycin displaying specificity for N2a cells. We have also examined the modulatory power of NP-1 on the cytotoxicity of the other three compounds and found that at a permissible concentration (125 µg/mL), NP-1 sensitized N2a and MCF-7 cells to Euglenophycin and Glycolipid 652 induced cytotoxicity.     

Omega-3 Fatty Acids Modulate Weibel-Palade Body Degranulation and Actin Cytoskeleton Rearrangement in PMA-Stimulated Human Umbilical Vein Endothelial Cells

Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.     

Characterization of Molecular Species and Anti-Inflammatory Activity of Purified Phospholipids from Antarctic Krill Oil

The phospholipids (PLs) from Antarctic krill oil were purified (>97.2%) using adsorption column chromatography. Forty-nine PL molecular species were characterized by ultrahigh-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Most of molecular species contained eicosapentaenoic acid (EPA, 20:5), docosahexaenoic acid (DHA, 22:6), docosapentaenoic acid (DPA, 22:5), and arachidonic acid (AA, 20:4). Notably, a special species PC (20:5/22:6) (1298.17 nmol/g) and many ether PLs were detected. The Antarctic krill PL liposome (IC₅₀ = 0.108 mg/mL) showed better anti-inflammatory activity than crude Antarctic krill oil (IC₅₀ = 0.446 mg/mL). It could block NF-κB signaling pathway via suppression of IκB-α degradation and p65 activation and dose-dependently reduce the cellular content of inflammatory mediators including nitric oxide (NO), reactive oxygen species (ROS), and inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In addition, it can suppress carrageenan-induced mouse paw swelling. Results from the present study could provide a reference for better evaluation of nutritional and medicinal values of Antarctic krill oil.     

北美大豆猝死综合症病菌厚垣孢子的液体培养条件

用液体培养的方法,研究培养基、pH值、温度以及氧气等因素对北美大豆猝死综合症病菌(Fusarium virguforme sp.nov.)菌株F-171厚垣孢子产生的影响.结果表明:菌株F-171产生厚垣孢子的最适液体培养条件为7500r·min -、2 min匀浆断裂菌丝,在250 mL三角瓶装入80 mL大豆茎秆或...     

Optimization of process parameters influencing the submerged fermentation of extracellular lipases from Pseudomonas aeruginosa, candida albicans and Aspergillus flavus

The present study was aimed at optimization, production and partial purification of lipases from Pseudomonas aeruginosa, Candida albicans and Aspergillus flavus. Various nutritional and physical parameters affecting lipase production such as carbon and nitrogen supplements, pH, temperature, agitation speed and incubation time were studied. Refined sunflower oil (1% v/v) and tryptone at a pH of 6.2 favored maximum lipase production in Pseudomonas at 30 degrees C and 150 rpm, when incubated for 5 days. In C. albicans refined sunflower oil (3% v/v) and peptone resulted in maximum lipase production at pH 5.2, 30 degrees C and 150 rpm, when incubated for 5 days. In A. flavus coconut oil (3% v/v) and peptone yielded maximum lipase at pH 6.2, 37 degrees C, 200 rpm after an incubation period of 5 days. The lipases were partially purified by ammonium sulphate precipitation and dialysis. In P. aeruginosa enzyme activity of the dialyzed fraction was found to be 400 U mL-' and for C. albicans 410 U mL(-1). The dialysed lipase fraction from A. flavus demonstrated an activity of 460 U mL(-1). The apparent molecular weights of the dialyzed lipases were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The dialyzed lipase fraction obtained from P. aeruginosa revealed molecular weights of 47, 49 and 51 kDa, whereas, lipases from C. albicans and A. flavus demonstrated 3 bands (16.5, 27 and 51 kDa) and one band (47 kDa), respectively. These extracellular lipases may find wide industrial applications.     

The effect of manipulations of incubation temperature on embryonic and post-hatching growth of native Saudi chickens

Embryonated egg is an ideal, environment in which to investigate the effects of incubation temperature on the development of the chick embryo. The objective of the current study were to investigate the effect of increasing the incubation temperature of chick embryo by 1.2 degrees C for 7 days (ED4 to ED11) on the body movement and mass of native saudi chick embryo. This objective was extended to examine the influence of pre-hatching temperature on post-hatching growth. Therefore, a total of 180 hatching egg of native Saudi chicken divided into two equal groups incubated at temperature 37.5 degrees C. The incubation temperature was raised to 38.7 degrees C from ED4 to ED11 in treated group before being returned to the control group incubation temperature (37.5 degrees C). The study revealed that elevating the incubation temperature of the eggs of native Saudi chicken by just 1.2 degrees C, from 37.5 to 38.7 degrees C, during embryonic days (ED) 4-11 causes significant increase in embryonic movement as demonstrated in day 8 in the chicks incubated at 38.7 degrees C together with an increase in embryonic development, the embryos incubated at higher temperature were heavier in weights and exhibit significantly longer legs than the controls in ED12 and 15. The increase in pre-hatching incubation temperature (38.7 degrees C) did not reveal any significant effects on post-hatching growth or of feed conversion efficiency.     

Synthesis and Bioactivity of Ancorinoside B,a Marine Diglycosyl Tetramic Acid

The sponge metabolite ancorinoside B was prepared for the first time in 16 steps and 4% yield. It features a β-d-galactopyranosyl-(1→4)-β-d-glucuronic acid tethered to a d-aspartic acid-derived tetramic acid. Key steps were the synthesis of a fully protected d-lactose derived thioglycoside, its attachment to a C₂₀-aldehyde spacer, functionalization of the latter with a terminal N-(β-ketoacyl)-d-aspartate, and a basic Dieckmann cyclization to close the pyrrolidin-2,4-dione ring with concomitant global deprotection. Ancorinoside B exhibited multiple biological effects of medicinal interest. It inhibited the secretion of the cancer metastasis-relevant matrix metalloproteinases MMP-2 and MMP-9, and also the growth of Staphylococcus aureus biofilms by ca 87% when applied at concentrations as low as 0.5 µg/mL. This concentration is far below its MIC of ca 67 µg/mL and thus unlikely to induce bacterial resistance. It also led to a 67% dispersion of preformed S. aureus biofilms when applied at a concentration of ca 2 µg/mL. Ancorinoside B might thus be an interesting candidate for the control of the general hospital, catheter, or joint protheses infections.     

Agarose-Degrading Characteristics of a Deep-Sea Bacterium Vibrio Natriegens WPAGA4 and Its Cold-Adapted GH50 Agarase Aga3420

Up until now, the characterizations of GH50 agarases from Vibrio species have rarely been reported compared to GH16 agarases. In this study, a deep-sea strain, WPAGA4, was isolated and identified as Vibrio natriegens due to the maximum similarity of its 16S rRNA gene sequence, the values of its average nucleotide identity, and through digital DNA–DNA hybridization. Two circular chromosomes in V. natriegens WPAGA4 were assembled. A total of 4561 coding genes, 37 rRNA, 131 tRNA, and 59 other non-coding RNA genes were predicted in the genome of V. natriegens WPAGA4. An agarase gene belonging to the GH50 family was annotated in the genome sequence and expressed in E. coli cells. The optimum temperature and pH of the recombinant Aga3420 (rAga3420) were 40 °C and 7.0, respectively. Neoagarobiose (NA2) was the only product during the degradation process of agarose by rAga3420. rAga3420 had a favorable stability following incubation at 10–30 °C for 50 min. The Km, Vmax, and kcat values of rAga3420 were 2.8 mg/mL, 78.1 U/mg, and 376.9 s⁻¹, respectively. rAga3420 displayed cold-adapted properties as 59.7% and 41.2% of the relative activity remained at 10 3 °C and 0 °C, respectively. This property ensured V. natriegens WPAGA4 could degrade and metabolize the agarose in cold deep-sea environments and enables rAga3420 to be an appropriate industrial enzyme for NA2 production, with industrial potential in medical and cosmetic fields.     

Development of a formulation of Trichoderma asperellum to control black rot disease on pineapple caused by (Thielaviopsis paradoxa)

A local isolate of Trichoderma asperellum was tested for its antagonistic activity against Thielaviopsis paradoxa (telemorph = Ceratocyctis paradoxa). The highest antagonistic activity was achieved when the concentration of T. asperellum conidia was 1 × 10⁷ conidia/mL. The highest biomass and number of colony forming unit/mL of the T. asperellum peaked at 144 h after incubation in yeast waste residue medium. The minimum inhibition concentration value of the formulation was observed as 1% on growth of Th. paradoxa incubated at 28 ± 2 °C for 10 d. In the soil fungicide-screening test, the effect of concentrations 100-1600 μg/mL on mycelia growth was not significant (P < 0.05). Complete mycelial growth inhibition occurred at concentration above 52,600 μg/mL. Results of the fruit application tests clearly showed that all treated fruits were free of disease at the end of the incubation period. No significant differences (P > 0.05) in pH, total soluble solids and titratable acidity were observed between fruits treated with formulation of T. asperellum and the control formulation treated pineapples.     

Latent infection of Botrytis cinerea in rose flowers and combined chemical and physiological control of the disease

A high percentage of rose flowers collected during winter from greenhouses around Israel developed grey mould disease when incubated in the laboratory in favourable conditions. Symptomless flowers of various cultivars were incubated in a humidity chamber after surface disinfection. More than 50% of the symptomless flowers yielded the pathogen Botrytis cinerea Pers., thus showing the presence of latent infection which probably occurred 3–8 days earlier in the commercial greenhouses. Initially, the symptoms which occurred on affected petals were mainly restricted lesions. However, when flowers were incubated under favourable conditions, necrosis developed and spread. Chemical control of grey mould was tested on cut rose flowers. Partial control of the disease was correlated with reduction in necrosis and a relative increase in restricted lesions (<2 mm in diameter). Of the 18 fungicides which were tested, the following were the most efficient during incubation at 20°C: metomeclan, dichlofluanid, myclozolin, N-phenylcarbamate plus carbendazim (NPC + MBC), polyoxin D, prochloraz and iprodione. When incubated at 10°C, the most effective fungicides against grey mould of rose flowers were NPC + MBC, polyoxin D, chlorothalonil, the detergent Tween 20, and the inhibitor of ethylene activity, silver thiosulphate (STS). Diphenylamine was more effective at 4°C than at 10° or 20°C. STS in combination with each of the fungicides chlorothalonil, polyoxin B, myclozolin or merpan was additively more effective than STS or each of the respective fungicides alone.     

Characterization of pectic substances from two potato cultivars with different sensitivities to prewarming

Summary Potato cultivars Chieftain and Kennebec were prewarmed at 70°C for 15 min. prior to heating at 100°C for 18 min. in a water bath. Chieftain exhibited a large increase in fracturability upon treatment while Kennebec showed a smaller increase. Pectic substances of the two cultivars were sequentially extracted in water, sodium hexametaphosphate (0.5%), hydrochloric acid (0.05M, 50°C), and sodium hydroxide (0.05M, 5°C). The largest fraction isolated from both cultivars consisted of hydroxide-soluble pectic substances, and was followed by the acid-soluble fraction. Chieftain contained significantly greater amounts of the hydroxide-soluble fraction than Kennebec. Ion exchange chromatography revealed that the extracted pectic substances from the two major fractions consisted of a charged pectin chain with attached neutral sugars. Gel filtration chromatography revealed wide molecular size distributions for the isolated pectic substances. Pectic neutral sugar content for the hydroxide-soluble fraction (50% of total pectic substances) was greater for Chieftain than for Kennebec.     

Effect of esophageal distention on basal and stimulated gastric acid secretion in rats

BACKGROUND: It is well established that the esophageal distention (ED) leads to gastric relaxation, partly by vago-vagal reflex, but till now, the effect of ED on gastric acid secretion has not been investigated. The aim of this study was to investigate the effect of ED on basal and stimulated gastric acid secretion. METHODS: Adult male Wistar rats (200-240 g) were deprived of food but not the water 24 h before the experiments. Under urethane anesthesia (1.2 g/kg, i.p.), animals underwent tracheostomy and laparotomy. A catheter was inserted in the stomach through duodenum for gastric distention and gastric washout and the esophagus was cannulated with a distensible balloon orally to distend esophagus (0.3 ml, 10 min). Gastric acid secretion was stimulated by gastric distention, carbachol (4 microg/kg, i.p.) or histamine (5 mg/kg, s.c.). Effects of vagotomy, NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v.) and also hexamethonium were investigated. RESULTS: Basal and gastric distention- and carbachol, histamine-stimulated acid secretion were reduced by the ED (P<0.05, P<0.0001, P<0.01 and P<0.02, respectively). L-NAME (10 mg/kg, i.v.) elevated the acid output (P<0.002). Vagotomy reduced the inhibitory effect of the esophagus distention on gastric distention-induced acid secretion (P<0.01). CONCLUSION: These results indicate that the vagus nerves are involved in the inhibitory effect of the ED on the basal and stimulated gastric acid secretion. Furthermore, nitric oxide could be involved.     

Enumeration of lactobacilli in the fecal flora of infant using two different modified de-Man Rogosa Sharpe media under aerobic and anaerobic incubation

Regarding the importance of the presence of intestinal lactobacilli and their population in infants, four different treatments were evaluated for Lactobacillus isolation efficiency via reduction in the growth of other groups of bacteria capable of growing on de-Man Rogosa Sharpe (MRS) medium from fecal samples of 11 Iranian infants. MRS-Vancomycin (1 mg L(-l)) was used as a base medium and application of lactic acid and aerobic incubation of inoculated plates were performed as selective factors. Each fecal sample was cultivated as duplicate on to the base medium with or without lactic acid to reduce the pH to 5.4 +/- 0.2. Half of the plates were incubated aerobically and the rest of them incubated under 10% CO2 concentration. Total count and Lactobacillus count of all samples were recorded according to the age differences of infants. The counts of false positive colonies were recorded with respect to their cell morphology and gram reaction in all treatments. Anaerobic incubation of lactic acid modified MRS-Vancomycin gave the most Lactobacillus percentage coverage, about 93% among the Lactobacillus positive samples. Using this treatment, the median Lactobacillus count yielded 8.29 log10 cfu g(-1) in the younger and 5.70 log10 cfu g(-1) in the elder group. It could be concluded that lactic acid might be a proper pH reducing agent when enumeration of lactobacilli from fecal samples is of interest.     

Extraction ofclaviba cter michiganensis subsp.sepedonicus from composite samples of potato tubers

The presence ofClavibacter michiganensis subsp.sepedonicus was detected by immunofluorescence and ELISA in diluent in which cores from 200 potato tubers had been incubated on a rotary shaker. By this procedure the ring rot pathogen was detected in composite samples from naturally infected tubers and tuber samples “spiked” with infected tissue or a pure culture of the pathogen. Populations of saprophytic bacteria increased about 100-fold in the diluent during 18 h of incubation but did not interfere with serological detection of C.m. sepedonicus. Some of the results suggest thatC. m. sepedonicus cells multiply during incubation of tuber samples. Extraction of ring rot bacteria from potato tuber tissue by incubation on a rotary shaker requires little work and minimizes the amount of plant tissue in the extract compared to the standard extraction procedure involving maceration and differential centrifugation.     

Method and quantity of liquid formulation of phosphobacteria required for seed inoculation

The survival of vegetative and sporulated cells of the Bacillus cultures on the seeds of the crop plants was tried in different combinations. One milliliter inoculum with 1 mL adhesive combination or sterile water showed better results followed by 1.5 mL inoculum with 0.5 mL adhesive or sterile water. The population of 5.5x10(5) cfu seed(-1) on black gram, 10.5x10(5) cfu seed(-1) on soybean and 6.5x10(5) cfu seed(-1) on maize were observed after 12 h of incubation in 1 mL sporulated inoculum mixed with 1 mL of rice gruel. The sporulated inoculum along with rice gruel favoured the adherence of the regenerated cells as rice gruel is rich in nutrient content.