Effect of Bacillus subtilis,Enterococcus faecium,and Enterococcus faecalis supernatants on serotonin transporter expression in cells and tissues

BACKGROUND Bacillus subtilis(B.subtilis),Enterococcus faecium(E.faecium),and Enterococcus faecalis(E.faecalis)are probiotics that are widely used in the clinical treatment of irritable bowel syndrome(IBS).Whether the supernatants of these three probiotics can improve gastrointestinal sensation and movement by regulating the serotonin transporter(SERT)expression needs to be clarified.AIM To investigate whether B.subtilis,E.faecium,and E.faecalis supernatants can upregulate SERT expression in vitro and in vivo.METHODS Caco-2 and HT-29 cells were stimulated with probiotic culture supernatants for 12 and 24 h,respectively.A male Sprague-Dawley rat model of post-infectious irritable bowel syndrome(PI-IBS)was established and the rats were treated with phosphate-buffered saline(group A)and three probiotics culture supernatants(groups B,C,and D)for 4 wk.The levels of SERT were detected by quantitative PCR and western blotting.RESULTS The levels of SERT at post-treatment 12 and 24 h were significantly elevated in Caco-2 cells treated with B.subtilis supernatant compared with those in the control group(aP<0.05).Those levels were markedly upregulated in Caco-2 cells stimulated with E.faecium and E.faecalis supernatants at 24 h(aP<0.05).In addition,SERT expression in groups B,C,and D was significantly higher than that in group A in the 2nd wk(aP<0.05).Increased SERT expression was only found in group D in the 3rd wk(aP<0.05).However,there was no significant difference in SERT expression between the groups in the last week(P>0.05).CONCLUSION The supernatants of B.subtilis,E.faecium,and E.faecalis can upregulate SERT expression in intestinal epithelial cells and the intestinal tissues in the rat model of PI-IBS.

Effect of Lactobacillus rhamnosus GG supernatant on serotonin transporter expression in rats with post-infectious irritable bowel syndrome

AIM To evaluate the effect of Lactobacillus rhamnosus GG supernatant(LGG-s) on the expression of serotonin transporter(SERT) in rats with post-infectious irritable bowel syndrome(PI-IBS).METHODS Campylobacter jejuni 81-176(1010 CFU/m L) was used to induce intestinal infection to develop a PI-IBS model. After evaluation of the post-infectious phase by biochemical tests, Dn A agarose gel electrophoresis, abdominal withdrawal reflex(AWR) test, and the intestinal motility test, four PI-IBS groups received different concentrations of LGG-s for 4 wk. The treatments were maintained for 1.0, 2.0, 3.0 or 4.0 wk during the experiment, and the colons and brains were removed for later use each week. SERT m Rn A and protein levels were detected by real-time PCR and Western blot, respectively.RESULTS The levels of SERT m Rn A and protein in intestinal tissue were higher in rats treated with LGG-s than in control rats and PI-IBS rats gavaged with PBS during the whole study. Undiluted LGG-s up-regulated SERT m Rn A level by 2.67 times compared with the control group by week 2, and SERT m Rn A expression kept increasing later. Double-diluted LGG-s was similar to undiluted-LGG-s, resulting in high levels of SERT m Rn A. Triple-diluted LGG-s up-regulated SERT m Rn A expression level by 6.9-times compared with the control group, but SERT m Rn A expression decreased rapidly at the end of the second week. At the first week, SERT protein levels were basically comparable in rats treated with undiluted LGG-s, double-diluted LGG-s, and triplediluted LGG-s, which were higher than those in the control group and PBS-treated PI-IBS group. SERT protein levels in the intestine were also comparable in rats treated with undiluted LGG-s, double-diluted LGG-s, and triple-diluted LGG-s by the second and third weeks. SERT m Rn A and protein levels in the brain had no statistical difference in the groups during the experiment.CONCLUSION LGG-s can up-regulate SERT m Rn A and protein levels in intestinal tissue but has no influence in brain tissue in rats with PI-IBS.

Bioactivities of Culture Supernatants from Retroviral Packaging Cells Carrying the Mouse Fas Ligand Gene

The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated. FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology, PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine 2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels of mFasL protein on NIH3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3 cells membrane was investigated by the inducing apoptosis of Fas + Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately co-cultured with Yac-1 cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants from twelve resistant clones was 8.5×10 5 colony-forming-unit (CFU)/ml. The NIH3T3 cells transfected by above supernatants had a higher level of mFasL (53.81±6.9 %), and significantly induced the apoptosis of Fas + Yac-1 cells (56.78±4.5 %), as both were cocultured for 5 h at 1∶1 ratio, whereas it is 7.08±3.4 % in control group (P<0.01). Supernatant from PLFIN-PA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.

Rheological properties in supernatant of peach gum from almond(Prunus dulcis)

The rheological properties in the supernatant of peach gum from Prunnus dulcis were discussed in order to provide more scientific technical parameters and references for developing peach gum as a kind of medicinal gum.The rheological properties in the supernatant of peach gum were comparatively studied in different material ratios,temperatures,shaking times,pH values and salinities.The results show that,1) the mathematical model of shear rate with material ratio and shear stress is Y=0.069X12+0.035X2 -1.174,R2=0.942;2) the mathematical model of shear rate with temperature and shear stress is Y=4.936X12+0.023 2X2-1.688,R2=0.937;3) the mathematical model of shear rate with shaking time and shear stress is Y=0.005 192 X13-0.140 73X12+1.249 045X1+ 0.036 546 X2-3.644 29,R2=0.954 3;4) the effects of pH value on the rheological properties in the supernatant of peach gum are comparatively complicated with a varying range of 3-11 and the shear rate shows a change trend of saddle model;5) the mathematical model of shear rate with the concentration of NaCl and shear stress is Y=-0.037 44X1+0.012 93 X2,R2=0.998;6) the mathematical model of shear rate with the concentration of CaCl2 and shear stress is Y=0.025 789X1+0.016 19X2,R2 =0.999;and 7) the mathematical model of shear rate with the concentration of sorbic acid potassium and shear stress is Y=0.079 5X1+0.017 3X2,R2=0.998.The results show that the material ratio,temperature,shaking time,pH value significantly affect the rheological properties in the supernatant of peach gum,and the concentrations of NaCl and CaCl2 also significantly affect the rheological properties expect the concentration of sorbic acid potassium.

Antifungal Activity against <i>Aspergillus parasiticus</i>of Supernatants from Whey Permeates Fermented with Kefir Grains

Aspergillus parasiticus, a common fungal contaminant in food, produces aflatoxin B1, which is classified as human carcinogen. Kefir is an ancient fermented beverage obtained by the fermentation of different substrates with kefir grains. A very important waste produced by the dairy cheese industry is the whey permeate, which nowadays is a strong ambient contaminant. The aim of this work was to assess the effect of whey permeates fermented with kefir grains against A. parasiticus growth, aflatoxin B1 biosynthesis, and the kefir microorganisms protection against the cell damage produced by aflatoxin B1. It was observed that kefir-cell-free-supernatants (CFS) produced fungal inhibition. A fungicidal effect was observed with 65% v/v of CFS in the culture medium (final pH 4.55 and total undissociated lactic and acetic acid concentration 34.08 mM). Under these conditions, aflatoxin production was not detected. Finally, it was found that non-viable kefir microorganisms protected HepG2 cells from the damage produced by aflatoxin B1.

PROLIFERATION OF ANTI－CD_3 McAb AND IL－2 INDUCED SPLENOCYTES AND ANTITUMOR EFFECT OF THEIR CULTURE SUPERNATANTS

The proliferation of splenocytes from health adults was induced by anti-CD3 McAb and IL-2.The proliferative potential of the splenocytes and antitumor activity of their culture supernatants of splenocytes were studied.The results showed that anti-CD3 McAb not only enhanced the proliferation of the splenocytes directly,but also enhanceil that of induced by IL-2.Their enhancing effect was more significant when the incubation time in vitro was prolonged.The culture supernatants of anti-CD3 and IL-2 induced splenocytes also had the antitumor activity and enhancing capability to the antitumor activity of LAK tells.The results suggested that LAK cells could secret lymphokine,and this effect would be synergically promoted when anti-CD3 and IL-2 were simultaneously used.

Fast start-up anammox process using Acyl-homoserine lactones(AHLs) containing supernatant

N-dodecanoyl homoserine lactone（C（12）-HSL）was detected in the supernatant of an anammox granular sludge reactor（AGSR）.C（12）-HSL could enhance the specific anammox activity of anammox biomass.Adding C（12）-HSL-containing AGSR supernatant into the continuously stirred tank reactors reduced the start-up time of the anammox process from80 to 66 days.Moreover,the nitrogen loading rate was also enhanced to 1.6 times that of the control reactor.AHLs could increase the secretion of extracellular polymeric substances and anammox obtained better enrichment with the addition of AHLs-containing AGSR supernatant.Denaturing gradient gel electrophoresis analysis further revealed that AHLs played a role in mediating microbial community parameters.In conclusion,adding AHL-containing supernatant could be an effective and economical way to accelerate the start-up of anammox.

Acaricidal activities of whole cell suspension,cell-free supernatant,and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.)

Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp.,a mushroom mite endemic in Thailand.To develop an effective formulation of Xenorhabdus stokiae,treatments using different parts of X.stokiae isolate PB09 culture,including whole cell suspension,cell-free supernatant,and crude cell extract,were performed.The results show that different parts of X.stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity.Application with cell-free supernatant of X.stokiae culture resulted in both the highest mite mortality rate [(89.00±3.60)%] and the lowest mite fecundity [(41.33±23.69) eggs/gravid female].Whole cell suspen-sion of X.stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant,suggesting that X.stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media.Crude cell extract of X.stokiae was not effective against mites.Cell-free supernatant of X.stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

The performance of a combined nitritation–anammox reactor treating anaerobic digestion supernatant under various C/N ratios

A combined nitritation–anammox reactor was developed to treat the digestion supernatant under various C/N ratios. Due to the difficulties for heterotroph to utilize the refractory organics, the reactor presented relatively stable performance with increasing supernatant addition. Nevertheless, the adverse effects of supernatant would accumulate during the long-term operation and thus weakened the activity and shock resistance of microbes,which further led to the gradual decrease of reactor performance after 92 days＇ operation.Under this circumstance, supernatant with volatile fatty acids（VFAs） residuals was further introduced into the reactor to investigate the performance of combined nitritation–anammox process with VFA addition. With the appearance of VFAs, the nitrogen removal performance gradually restored and the reactor finally achieved stable and efficient performance with C/N ratio of 0.35. The VFA residuals within 150 mg/L in the supernatant served as the extra electron donors and stimulated the heterotrophic denitrification process, which was vital for the enhancement of reactor. The nitrogen removal rate and total nitrogen removal efficiency reached 0.49 kg N/（m^3·day） and 88.8% after 140 days＇ operation, respectively. The combined nitritation–anammox reactor was proved suitable to treat digestion supernatant.

Differentiation of bone marrow stem cells into the cardiomyocyte-like cells induced by fetal cardiac supernatant with 6-bromoindirubin-3-oxime

Background Our previous study showed the 150 mg/mL fetal cardiac supernatant （FCS） could induce differentiation of BMSCs into cardiomyocye-like cells without cardiomyocyte touch,but differentiation efficiency is not high enough.Inhibition of glycogen synthase kinase-3 enhanced the proliferation and survives of stem cells.We tested if 6-bromoindirubin-3-oxime （BIO,glycogen synthase kinase-3 inhibitor） enhances the effects of FCS on differentiation of BMSCs and explore the growth factors in FCS.Methods BMSCs were isolated from the femur and tibia of four-week-old male Sprague-Dawley rats and co-cultured with FCS （150 mg/mL） that was made from fetal hearts from nineteen-day pregnant Wistar rats.BIO with different concentration （0,1,10,and 100 nM） was introduced in culture dishes.Transforming growth factor beta 1 （TGF-β1）,bone morphogenetic protein 2 （BMP-2） and Akt in cardiac supernatant and culture medium were assayed with ELISA methods.Results After co-culturing with FCS,beating myotubes were observed in 25.9 % BMSCs dishes after 1 to 2 weeks＇ culture.The levels of TGF-β1 and BMP-2 in FCS concentrations were no more than that in young and adult cardiac supernatant.All BIO groups significantly enhanced the effects of FCS on differentiation of BMSCs into the cardiomyocyte-like cells （1 nM,83 %;10 nM,73 %;100 nM,100 %）.Akt levels were higher in BMSCs cultural medium with FCS.Conclusions FCS could induce the differentiation of BMSCs into the cardiomyocyte-like cells.TGF-β1 and BMP-2 might not play a role in the differentiation of BMSCs induced by FCS.BIO enhanced the effects of FCS on the differentiation of BMSCs into cardiomyocyte-like cells,which might involve the Akt pathway.

STUDY ON PREVENTIVE AND CURATIVE EFFECTS OF LIU WEI DI HUANG TANG ON TUMORS

Liu Wei Di Huang Tang (LWDHT), a Chinese Prescription for strengthening the body re-sistance, restoring the normal functions of the body to consolidate the constitution, and nour-ishing and invigorating the kidney yin, has been used to prevent and treat severe hypeplasiaof esophageal epithelium for many years. The results of this experiment show that LWDHTcan increase markedly the number of lymphocytes, mainly T lymphocytes, in tumor--bearingmice. Free--flow electrophoresis shows that in tumor--bearing mice the electrophoretic char-acteristics of T lymphocytes are changed, i.e., reduction of the number of T lymphocyteswith a higher electrophoretic rate, but LWDHT can alleviate this disorder. Study on the cellmembrane fluidity of carcinoma cells in EAC mice demonstrates that LWDHT can decreasethe cell membrane fluidity. suggesting that it can inhibit division of carcinoma

Neodymium Oxide Induces Cytotoxicity and Activates NF-κB and Caspase-3 in NR8383 Cells

We investigated whether Nd_2O_3 treatment results in cytotoxicity and other underlying effects in rat NR8383 alveolar macrophages.Cell viability assessed by the MTT assay revealed that Nd_2O_3 was toxic in a dose-dependent manner, but not in a time-dependent manner. An ELISA analysis indicated that exposure to Nd203 caused cell damage and enhanced synthesis and release of inflammatory chemokines. A Western blot analysis showed that protein expression levels of caspase-3, nuclear factor-KB （NF-KB） and its inhibitor IKB increased significantly in response to Nd203 treatment. Both NF-KB and caspase-3 signaling were activated, suggesting that both pathways are involved in Nd203 cytotoxicity.

The Role of NF mRNA and Calpain in NF Reduction of Acrylamide Neuropathy

The purpose of this study was to study the role of neurofilament （NF） mRNA and calpain in NF reduction of acrylamide （ACR） neuropathy. Male Wistar adult rats were injected i.p. every other day with AER （20 mg/kg.bW or 40 mg/kg.bW） for 8 weeks. NF mRNA expression was detected using RT-PCR and the calpain concentration was determined using western blot analysis. The NF mRNA expression significantly decreased while the level of m-calpain and μ-calpain significantly increased in two ACR-treated rats groups regardless of the ACR dose. The light NF （NF-L） protein expression was significantly correlated with NF-L mRNA expression. Combined with previous data, the concentrations of three NF subunits were negatively correlated with the calpain levels. These findings suggest that NF-L mRNA and calpain mediated the reduction in NF of AER neuropathy.

Authentication of <i>Rothmannia whitfieldii</i>Dye Extract with FTIR Spectroscopy

Extraction of dye from dry fruit of Rothmannia whitfieldii was carried out using four different extraction methods. Solvent and acid extraction methods gave a colourless supernatant solution after extraction time of 45 minutes at 60°C. The alkali method gave a deep brown coloured supernatant solution while the aqueous method gave a dark coloured supernatant solution after extraction under the same conditions. From the result of the FTIR spectroscopy characterization of the coloured solutions and the dry powder of Rothmannia whitfieldii fruit, it was observed that only the alkali method extracted what can be called a dye with likely presence of tannins. The result also showed that the possible functional groups present in the supernatant solution after aqueous extraction are same with the functional groups present in the dry pulverized Rothmannia whitfieldii fruit. Hence, aqueous method did not extract any dye. Similarly, a mixture of the solution after aqueous extraction with drops of alkali solution produced a deep brown coloured solution indicating solubility of the dye component in alkali media.

In vitro and in vivo trypanocidal action of aescin and aescin liposomes against Trypanosoma evansi in experimental mice

Objective:To verify the trypanocidal effectiveness of aescin and aescin liposomes against Trypanosoma evansi in vitro and in vivo.Methods:Aescin and aescin liposomes were used in vitro on trypomastigotes at different concentrations(0.5%,1.0%and 2.0%) and exposure times(0,1,3,6 and 9 h).In vivo tests were performed using mice as the experimental model.Trypanosome evansi infected mice were treated with aescin and aescin liposomes with doses of 60 and 100 mg/kg during 4 d.Results:The three concentrations tested in free form and nanoencapsulated showed trypanocidal activity in vitro,completely eliminating the parasites in small concentration after6 h of assay.Animals treated with aescin(100 mg/kg) and aescin liposomes(100 mg/kg)showed increase in longevity,however without curative effect.Conclusions:Active compounds present in natural products,such as aescin,may potentiate the treatment of trypanosomosis when used in association with other trypanocidal drugs.

Effects of myoblast transfer of human erythro-poietin gene on the erythropoiesis in a rat model of renal failure

Anemia is an invariable symptom of end-stage renalfailure. Erythropoietin (Epo) has been used to improvethe quality of life of patients with end-stage renal failure,but repeated injection of Epo requires patients frequenthospital visits and easily spreads the infectious diseases.In order to investigate whether Epo gene therapy iseffective to renal anemia, we conduct this research work.Firstly, the retrovirus vector (pLEpoSN)

Propagation of Low Temperature-Adapted Hepatitis A Virus in BSC-1 Cell Culture

Hepatitis A virus, HM-175 strain, adapted to low temperature was cultivated in BSC-1 cell culture.Replication of the virus was detected by radioimmunoassay(RIA), radioimmunofocus assay (RIFA) and dot blot hybridization. The results showed that, of the 7 low temperature-adapted(LTA) strains studied, strain E, F and D gave the highest titer in BSC-1 cell culture. This culture system was proved to be suitable for the study of the characteristic of temperature mutants of HAV.

Transfer and expression of rhSCF gene in human hematopoietic stem/progenitor cells

Stem cell factor (SCF) is a novel growth factor thatinfluences the growth and development of hematopoieticcells, germ cells and melanocytes. To explore

Changes of tumorigenicity of the B16 melanoma cells transfected with MIP-1α gene

Macrophage inflammatory protein-l, a recentlycharacterized chemokine, consists of two chains (αand β). MIP-lα has been shown to exert strongchemotactic effect on neutrophils, monocytes and Tlymphocytes. In the present study, the B16 melanomacells were transfected with recombinant adenoviruscontaining MIP-lα gene. The biological characteri-zation of the MIP-1α gene transfected B16 melanomacells was investigated. The level of MIP-1α in thesupernatant of gene-transfected melanoma cells was368±24 ng/ml/10~6/24hr.. By using Boyden chambersystem, this supernatant showed strong chemotacticactivity for NK cells, CD4^+ T cells, CD8^+ T cells orthe freshly isolated peritoneal macrophages in vitro.Though the in vitro growth of the gene-transfected B16 melanoma cclls was not aftered, the in vivogrowth of the tumor cells subcutaneously inoculatedwas significantly inhibited. The infiltration ofinflammatory cells into the tumor mass formed bygene-transfected B16 cells was much more obviousthan that by