Inhibition of cyclooxygenase-2 by diallyl sulfides (DAS) in HEK 293T cells

Cyclooxygenase (COX) is involved in modulating inflammatory response through the synthesis of prostaglandins. The inducible isoform of the enzyme, COX-2, is overexpressed in some malignant and premalignant lesions. Several preclinical and clinical studies have reported COX-2 inhibition as an effective strategy for chemoprevention. Nonsteroidal anitinflammatory drugs (NASIDs) such as celecoxib, are the most widely investigated COX-2 inhibitors. The oil-soluble diallyl sulfides (DAS) include monosulfides (DAMS), disulfides (DADS) and trisulfides (DATS). They were found to be effective against canine and human tumors, the mechanism of which remains unresolved. We attempted a comparative evaluation of the antiproliferative effect of DAS in HEK 293T cells. The cells were treated with increasing concentrations of DAMS, DADS and DATS. There were significant differences between the IC50 values of DAMS, DADS and DATS. RT-PCR was performed and the expression of COX-2 was compared with that of b actin. DATS inhibited COX-2 gene expression significantly stronger than DAMS and DADS. The data are suggestive of antineoplastic effect of DAS, mediated by controlling COX-2 expression.     

实时荧光定量PCR法研究结直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化

目的通过研究结、直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化,揭示肠道相关菌群改变在大肠癌发病中的作用及意义。方法收集术前结、直肠癌患者粪便标本40例及正常对照标本40例,根据细菌的靶基因序列设计特异性引物。提取待测粪便标本细菌DNA,应用SYBR Green I实时荧光定量PCR测定不同细菌的数量。结果正常对照组与实验组粪便中细菌数量分别为拟杆菌属(8.76±0.77;9.85±0.88)、梭杆菌属(7.94±1.25;10.0±1.65)、梭菌属(3.54±0.70;6.56±0.68),拟杆菌属中的脆弱拟杆菌为(2.12±0.48;4.07±1.77)、梭杆菌属中的坏死梭杆菌为(2.31±0.26;7.62±2.68)及梭菌属中的肉毒梭菌为(2.76±1.16;5.43±1.21),实验组数量均明显增多(P0.05)。结论结、直肠癌患者粪便中拟杆菌属、梭杆菌属和梭菌属的数量较正常对照明显增多,提示结、直肠癌的发生发展与肠道菌群有明显关系。     

Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 μM DAS and 50 μM DADS but slightly affected with 100 μM DADS. Treatment with 50 and 100 μM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 μM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca²⁺]_i, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFκB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.     

Gut microflora of two saltmarsh detritivore thalassinid prawns,Upogebia africana andCallianassa kraussi

The presence and digestive capabilities of bacteria associated with the digestive systems and habitats of two saltmarsh-burrowing detritivore thalassinid prawns (Upogebia africana andCallianassa kraussi) was examined.U. africana is a filter-feeding prawn inhabiting muddy deposits, whereasC. kraussi, a deposit feeder, inhabits coarser more sandy deposits. Scanning electron microscopy was used to examine the gut lining and associated microflora and the nature of the ingested food of both prawns. The gut contents of both prawns included plant fragments, fragmented diatoms, partially degraded protozoa, and bacteria attached to organic matter. In bothU. africana andC. kraussi the midgut walls and gut contents were extensively coated by filamentous bacteria which were absent in the hindgut. The hindgut epithelium ofU. africana was coated by mats of rodshaped bacteria, not reported in marine invertebrates previously. The digestive glands of both species contained bacteria in the lumen. Isolation of gut and habitat bacteria suggests that bothU. africana andC. kraussi maintain a gut microflora distinct from the habitat microflora. Bacteria isolated from the guts of both species of prawn differed from those isolated from their respective habitats with regards to both the genera isolated and their digestive capabilities. The dominant genera isolated from the guts of bothU. africana andC. kraussi wereVibrio andPseudomonas, with an unidentified fermenter andPseudomonas, respectively dominating in the digestive glands. Bacteria of the genusAcinetobacter dominated the isolates from the habitats of both species of prawn. Resident gut bacteria isolated from the guts of both species of prawn exhibited lipase, protease, chitinase, and lysozyme, but not cellulase activity, and may contribute to nitrogen aquisition by the prawns. Isolates from the prawns' habitat exhibited alginase, gelatinase, and lipase activity, a few (3%) fromU. africana habitat having cellulases. In this study a distinction between resident gut bacteria and transient gut bacteria was made. Results suggest that some habitat bacteria remain viable in the guts ofU. africana, but not inC. kraussi.     

Alterations in the gut microbiota of patients with acquired immune deficiency syndrome

Acquired immune deficiency syndrome (AIDS), caused by infection with human immunodeficiency virus (HIV), is associated with gastrointestinal disease, systemic immune activation and changes in the gut microbiota. Here, we aim to investigate the gut microbiota patterns of HIV‐infected individuals and HIV‐uninfected individuals in populations from South China. We enrolled 33 patients with HIV (14 participants treated with highly active antiretroviral therapy [HAART] for more than 3 months; the remaining 19 individuals had not received treatment) and 35 healthy controls (HC) for a cross‐sectional comparison of gut microbiota using stool samples. Gut microbial communities were profiled by sequencing the bacterial 16S rRNA genes. Dysbiosis was more common among patients with AIDS compared with healthy individuals. Dysbiosis was characterized by decreased α‐diversity, low mean counts of Bacteroidetes, Faecalibacterium, Prevotella, Bacteroides vulgatus, Dialister and Roseburia inulnivorans, and high mean counts of Proteobacteria, Enterococcus, Streptococcus, Lactobacillus, Lachnociostridium, Ruminococcus gnavus and Streptococcus vestibularis. Increased abundance of Bacilli was observed in homosexual patients. Proteobacteria were higher among heterosexual patients with HIV infections. Tenericutes were higher among patients with history of intravenous drug abuse. Restoration of gut microbiota diversity and a significant increase in abundance of Faecalibacterium, Blautia and Bacteroides were found in patients receiving HAART compared to those who did not receive. HIV infection‐associated dysbiosis is characterized by decreased levels of α‐diversity and Bacteroidetes, increased levels of Proteobacteria and the alterations of gut microbiota correlate with the route of HIV transmission. The imbalanced faecal microbiota of HIV infection is partially restored after therapy.     

Some effects of ethyl alcohol on the growth of rumen bacteria

The effects of ethyl alcohol on the growth rate and yield of representative isolates of obligately anaerobic rumen bacteria were studied. Yield was monitored as turbidity at 600 nm until stationary phase was obtained and quantitated as protein per milliter. Rate was determined as the fastest rate of turbidity doubling. With very few exceptions, within the concentration range allowing growth, no effects of alcohol concentration were observed on rate, but substantial effects were found on yield. Growth of most of the organisms studied was completely inhibited at an alcohol concentration between 3% and 7% vol/vol, but the growth ofBacteroides amylophilus andRuminococcus flavefaciens was drastically reduced or eliminated at a 1% alcohol concentration. It is likely that major physiological and biochemical differences exist among anaerobic rumen bacteria, and taxonomically similar organisms, as a function of alcohol exposure.     

Structure-activity relationships of organosulfur compounds as inhibitors of cytochrome P450 1-mediated activation of benzo[a]pyrene in a human cell model

Twelve naturally-occurring organosulfur compounds were investigated as inhibitors of cytochrome P450 1 (CYP450 1)-mediated activation of benzo[a]pyrene (B[a]P) in human hepatoma (HepG2) cells. Inhibition depended on the presence of a diallyl group and the number of S atoms. Diallyl trisulfide (DATS), with a diallyl group and three S atoms, had the highest activity with an IC₅₀ of 0.4 mM, and 1.5-fold higher potency than diallyl disulfide (DADS) containing a diallyl group and two S atoms. Organosulfur compounds containing an alkyl group were less effective, or even ineffective, inhibitors of both CYP450 1 and B[a]P-induced cytotoxicity than DADS and DATS. Alliin and S-allyl cysteine containing the S-cysteinyl group had no inhibition.     

Inactivation of anaerobic bacteria by various photosensitized porphyrins or by hemin

The photodynamic effects of deuteroporphyrin (DP), hematoporphyrin derivative (HPD), hematoporphyrin (HP), or protoporphyrin (PP) on a variety of anaerobic microorganisms were examined in this study. The majority of the species, among the 350 strains tested, were inhibited by concentrations of 2.5 g/ml of light-activated DP. Species found to be resistant to this treatment includedBilophila wadsworthia, Fusobacterium mortiferum, Fusobacterium varium, andBacteroides gracilis. These species were inhibited by concentrations of >60 g/ml of DP. The porphyrin-producing species,Porphyromonas andPrevotella spp, were all inhibited by 2.5 g/ml DP and light. Comparing the photodynamic activity of the porphyrins used onPorphyromonas strains resulted in the following pattern: DP>HPD>HP>PP.Porphyromonas spp., Gram-positive cocci, and many Gram-positive rods (excluding clostridia) were inactivated by hemin (a metal-containing porphyrin) at 10–20 g/ml. Hemin inhibitory action was not affected by light. Binding and insertion of DP into bacteria (both inactivated and non-inactivated strains by DP and light) were monitored by the characteristic fluorescence band of bound DP at 622 nm.Porphyromonas spp. bound DP tightly, whereas only low binding was seen withB. wadsworthia and other DP-resistant species. High binding of DP toB. wadsworthia can be achieved by pretreatment of the bacteria with imipenem or cefoxitin, -lactam agents known to interfere with the integrity of the cell wall. If cell wall integrity is disturbed (e.g., by these agents), inactivation ofB. wadsworthia by DP can occur.     

Diallyl sulfide, diallyl disulfide and diallyl trisulfide affect drug resistant gene expression in colo 205 human colon cancer cells in vitro and in vivo

To elevate chemo-resistance of human cancer cells is a major obstacle in the treatment and management of malignant cancers. Diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are presented in the Alliaceae family particularly in garlic. Although DAS, DADS and DATS have been shown to exhibit anticancer activities, there is little information on effects of these compounds on drug resistant genes in human colon cancer cells in vitro and in vivo. Herein, we are the first to show that DAS, DADS and DATS at 25 μM for 24-h and 48-h incubations promoted expression of drug resistant genes in colo 205 human colon cancer cells. In vitro experiments indicated that DATS promoted gene expression of multidrug resistant 1 (Mdr1) (p<0.05), and DAS and DADS promoted MRP3 gene expression and DATS alone stimulated gene expression of multidrug resistance-associated protein-1 (MRP1) (p<0.05) in colo 205 cells. In vivo studies demonstrated that DADS and DATS induced Mdr1 and MRP1 gene expression (p<0.05). DADS promoted MRP3 gene expression (p<0.05) as well as DADS and DATS increased MRP4 and MRP6 gene expression (p<0.05) in the colo 205 xenograft mice. Based on our in vitro and in vivo results, diallyl polysulfides (DAS, DADS and DATS) affected the gene expression of the multidrug resistance in colo 205 human colon cancer cells in vitro and in vivo.     

基于16S rRNA基因高通量测序的 太原地区健康成年人肠道菌群结构分析

目的探讨太原地区健康成年人的肠道菌群结构及多样性。方法采集太原地区20份健康成年人的粪便样本,提取DNA、构建菌群16S rRNA基因克隆文库、高通量测序并进行生物信息学分析。结果测序获得优质序列1 383 680条,平均序列为69 184条±551条,归类于455个OTUs;所有样本共含有10个菌门,101个菌属,141种菌;其中核心菌门3个,依次为拟杆菌门(63.13%)、厚壁菌门(31.14%)和变形菌门(5.10%),占所有样本微生物总量的99.37%;丰度最高的前10个菌属依次为拟杆菌属(43.34%)、普氏菌属(15.51%)、栖粪杆菌属(4.92%)、罗氏菌属(4.73%)、毛螺菌属(3.27%)、萨特菌属(2.50%)、粪球菌属(2.38%)、布劳特菌属(2.31%)、瘤胃球菌属(2.18%)和副杆状菌属(1.61%),占样本微生物总量的82.75%,未分类的菌属占6.84%。结论健康成年人肠道微生物群落复杂,但主要菌群相对稳定,为进一步研究人体肠道菌群结构与功能提供了参考依据。     

Vitamin K composition of anaerobic gut bacteria

The menaquinone (vitamin K₂) composition of a wide range of obligately anaerobic bacteria was investigated using chromatographic and physicochemical techniques. Members of the genera Bifidobacterium, Butyrivibrio, Clostridium, Fusobacterium, Lachnospira, Megasphaera, Propionispira, Ruminococcus, Selenomonas, Succinovibrio and Succinomonas were found to lack menaquinones, In contrast, all species of the genera Actinomyces, Arachnia, Bacteroides, Propionibacterium, Veillonella and Wolinella examined possessed menaquinones. Considerable variation in length and degree of saturation of the C-3 isoprenyl side-chains of the menaquinones isolated from these taxa were evident. In addition to menaquinone, strains of Wolinella and Eubacterium lentum contained methylated menaquinones. The possible contribution of bacterial menaquinones to the total pool of vitamin K in the human gut is discussed.     

Indigenous microbes and their soluble factors differentially modulate intestinal glycosylation steps in vivo

It has been shown that Bacteroides thetaiotaomicron, a representative member of the gut microflora, signals intestinal epithelial cells both in vivo and in vitro and modulate specific glycosylation processes that may mediate intestinal functions. However it is not known whether these modulations depend on the presence of live bacteria or may be elicited by soluble factors produced in vitro by this bacterium. We used lectins and an histochemical approach to survey tissue sections prepared from various cellular compartments of the small and large intestine of NRMI/KI mice grown under gnotobiotic conditions. We compared the results obtained with bacterial culture supernatant and live B. thetaiotaomicron to those obtained from germ-free mice or mice having a conventional microflora. This approach allowed us to conclude that (1) a small but specific number of glycan patterns were restored after treatment with bacterial culture supernatant and (2) the B. thetaiotaomicron associated mice restored a larger number of patterns, however, the complete conventional mice pattern must be a function of the whole microflora in the gut. The possibility to modulate this complex glycosylation pattern by introducing exogenous bacteria and bacterial products should be considered as a promising approach towards understanding the molecular basis of microbial-host interactions.     

草鱼肠道黏膜厌氧细菌的分离与鉴定

研究以草鱼(Ctenopharyngodon idellus)为实验对象, 运用厌氧培养的方法, 研究了饥饿状态下草鱼肠道黏膜固有微生物的类群及其在不同肠段的分布。实验结果显示草鱼前肠、中肠与后肠中细菌的数量分别是3.17×103、1.63×104和1.79×107 cfu/g。研究共分离到274株单菌落, 经16S rRNA鉴定, 分别属于拟杆菌属(Bacteroides spp.)、鲸杆菌属(Cetobacterium spp.)、梭形杆菌属(Fusobacterium spp.)、气单胞菌属(Aeromonas spp.)、希瓦氏菌属(Shewanella spp.)、芽孢杆菌属(Bacillus spp.)、泛菌属(Pantoea spp.)和柠檬酸杆菌属(Citrobacter spp.)8个种类, 其中专性厌氧细菌的数量占9.1%, 兼性厌氧细菌的数量占90.9%。进一步分析发现, 前肠中只分离到兼性厌氧细菌, 中肠与后肠专性厌氧细菌和兼性厌氧细菌都有分布。专性厌氧细菌Bacteroides paurosaccharolyticus和Bacteroides luti在中肠与后肠都有分布, 而Cetobacterium somerae和Fusobacterium ulcerans只在后肠有发现。兼性厌氧细菌是草鱼肠道黏膜的优势菌群, 其中嗜水气单胞菌Aeromonas hydrophila占73.7%。草鱼肠道不同部位固有厌氧微生物组成存在差异, 细菌数量也明显不同, 后肠中具有更高的细菌丰度和多样性。     

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132^T and B. longum JCM1217^T) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.     

Influence of organosulphur compounds from garlic on the secretion of matrix metalloproteinases and their inhibitor TIMP-1 by cultured HUVEC cells

Organosulphur compounds from garlic, especially diallyl disulphide (DADS) at non-toxic concentrations, affected production and secretion of some matrix metalloproteinases (MMPs) and of tissue inhibitor of metalloproteinase-1 (TIMP-1), one of their inhibitors, by human umbilical vein endothelial cells. Addition of DADS to the culture medium resulted in a concentration-dependent reduction of secreted MMP-2 protein and activity as well as TIMP-1 protein. In the presence of inducers (phorbol 12-myristate 13-acetate, forskolin and tumor necrosis factor alpha) addition of DADS caused a distinct concentration-dependent decrease of MMP-9 and TIMP-1 secretion, while not affecting MMP-9 mRNA levels. Intracellular protein levels remained low and were not affected. Other organosulphur compounds like allyl mercaptan and S-allylcysteine showed no or less clear effects on MMP-secretion or TIMP-1-secretion. These results suggest that DADS may mediate some of the biological effects ascribed to garlic preparations through affecting MMP-TIMP balance.     

The Inhibitory Effects of Garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) and Diallyl Trisulfide on <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> and other Harmful Algal Species

Using cell suspension ability as an indicator, we studied the inhibitory effect of garlic (Allium sativum) and diallyl trisulfide on six species of red tide causing algae. This included: the inhibition by 0.08% garlic solution of five algal species — Alexandrium tamarense, Scrippsiella trochoidea, Alexandrium catenella, Alexandrium minutum and Alexandrium satoanum; the effects of garlic concentration on the inhibition of A. tamarense, S. trochoidea and Chaetoceros sp.; the effects of inhibitory time on the rejuvenation of algal cells; and the effects of heating and preservation time on algal inhibition by garlic solution. In addition, whether or not the ingredients of garlic solution had a possible algicidal effect was studied by comparing inhibition of A. tamarense by garlic solution and man-made diallyl trisulfide. The results showed that 1) inhibition by garlic solution was significant on A. tamarense, A. satoanum, A. catenella and S. trochoidea, and the least effective was a concentration of 0.04% on A. tamarense and S. trochoidea. Moreover, the higher the concentration, the stronger was the inhibition, and a high inhibitory rate (IR) could be maintained for at least three days when the garlic concentration was above 0.04%. For A. tamarense, it was also found that the longer the inhibitory time and the higher the concentration, the lower was the rate of resumed cell activity. On the contrary, garlic solution could not inhibit A. minutum or Chaetoceros sp.; 2) The IR to A. tamarense was reduced slightly as the heating time of the garlic solution was lengthened, but the average IR was still above 80%. There was no significant difference between the IR of the supernatant and sediment of the garlic solution. Furthermore, no change of algal inhibition was found when the garlic solution was preserved at 20°C for several days; 3) As with garlic solution, diallyl trisulfide inhibited A. tamarense strongly; the IR was above 93% and was maintained for at least three days, as long as the concentration was 3.2–10.0 mg L⁻¹. Thus, diallyl trisulfide may have been the major ingredient in garlic solution which inhibited the algae but, in addition, more than one ingredient may have been inhibiting the algae. In conclusion, garlic was a good algal inhibitor with many advantages, such as being common, cheap, non toxic and with high efficiency, and diallyl trisulfide, one of the components of garlic, was similarly effective in algal inhibition. It would be useful, therefore, to further study garlic as an environmentally friendly algal inhibitor.     

Yeast mannan increases Bacteroides thetaiotaomicron abundance and suppresses putrefactive compound production in in vitro fecal microbiota fermentation

ABSTRACT

Yeast mannan is a part of yeast cell wall and can potentially affect gut microflora as a soluble dietary fiber. We demonstrated that yeast mannan suppressed putrefactive production and increased the relative abundance of Bacteroides thetaiotaomicron in in vitro fecal fermentation. These results suggest that yeast mannan can be used as a novel prebiotic food ingredient.     

Characterization of some members of the genusThermomonospora

Twenty strains ofThermomonospora, isolated from the environments of patients with hypersensitivity pneumonitis or received from culture collections or other workers, were studied for their detailed morphological, physiological, and immunological characteristics. The species studied includedThermomonospora fusca, T. alba, T. curvata, T. mesophilia, andT. mesouviformis. The results indicate that several species can be differentiated by various criteria studied; however, two species,T. fusca andT. alba, could not be separated from one another by any of the tests or methods used in the study.     

Inhibition of the benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene-induced toxicity by allyl sulfides in human epidermal keratinocytes

Diallyl sulfide (DAS) and diallyl disulfide (DADS) at 25 g ml^–1decreased the benzo[a]pyrene (B[a]P)-induced colony growth inhibition of human epidermal keratinocytes. DAS and DADS decreased B[a]P-DNA and B[a]P-protein adducts by 65% and 49–55%, respectively. The B[a]P-induced ethoxyresorufin O-deethylase activity, a marker enzyme for cytochrome P450 1, was decreased from 3 to 1.7–1.9 nmol min^–1 mg^–1 microsomal protein by DAS and DADS treatments. The activity of glutathione S-transferase, a detoxifying enzyme for B[a]P, but was decreased by DADS, but was unaffected by DAS.